icax sae he a { os eh a . ose eA De pree Re 4s & tacn 96 ~ 7 a ed - * ae — . 7 : Las ie af , a outs . 7.) = e's « * O awe ot o% os &f . rl vets 7 > *™ | ,. Dawes 45755, 2 ALBERT R. MANN LIBRARY NEW YorK STATE COLLEGES OF AGRICULTURE AND HoME ECONOMICS AT CORNELL UNIVERSITY ‘ornell Universit anual of bacteriology, Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003240433 MANUAL OF BACTERIOLOGY PUBLISHED BY THE JOINT COMMITTEE OF HENRY FROWDE AND HODDER & STOUGHTON AT THE OXFORD PRESS WAREHOUSE FALCON SQUARE, LONDON, E.C.1 MANUAL OF BACTERIOLOGY BY ROBERT MUIR, M.A., M.D., Se.D., F.R.S. PROFESSOR OF PATHOLOGY, UNIVERSITY OF GLASGOW AND JAMES RITCHIE, M.A., M.D., F.R.C.P.(Ep.) IRVINE PROFESSOR OF BACTERIOLOGY, UNIVERSITY OF EDINBURGH FORMERLY FELLOW OF NEW COLLEGE, OXFORD SEVENTH EDITION WITH TWO HUNDRED ILLUSTRATIONS IN THE TEXT AND SIX COLOURED PLATES LONDON HENRY FROWDE HODDER & STOUGHTON Oxrorp University Press Warwick Square, E.C. 1919 PRINTED IN GREAT BRITAIN BY Morrison & Grips Lav., EDINBURGH C® 148 44 PREFACE TO THE SEVENTH EDITION. — THE outstanding feature of the interval that has elapsed since the sixth edition of this work appeared is the impetus given to bacteriological research by the urgent requirements of practical medicine and surgery in the war. The success which has attended the efforts put forth is shown in the additions we have made to the chapters dealing with cerebro-spinal fever, with intestinal infections,—both bacterial and protozoal,—with tetanus, and with the grave conditions occurring in wounds. It is manifest likewise in the inclusion of new sections on infective jaundice and also on trench fever, which observations in both the field and the laboratory have differentiated from allied affections. Apart from aspects of bacteriology brought into prominence through the war, the whole book has _ been thoroughly reviséd, a number of new methods have been described, and new illustrations have been added. October 1918. PREFACE TO THE FIRST EDITION. —+—. Tue 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. 174-179 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 BIoLoey. InrRopucrory—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 A ; : c CHAPTER II. MetHops oF CULTIVATION OF 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 CHAPTER III. Microscopic MrtHops. The microscope—Examination of hanging-drop cultures—Film pre- parations—Examination of bacteria in tissues—The cutting of sections—Staining principles—Mordants and decolorisers —Formule of stains—Gram’s method and its modifications —Stain for tubercle and other acid-fast bacilli—Staining of spores, capsules, and flagella—The Romanowsky stains ix PAGE 26 89 x CONTENTS CHAPTER IV. EXAMINATION OF SERUM—PREPARATION OF VACCINES— GENERAL BactTeRioLocicaL D1acNosis—INocULATION oF ANIMALS. PAGE Observation of agglutination and sedimentation—Opsonic methods —Method of measuring the phagocytic capacity of the leuco-. cytes—Bactericidal methods—Hemolytic tests—Fixation and deviation of complement—Wassermann reaction— Preparation of vaccines—Methods of counting bacteria in dead cultures— General bacteriological diagnosis—Inoculation of animals— Autopsies on animals . | 7 : : . 2 5 | | CHAPTER V. Bacteria In Arr, Sort, WaTER, MiLK—ANTISEPTICS. Atk: Methods of examination. So1z: Methods of examination— Varieties of bacteria in soil. Water: Methods of examination —Bacteria in water—Bacteriology of sewage. MuiLx: Souring of milk—Pathogenic organisms in milk—Sterilisation of milk. ANTISEPTICS: Methods of investigation—The action of anti- septics—Certain particular antiseptics . é ‘ » 143 CHAPTER VIL. RELATIONS OF Bacteria To DisEASE—THE PRropucTIoN or Toxins By Bacteria. Introductory — Conditions modifying pathogenicity — Carriers — Modes of bacterial action—Tissue changes produced by bacteria —Local lesions—General lesions—Disturbances 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 F 5 : - 174 CHAPTER VII. INFLAMMATORY AND SUPPURATIVE ConpITIONS, 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—Ulecerative endocarditis—Acute suppur- CONTENTS xi PAGE ative periostitis — Erysipelas — Conjunctivitis — Acute rheu- matism—Vaccination treatment of infections by the pyogenic cocci—Methods of examination in inflammatory and suppur- ative conditions . . : : 2 > . 197 CHAPTER VIII. . INFLAMMATORY AND SUPPURATIVE CONDITIONS, continued : Tue Acute Pneumonias, EPIDEMIC CEREBRO-SPINAL MENINGITIS. Introductory—Historical—Fraenkel’s pneumococcus—Experimental inoculation—Strains of pneumococeus—Pathology of pneumonia —Methods of examination —Friedlander’s pneumobacillus. Eprpemic CEREBRO-SPINAL MENINGITIS—Serum reactions— Allied diplococei < ; : : : : . 225 CHAPTER IX. GoNORRH@A AND Sort Sore. The gonococcus — Microscopical characters — Cultivation — Com- parison with -meningococcus—Relations to the disease—Its toxin—Distribution—Gonococeus in joint affections—Methods of diagnosis. Sorr SorE: Microscopical characters and culti- vation of bacillus : . : : , . 255 CHAPTER X. 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-—Specific reactions of the tubercle bacillus —Tuberculins—Phenomena of supersensitiveness—Tuberculin reactions — Toxins of the tubercle bacillus —- Immunity phenomena in tuberculosis—Therapeutic application of the tubereulins — Active immunisation associated with opsonic observations—Antitubercular sera—Methods of examination . 266 xii CONTENTS?’ CHAPTER XI. LEPRosY. PAGE a Pathological changes—Bacillus of leprosy—Position of the bacilli —Relations to the disease—Methods of diagnosis 2 . 299 CHAPTER XII GLANDERS AND RHINOSCLEROMA. Guanpers: The natural disease—The glanders bacillus—Cultiva- tion of glanders bacillus—Powers of resistance—Experimental inoculation—Action on the tissues—Mode of spread—Serum reactions—Mallein and its preparation—Methods of examina- tion—RHINOSCLEROMA . ‘ : z . 309 CHAPTER XIII. ACTINOMYCOSIS AND ALLIED DIsHASES. 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. é . 3820 CHAPTER XIV. ANTHRAX, Historical summary—Bacillus anthracis—Appearauces 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 . 834 CHAPTER XV. TypHoID Fever—Baciiii ALLIED To THE TyPHoID Baciiuvs. Introductory — Bacillus coli communis — Culture reactions — Isolation and recognition of B. coli—Pathogenic properties— Bacillus typhosus—Isolation and appearances of cultures— CONTENTS xiii PAGE Biological reactions—Pathological changes in typhoid fever— Immunisation of animals—Etiological relationships of bacillus typhosus—Epidemiology of typhoid fever—Typhoid carriers — —Serum diagnosis of typhoid fever—Vaccination against typhoid—Methods of examination—Paratyphoid fever—The bacillus paratyphosus—Bacillus enteritidis (Gaertner)—The psittacosis bacillus—Danysz’s bacillus and rat viruses—Bacillary dysentery—Summer diarrhea—Differentiation of coli-typhoid group by culture and agglutination—Varieties of B. coli— Mutation in coli-typhoid bacilli : 5 . . 3853 CHAPTER XVI DIPHTHERIA. Historical — General facts— Bacillus diphtheria — Microscopical characters — Distribution — Association with other organisms —Cultivation—Powers of resistance—Inoculation experiments —The toxins of diphtheria—Variations in virulence of bacilli— Bacilli allied to the diphtheria bacillus—Summary of ee genic action—Methods of diagnosis é . . 398 CHAPTER XVII. TrTanus—OTHER ANAEROBIC BACILLI. 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 cedema—Characters of bacillus—Experimental inoculation— Methods of diagnosis—ANAEROBES IN INFECTED WounDs— Bacillus botulinus — Quarter - evil —FustrorM ANAEROBIC BaciLul . : : : . . i . 419 CHAPTER XVIII. CHOLERA. In roductory—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— Metchnikoff's spirillam—Finkler and Prior’s spirillum—Deneke’s spirillum . 460 xiv CONTENTS CHAPTER XIX. InrLuENza, WHoopine-Coveu, PLacugz, Mata FEvER. INFLUENZA BACILLUS : Microscopical characters—Cultivation—Dis- tribution—Experimental inoculation—Methods of examina- tion. Wxoopinc-coucH BaciLuus : Microscopical characters— Pathogenic effects—Methods of examination. BaciLLus OF PuLaGuE: Microscopical characters—Cultivation—Anatomical changes produced and distribution of bacilli—Experimental inoculation—Paths and mode of infection—Toxins, immunity, etc.—Preventive inoculation—Anti-plague sera—Methods of diagnosis. Matra Fever: Micrococcus melitensis—Relations to the disease—Mode of wigan of the disease—Methods of diagnosis : . . . CHAPTER XX. DISEASES DUE TO SPIROCHATES—THE RELAPSING FEVERS, SYPHILIS, AND FRAMB@SIA. ReLapsine Fever: Characters of the spirochete—Relations to the disease—Immunity. AFRICAN Tick FrvEer: Transmission of the disease. SypuHitis: Microscopic characters of spirochete pallida — Distribution — Cultivation — Transmission of the disease—Serum diagnosis— Wassermann reaction. FRAMB@SIA or Yaws. INFECTIVE JAUNDICE. RAT-BITE FEVER CHAPTER XXL PatHoGENICc FUNGI. Botanical description — Methods — Microspora — Trichophyta— , Achoria—Thrush— Aspergillosis — Sporotrichosis — Blastomy- cosis—Microsporon furfur CHAPTER XXII. Immunity. Introductory—Acquired immunity—Artificial immunity—Varieties —Active immunity—Methods of production—Attenuation and exaltation of virulence—Properties of immune-sera—Antitoxic serum—Standardising of toxins and of antisera—Nature of PAGE 506 530 CONTENTS antitoxic action—Ehrlich’s theory of the constitution of toxins— Antibacterial serum—Bactericidal and lysogenic action— Hemolytic and other sera—Methods of ‘the hemolytic tests— Opsonic action — Agglutination — Precipitins — Therapeutic effects of anti-sera—Theories as to acquired immunity—Ehrlich’s side-chain theory—Theory of phagocytosis—Natural immunity — Natural bactericidal powers— Natural susceptibility to toxins—Supersensitiveness or anaphylaxis—The serum disease in man—Desensitisation APPENDIX A. SMALLPOX AND VACCINATION. Jennerian vaccination—Relationship of smallpox to cowpox— Virus of smallpox—The nature of vaccination - APPENDIX B. HYDROPHOBIA Introductory—Pathology—The virus of i a —Antirabic serum—Methods—Chlamydozoa e APPENDIX C. MALARIAL FEVERS. The malarial parasite—The cycle of the malarial parasite in man —The cycle in the mosquito—Varieties of the malarial parasite — General considerations—The pathology of malaria—Methods of examination , : . APPENDIX D. Ama@pic DYSENTERY. Amebic dysentery—Characters of the amcebe—Cultivation of the amcebe-—Distribution of the amcebe—Experimental inocula- tion—Methods of examination . Xv PAGK 552 604 611 624 641 xvi CONTENTS APPENDIX E. TRYPANOSOMIASIS— LEISHMANIOSIS— PIROPLASMOSIS. THE PaTHocENIC TRYPANOSOMES: Morphology and biology of the trypanosomata—Trypanosoma lewisi—Nagana or tse-tse fly disease—Trypanosome of sleeping sickness—Trypanosoma rhodesiense—Trypanosoma cruzi. LyisHmMawiosis : Leishmania donovani—Leishmania infantum—Leishmania tropica—Husto- plasma capsulatum. PrRoPLASMOSIS APPENDIX F. YELLOW FEVER. i ‘ . : 5 APPENDIX @G. EpripemMic PoLIOMYELITIS . APPENDIX H. PHLEBOTOMUS FEVER APPENDIX J. TypHus FEVER APPENDIX K. TRENCH FEVER BIBLIOGRAPHY INDEX . PAGE’ 651! 679 684 692 694 697 701 735 co Oo N D 10. 11. 12, 18. 14. 15. PF ON HS LIST OF COLOURED PLATES. —~ PLATE I. . Film of pus, containing staphylococci and streptococci. . Fraenkel’s pneumococcus in sputum. . Meningococcus in epidemic cerebro-spinal fever. . Film from a scraping of throat in Vincent’s angina, showing fusiform bacilli and spirochetes. . Gonorrheal pus, showing gonococci and staphylococci. PLATE II. . Spirochete pallida, case of congenital syphilis. . Tubercle bacillus and other bacterium in sputum. . Leprous skin, showing clumps of bacilli in the cutis. . Leprous granulation tissue, showing bacilli. PLATE III. Streptothrix actinomyces. Anthrax bacilli. Bacillus diphtheria. Bacillus diphtherie (involution forms). Hofmann’s pseudo-diphtheria bacillus. xvii Typhoid bacillus, showing flagella. 6 xviii LIST OF COLOURED PLATES PLATE IV. FIG. 16. Negri bodies in nerve cells in rabies. 17. Bacillus pesti8 (involution forms). 18. Spirochete of relapsing fever. 19. The cholera spirillum, showing flagella. 20. Bacillus tetani, showing spores, PLATE V. 21. The parasite of mild tertian malaria. Cycle I, (Schizogony). Asexual cycle in the human blood. Cycle II. (Sporogony). Sexual cycle in the mosquito. 22. The parasite of malignant malaria. PLATE VI. 23. Entameeba histolytica in pus, from tropical abscess of liver. 24, Leishman-Donovan bodies, from a case of kala-dzar. 25. Trypanosoma gambiense. LIST OF ILLUSTRATIONS IN TEXT. —-— Fie. PAGE 1. Forms of bacteria 3 : ‘ : » 13 2. Hot-air steriliser 2 i : ‘ . ¢ 28 3. Koch’s steam steriliser . i : : ‘ « 28 4, Autoclave 3 : ‘ : 7 » 80 5. Steriliser for blood serum 3 : . 8l 6. Meat press 3 < Z ! : - 82 7. Hot-water funnel . ‘ ‘ A ‘ . 87 8. Blood serum inspissator . : ‘ x @1 9. Cylinder of potato cut obliquely ' . 47 10, Ehrlich’s tube, containing piece of potato a i . 47 11. Apparatus for filling tubes . " ; . 55 12, Tubes of media . - ‘ . . 55 13. Platinum wires in glass handles: Pa . . . 56 14. Method of inoculating solid tubes : : . » BF 15. Rack for platinum needles ‘ , : : . 57 16. Petri’s capsule . ‘ . 58 17. Apparatus for supplying hydrogen for anaenabie ugttares - 62 18. Bulloch’s apparatus for anaerobic plate cultures - - 63 19. Lid of M‘Intosh and Fildes anaerobic jar f si . 64 20. M‘Leod’s capsule for anaerobic plating . : . 65 21. Henry’s apparatus ‘ : . . 65 22. Flask for anaerobes in liquid reli . ‘ . 67 28, Flask arranged for culture of anaerobes which develop gas. 68 24, Tubes for anaerobic cultures on the surface of solid media . 69 25. Slides for hanging-drop cultures : é , . 70 26. Geissler’s vacuum pump for filtering cultures . = #4 27. Chamberland’s candle and flask arranged for cation, 74 28. Chamberland’s bougie with lamp funnel . : ee) 29. Bougie inserted through rubber stopper : e . 75 30. Muencke’s modification of Chamberland’s filter ‘ 2 46 31. Flask for filtering small quantities of fluid : ; 5 UE 32. Tubes for demonstrating gas-formation by bacteria. 80 xix xx LIST OF ILLUSTRATIONS IN TEXT FIG. 33. Geryk air-pump for drying tn vacuo. " . 34, Reichert’s gas regulator “ é 7 . . 35. Hearson’s incubator for use at 37°C. . . c f 36. Cornet’s forceps for holding cover- glasces r 87. Needle with square of paper on end for nfuriipalating paraifin sections : 38. Siphon wash- bottle for distilled anheee 39. Wright’s 5 c.mm. pipette : . 40. Tubes used in testing eee aa sedimenting properties of serum n : F ‘ 41. Wright’s blood- eapante 42. Test-tube and pipette arranged for chidinings fluids contaitiling bacteria : . ; E ‘ . 43, Petri’s sand filter 44, Staphylococcus pyogenes incu vouny suftune on agar, x 1000 : , 45. Two stab cultures of staphgtoconms ipasation aureus in ngalatin 46. Streptococcus pyogenes, young culture on agar. x 1000 47. Culture of the streptococcus pyogenes on an agar plate 48. Micrococcus tetragenus ; young culture on agar. x 1000 49. Bacillus pyocyaneus ; young culture on agar. x 1000 ‘50. Streptococci in acute suppuration. x1000 . z 51. Minute focus of commencing suppuration in brain. x 50 52. Secondary infection of a.glomerulus of kidney by the staphylo- coccus aureus. x 800 5 : m 58. Section of a vegetation in ulcerative silent x 600 54, Film preparation from a case of acute conjunctivitis, nee the Koch-Weeks bacilli. x 1000 55. Koch-Weeks bacillus from a young culture on blood sate x 1000 56. Film preparation of senjanetioral sexe bien) showing the aiplo bacillus of conjunctivitis. 1000. 57. Film preparation of pneumonic sputum, showing numerous ' pneumococci (Fraenkel’s). x 1000 58. Fraenkel’s pneumococcus in serous exudation. x 1000 59. Stroke culture of Fraenkel’s pneumococcus on blood agar 63. 64, . Fraenkel’s aaa from a pure culture on blood agar. x 1000 . Capsulated pabinsende! in Blood iuken roan the heat of a a rabbit. x1000.—«. . Friedlander’s pneumobacillus, from enidats in a case of pneumonia. x 1000 Stab culture of Friedlander’s pheumclinedlie Friedlinder’s pneumobacillus, from a young culture on agar, x 1000 . . . . : . LIST OF ILLUSTRATIONS IN TEXT . Film preparation of exudation from a case of meningitis. x 1000 . Two-day colonies of the meningococcus on Martin’s medium. xo, ri ‘8 . Pure culture of diplococeus intracellularis . Portion of film of gonorrheal pus. x 1000 . Colonies of gonococcus on serum-agar . : : . Gonococci, from a pure culture on blood-agar. x 1000 . Film preparation of pus from soft chancre, showing Ducrey’s bacillus. x 1500 Ducrey’s bacillus. x 1500 . Tubercle bacilli, from a pure culture on glycerin agar. 1000 . Tubercle bacilli in phthisical sputum. x 1000 . Cultures of tubercle bacilli on glycerin agar . Tubercle bacilli in section of human lung in acute phehiats x 1000 - . . Tubercle bacilli in giant-cells. x 1000 . Tubercle bacilli in urine. x1000 . . Bovine tubercle bacilli in milk. x 1000 . Cultures of bovine and human tubercle bacilli, 5 ‘eules old, on glycerin egg . . Moeller’s Timothy-grass , bacillus, x 1000 a : - . Cultures of acid-fast bacilli grown at room gi . Smegma bacilli. = 1000 ‘ : . Section through leprons skin, showing the masses of oéliadar grantlation: tissue in the cutis. x 80 ‘ i . Superficial part of leprous skin. x 500 . High-power view of portion of leprous nodule, showing the arrangement of the bacilli within the cells of the granula- tion tissue. 1100 . Kedrowski’s leprosy bacillus. 1000 : p . Glanders bacilli from peritoneal exudate of guinea-pig. x 1000 . Glanders bacilli. x 1000 ; 3 ‘ ‘ ; . Actinomycosis of human liver. x 500 . Actinomyces in human kidney. x 500 . Colonies of actinomyces. x 60 ‘ . Cultures of the actinomyces on glycerin agar . ‘ . Actinomyces, from a culture on glycerin agar. x 1000 . Shake cultures of actinomyces in glucose agar . : . Section of a colony of actinomyces from a culture in blood serum, x 1500 é # . Streptothrix madure. x 1000 : : . Surface colony of the anthrax bacillus on an ipa plate. BO «4 . Anthrax bacilli, arranged i in chains, from a twenty: four hours’ 1 culture on agar at 37°C. =x 1000 . xxi PAGE 245 246 247 256 257 257 264 264 268 269 271 275 276 277 279 280 284 285 286 300 302 308 304 311 312 322 323 324 326 327 328 329 331 336 337 Xxii FIG. 100. 101. 102. 108. 104. 105. 106. LIST OF ILLUSTRATIONS IN TEXT Stab culture of the anthrax bacillus in peptone-gelatin Anthrax bacilli containing spores. x1000 . : a Scraping from spleen of guinea-pig dead of anthrax. x 1000 Portion of kidney of a guinea-pig dead of anthrax. x 300 Bacillus colicommunis. 1000 7 ; A large clump of typhoid bacilliin a spleen. 500. Typhoid bacilli, from a young culture on agar, showing some filamentous forms. 1000 © . Typhoid bacilli, from a young culture on " agar, stepine flagella. 1000 . Culture of the typhoid bacillus and of he bacillus wale . Colonies of the typhoid bacillus on a gelatin plate. x15 . Film preparation from diphtheria membrane, showing numerous diphtheria bacilli. 1000 . Section through a diphtheritic membrane in ee show ing diphtheria bacilli. x 1000 . Cultures of the diphtheria bacillus on an agar plate . Diphtheria colonies, two days old, on agar. x8 . Diphtheria bacilli, from a twenty-four hours’ culture on en x 1000 . Diphtheria bacilli, Boer: a lives, days’ — exilinive: x 1000. : . Involution forms of the diphtheria bacillus. 1000. . Pseudo-diphtheria bacillus (Hofmann’s). x 1000 . Xerosis bacillus from a young agar culture. 1000. . Film preparation of discharge from wound in a case of tetanus, showing several tetanus bacilli of ‘‘dramstick” form. -x1000 . Tetanus bacilli, showing fagella, x 1000 é . Spiral composed of numerous twisted flagella of the sateen bacillus. x 1000 . Tetanus bacilli, some of which possess spnies: x 1000 . Stab culture of the tetanus bacillus in glucose gelatin . . Colonies of the tetanus bacillus on agar, seven days old. x 50 . Film taken from margin of spreading gas gangrene, showing b. welchii. x 1000 e . Film from necrosed muscle in gas gangrene. _ x 1000 . Film from a pure culture of b. welchii. x 1000 . Bacillus welchii, showing capsules. 1000 . . Film preparation from the affected tissues in a case af malignant edema. x 1000 . Bacillus of malignant cedema, showing spores. 1000 . Stab cultures in agar—tetanus bacillus, bacillus of nove cedema, and bacillus of quarter-evil . . B. sporogenes, pure culture, showing sub- joni ebotbe, x 1000 "PAGE 337 339 342 343 354 359 360 361 362 363 400 401 403 403 404 404 405 415. 416 421 422 423 423 424 425 443 443 444 445 449 450 451 455 Fie. 133. 134. 135. 186, 137. 138. 139. 140. 141. 142. 1438. 144, 145. 146. 147. 148. 149. 150. 151. 152. 1538, 154. 155. LIST OF ILLUSTRATIONS IN TEXT Bacillus of quarter-evil, showing spores. x 1000 Film preparation from a case of Vincent’s angina. x 1000 Cholera spirilla, from a culture on agar of twenty-four hours’ growth. x1000 . . Cholera spirilla stained to show the terminal flagella, x 1000 Cholera spirilla from an old agar culture. x 1000 Puncture culture of the cholera spirillum Colonies of the cholera spirillum on a gelatin plate Metchnikoff’s spirillum. 1000 - 2 Puncture cultures in peptone-gelatin Finkler and Prior’s spirillum. x 1000 : : Influenza bacilli from a culture on blood-agar. x 1000 3 Film preparation from a twenty-four hours’ culture of the whooping-cough bacillus. 1000. ‘ Film preparation from a plague bubo. x 1000 Bacillus of plague from a young culture on agar. x 1000 Bacillus of plague in chains. 1000. Culture of the bacillus of plague on 4 per cent. salt ager. x 1000 6 ‘ Section of a human anpibatie glenda in ‘planes, x 50 ‘ Film preparation of spleen of rat after inoculation with the bacillus of plague. 1000. 7 eS : Micrococcus melitensis. 1000 i : Spirocheates of relapsing fever in human Blood, x about 1000 Spirochete obermeieri in blood of infected mouse. 1000 Film of human blood containing spirochete of tick fever. x 1000 Spirillum of human tick fever (spirillum duttoni) in blood of infected mouse. 1000 . ei . . 156 and 157. Film preparations from juice of hard chanene, showing 158. 159. 160. 161. 162. 163, 164, 165. 166, 167. 168, spirochete pallida. 1000 ‘ Film preparation from juice of hard chanere, Sieiaaits spirochaete pallida. x 2000 Section of spleen from a case of congenital syphilis showing spirochete pallida. x 1000. ‘ Spirochete refringens. - x 1000 3 , Specimens of spirochete ictero-hemorrhagie. x 1000 Forms of fungi . : Hair infected with HGriesparen ‘autlestiad: x 500 Microsporon audouini on Sabouraud’s maltose agar Trichophyton crateriforme and eneaees rosaceum on Sabouraud’s medium . Hair infected with large-spored ringworm. 500 Favus hair, showing air channels left by mycelium. x 300 Achorion schénleinii on Sabouraud’s maltose agar, and cultures of Achorion quinckeanum . é . - xxiii PAGE 457 458 461 462 462 464 465 476 477 478 479 485 488 489 489 490 492 493 502 508 509 512 513 516 517 518 519 526 532 536 537 538 539 540 541 xxiv LIST OF ILLUSTRATIONS IN TEXT FIG, \ PAGE 169. Scraping from favus scutula, showing spores and mycelium. x 250. F , . « 542 170. Edge of living colony of Sperotrielion bourmann!, x200 . 546 171. Film from agar culture of Sporotrichon beurmanni. 1025. 547° 172. Growth of blastomyces in kidney of rabbit infected from human case. x 1000 - 548 178. Double-contoured bodies in tissues fare case ‘of Rixford and Gilchrist. _ , t ~S oie 4 Aes ee on nt” i ea fa | ese eS Bes = S , poe 13 Fra. 1: Fic. Fre 25 MANUAL OF BACTERIOLOGY > CHAPTER I. GENERAL MORPHOLOGY AND BIOLOGY. Introductory.—At the bottom of the scale of living things there exists a group of organisms to which the name of bacteria is usually applied. These are apparently of very simple structure, and may be subdivided into two sub-groups, a lower and simpler and a higher and better-developed. The lower forms are the more numerous, and consist of minute unicellular masses of protoplasm devoid of 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 & (sston inch). 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 coccz. (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 spiridia. The full description of the characters of these groups will be more conveniently taken later (p. 12). 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 filaments may be more or less septate, may be provided with a I 2 GENERAL MORPHOLOGY AND BIOLOGY 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 genéral words, such as germ, microbe, micro- organism, are used as synonymous with bacterium, though these are often made to include minute organisms belonging to other groups. While no formed living organisms lower than the bacteria are known (though, as will be seen later, the existence of life associated with matter in an ultra-microscopic state is probable), the upper limits of the group are difficult to define, and it is impossible at present to give other than a provisional classifica- tion 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.—The relations of the bacteria to the animal kingdom on the one hand and to the vegetable on the other constitute a difficult question. It is best to think of there being a group of small, unicellular organisms, which may be survivals of the most primitive forms of life before differentiation into animal and vegetable types had occurred and before in an individual cell nucleus had been differentiated from cytoplasm. This would include the flagellata and infusoria, the myxo- mycetes, the lower alge, and the bacteria. To the lower alge the bacteria show many similarities. These alge are unicellular masses of protoplasm, having generally the same shapes as the hacteria, and largely multiplying by fission. Endogenous sporulation, however, does not occur, nor is motility necessarily associated with the possession of flagella. Also their proto: plasm differs from that of the bacteria in containing chlorophyll and another blue-green pigment called phycocyan. From the morphological resemblances between these alge and the bacteria, and from the fact that fission plays a predominant part in the multiplication of both, they THE STRUCTURE OF THE BACTERIAL CELL 3 were formerly grouped together in one class as the Schizophyta or splitting plants. And of the two divisions forming these Schizophyta the splitting alge were denominated the schizophycee, while the bacteria or splitting fungi were called the schizomygetes. The bacteria were, there- fore, often spoken of as the schizomycetes. This classification in its reference to splitting fungi reflects the view, now practically abandoned, that the bacteria represent the last stage of a progressive degeneration which parasitism has entailed in the fungoid plants. GenERaL MorpHouocy oF THE BacTERia. The Structure of the Bacterial Cell.— When examined under the microscope, in their natural condition, ¢.g., in water, bacteria appear merely as colourless refractile bodies of the different shapes named. Spore formation and motility, when these exist, can also be observed, but little else can be made out. For their proper investigation advantage is always taken of 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. 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 overstaining a specimen with a strong aniline dye, when it will appear as a halo round the bacterium. This envelope may sometimes be seen to be of considerable thickness. Its innermost layer is probably of a denser con- sistence, and sharply contours the contained protoplasm, giving the latter the appearance of being surrounded by a membrane. It is only, however, in some of the higher forms that a definite membrane occurs. Sometimes the outer margin of the envelope is sharply defined, in which case the bacterium appears to have a distinct capsule, and is known as a capsulated bacterium (vide Fig. 1, 4; and Fig. 58). The cohesion of bacteria into masses depends largely on the character of the envelope. If the latter is glutinous, then a large mass of the same species may occur, formed of individual bacteria embedded in what appears to be a mass of jelly. When this occurs, it is known as a zoogloea mass. On the other hand, if the envelope has not this cohesive property the separation of individuals may easily take place, especially in a fluid medium in which they may float entirely free from one another. Many of the higher bacteria possess a sheath which has a much more definite structure than is found 4 GENERAL MORPHOLOGY AND BIOLOGY 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,—usually 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. In some cases the bacterial cell enlarges before division, in others the cell divides and each element then expands to its adult size. If, in the latter alterna tive, multiplication is proceeding rapidly, great variation in the size of the individuals may be observed, and this may give rise to anomalous appearances. From investigations by Graham-Smith and others, it appears that the consistence of the envelope may have an importance in modifying. the naked-eye and low-power appearances presented by bacterial colonies which constitute a feature in the identification of species (see p. 184). Graham-Smith, working with bacilli, differentiates four groups—a ‘‘loop- forming,” in which the envelope is so tough that, after division, rupture but rarely occurs (b. anthracis) ; a ‘‘ folding” group, in which the envelope is so flexible and extensile that the members of a chain can be folded on one another as successive divisions take place (b. pestis); a ‘‘ snapping” group, in which partial rupture of the envelope occurs on division (b. diphtheriz); and a ‘‘slipping” group, where the envelope readily breaks, and successively developed bacilli slip past each other (v. cholera), - When bacteria are placed in unfavourable conditions as regards food, etc., growth and multiplication take place with difficulty. In the great majority of cases this is evidenced by changes in the appearances 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} SPORE FORMATION 5 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 (Fig. 1, #1, 22). That these forms really betoken degenerate changes ig 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 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. 1t 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 (¢.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, s). In structure the spore consists of a mass of protoplasm surrounded by a dense membrane. This can be demonstrated by methods which will 6 GENERAL MORPHOLOGY AND BIOLOGY 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, it again assumes the original bacillary or spiral form. The capsule may dehisce 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 when 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 alge. 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 strain 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 SPORE FORMATION 7 among bacteria preferring the absence of oxygen for vegetative growth, the presence of this gas favours sporulation, Some facts relating to the cases in which two spores are formed in one bacterium have been adduced to support the view that sporulation may represent a degenerate sexual process. Here a partial fission of a cell has been observed, followed by a re- fusion of the protoplasmic moieties and the formation of a spore at each end of the rod. The second view with regard to sporulation is that a bacterium only forms a spore when its surroundings, especially its food supply, become unfavourable for vegetable 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. ften sporulation can be prevented from taking place for an indefinite time if a bacterium is constantly supplied with fresh food (the other conditions of life being equal). The presence of substances excreted by the bacteria themselves plays, however, a more important part in making the surroundings unfavourable than the mere exhaustion of the food supply. A living spore will always develop into a vegetative form if placed ina 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. 106); (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 is very difficult to determine. The Question of Arthrosporous Bacteria. —It is stated by Hueppe that. among certain organisms, ¢.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 8 GENERAL MORPHOLOGY AND BIOLOGY 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. 69). 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, 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 (wide Fig. 1, q; and Fig. 107). 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. Some- times complicated spiral tresses of free flagella are found in bacterial cultures; the development of these is difficult to explain. The nature of flagella has been much disputed. Some have held that, unlike what occurs in many alge, they are not actual prolongations of the bacterial protoplasm, but merely ap- pendages 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 (vede 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 MINUTER STRUCTURE OF BACTERIA 9 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, especially with reference to the existence of a differentia- tion into nucleus and cytoplasm and as to the intimate phenomena of division. Observations bearing on such points can only be made on certain large species, but even with these, the minuteness of the cells makes the interpretation of the appearances seen most difficult. While bacterial protoplasm generally exhibits a selective action for nuclear aniline dyes, the material thus picked out appears in certain bacteria not to be uniformly distributed through the cell, but to be deposited in certain parts, and controversy has turned on the interpretation of such appearances. Two main views are at present held by different schools. Some consider that the bacterial cell contains a formed nucleus and a cytoplasm ; at the same time it is questioned whether all the material giving the reaction of a nucleus is really part of such a central structure and not merely stored material. A modification of this view looks on the nucleus as an extended thread lying in the protoplasm,—in some bacillary types having a spiral or zigzag appearance. The other view is that the bacterial cell represents a vital unit in which differentiation into nucleus and cytoplasm has not yet occurred, and where the two main elements of higher cells are still intermingled with one another, the homologue of the cytoplasm being present in a close meshwork of nuclear material. With regard to the behaviour of the cell in division, amongst those who hold the former view some have figured appearances in the supposed nucleus which suggest the occurrence of mitosis, and others consider that before division there is a longitudinal splitting of ‘the nuclear threads. All that can at present be certainly stated is that there is frequently in the bacterial protoplasm material which reacts to nuclear dyes, and material which does not so react, and that granules occur which probably represent material in process of transformation for the purposes of cellular nutrition. Before bacteria exceeding, say, 1 to 1°5 uw in thickness were known, appearances analogous to those described had been recognised among the smaller forms, even when stained by ordinary methods. Occasionally irregular, deeply staining granules had been observed in the protoplasm, often, when they occurred in a bacillus, giving the latter the appearance of a short chain of minute cocci. These were called metachromatic granules from the fact that by appropriate procedure they could be stained with one dye, while the rest of the bacterial cell could be made to take on another colour. Such an appearance is well known as occurring in the diphtheria bacillus, especially when stained by Neisser’s method (p. 114). Incertain bacteria, for example the plague bacillus, the granules appear chiefly or solely at the poles and are often referred to as polar granules. It will be gathered from what has been said that at present it is impossible to interpret the significance of such granular structures. The appearances are present in certain bacteria under all circumstances, sometimes they are associated with growth in particular surroundings. In some species the presence of granules is an indication of lowered vitality. : i Whatever the composition and relationships of the essential parts of the bacterial protoplasm may be, there is, as has been said, reason for 10 GENERAL MORPHOLOGY AND BIOLOGY believing that even in the lower forms reserve material exists. This may consist of fat, glycogen, and other substances, amongst which may be mentioned volutin, as described by A. Meyer, a substance probably of proteid nature characterised by solubility in water, alkalies and acids, and by insolubility in alcohol. In perfectly healthy and young bacteria, appearances of granule formation and of vacuolation may be artificially produced by physical means from the occurrence of what 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 polar granules may thus be brought about. Plasmolysis in bacteria has been extensively investigated,} and has been found to occur in some species more readily than in others. Furthermore, it is often more readily observed in old or otherwise enfeebled cultures. Biitschli, 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 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. — The chemical structure of bacterial protoplasm has been investigated both by, micro- and macro-chemical methods,—the former being chiefly applicable to the larger forms. With iodine, granules staining brownish red or blue have been observed, and these are looked on as composed of substances allied to glycogen and starch respectively. Similarly, reactions with osmic acid, scharlach and similar dyes, have pointed to the presence of fats. While macro-chemical investigation has not thrown much light on the occurrence of carbohydrates, cellulose is said to be obtainable from certain bacteria. Bodies giving the reactions of fats have been isolated in bulk and have received much attention in the case of the tubercle bacillus group, whose special staining char- acteristics are probably due to bodies of this class, The substances mentioned are to be looked upon as reserve material or metabolic products in the life of the bacterial cell; but substances of a proteid nature have also been derived from bacterial protoplasm, and these are probably more intimately related to the vital structures of the organism. Chemically they are allied to, or are identical with, similar substances found 1Qonsult Fischer, ‘‘Untersuchungen iiber Bakterien,” Berlin, 1894; “Ueber den Bau der Cyanophyceen und Bakterien,” Jena, 1897. THE CHEMICAL COMPOSITION OF BACTERIA 11 in plant and animal tissues, for example, albumins, globulins, and phosphorised substances such as nucleins and nucleinic acid. There is also evidence that in the bacteria, as in the higher: cells, lipoidal bodies are intimately associated with the proteid elements. Further, various mineral salts, especially those of sodium, potassium, and magnesium, are constituents of bacterial protoplasm. All the constituents show great varia- tions, dependent not only on the species under investigation, but also on the composition of the culture media, on the temperature of growth, and on the age of the culture. Many species of bacteria, when growing in masses, 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, namely, the lipochromes. ‘These lipochromes, which get their name from the colouring matter of animal fat, include the colouring matter in the petals of Ranunculacee, 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. The Classification of Bacteria.—In what we have to say under this heading we shall chiefly confine ourselves to the characters of the pathogenic bacteria. There have been numerous schemes set forth for the classification of bacteria, the 12° GENERAL MORPHOLOGY AND BIOLOGY fundamental principle running through all of which has been the recognition of the two sub-groups and the type forms mentioned in the opening paragraph above. In the attempts to subdivide the group still further, 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 classifica- tion to be attempted. To prepare for the elaboration of the latter, there should be studied in every species the habitat, best food supply, condition as to gaseous environment, range of growth temperature, morphology, micro-chemical reactions, life- history, special properties, and pathogenicity (p. 136). 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, transitional forms have to be accounted for. In subdividing the bacteria further, the forms they assume con- stitute 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 subdivision are the planes in which fission takes place and the presence or absence of spores. The recogni- tion of actual species is often a matter of great difficulty. The points to be observed in this will be discussed later (p. 135). 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 Cocei.—In this group the cells range in different species from ‘5 y to 2 » in diameter, but most measure about 1 pi. Before division they may increase in size in all directions. The species are usually classified according to the method of division. If the cells divide only in one axis, and through the consistency of their envelopes remain attached, then a chain of cocci will be formed. A species in which this occurs is known as a 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 1 For the illustration of this and the succeeding systghnatie paragraphs, vide Fig. 1. i y (J \ ie. Fig. 1.—a-h. Different types of cocci. a. Single round cocci and simple diplococcal forms. b. Lancet-shaped cocei (type of pneumococcus). ¢. Biscuit cocci (gonococcus). a. Streptococci. e. Staphylococci. /. Tetrads (micrococcus tetragenus). g. Sarcina forms. h. Capsulated cocci. 71-17. Bacilli. i!-d3. Ordinary types of different shapes. 4, 75. Bacilli with granular or vacuolated protoplasm. ' 1%, 77. Large forms. k-n. Spirochetes. kl. Spiro- chete with open turns (spirochete refringens). 4&2. Possible longitudinal splitting of spirochete. £3. Two individuals separating. m. Spirochete with irregular turns. 1. Spirochete with closé turns (spirochete pallida). o. Mixed type of fusiform bacilli and spirilla (see Chapter XVII.). p. Spirilla. pl. Comma type. p2. Spirillary type. q. Differ- ent types of flagellum formation. g!. Terminal flagella. g?. Peritrichous formation. q®. Flagella on spirillum. gt Large flagellated spirillum. 71. Wreathed mass of flagella. 72, Detached flagellum. 73, Detached flagella assuming ring form. s. Types of sporula- tion. sl. Terminal. 82, 84. Mesial. s°. Subterminal. s°. Detached spores. 11, 2. Involu- tion forms (b? diphtheriz). wu. Hofmann’s bacillus. v1-v3- Involution forms (b. pestis). w. Streptothrix actinomyces. 2. Tigplseerle Mena y. Thiothrix tenuis. 14 GENERAL MORPHOLOGY AND. BIOLOGY 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 generally applied. _The individuals in a growth of micrococci often show a tendency to remain united in twos. These are spoken of as diplococct, 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 (mdcrococcus tetragenus), sometimes it is of great extent, its diameter being many times that of the coccus (streptococcus mesenterioides). In none of the cocci have endogenous spores been certainly observed. , The species of the streptococci and staphylococci differentiated number several hundreds. Usually included in this group are coccus-like organisms ‘which divide in three axes at right angles to one another.. These are referred to as sarcine. 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 sarcine 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. Bactili.—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. Several species are provided with sharply-marked capsules (b. pneumoniz). In many species endogenous sporula- tion occurs. . The spores may be central, terminal or subterminal, round, oval, or spindle-shaped. There is no doubt that among the bacilli in certain cases, ¢.g., in b. diphtheriz and b. tuberculosis, : the phenomenon of true branching may occur. Such instances form a connecting link between the bacilli and the higher. bacteria, e.g., streptothrices. 3 Great confusion in nomenclature has arisen in this group in con- sequence of the different artificial meanings assigned to the essentially synonymous terms bacterium and bacillus. Migula, for instance, applies the former term to non-motile species, the latter to the motile. Hueppe, on the other hand, calls those in which endogenous 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, THE LOWER BACTERIA 15 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, 4, m, »). 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 cholera microbe (Fig. 1, p). This latter type is of much more frequent occurrence. 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 proto- plasm. 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 (Fig. 1, g*). Division takes place as among the bacilli, but in some of the non-septate forms a longitudinal fission may occur. 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, spirochete, vibrio. Migula makes ‘‘vibrio” synonymous with ‘ microspira,” which he applies to members of the group which possess only one or two polar flagella ; ‘‘spirillum” he applies to similar species which have bunches of polar flagella, while ‘ spirochete ” is reserved for the long unflagellated spiral cells. Hueppe applies the term ‘‘spirochete” 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 ‘‘spirochete” and “‘spirillum” indiscriminately to any wavy or corkscrew form, and ‘‘vibrio” to forms where the undulations are not so well marked. It is thus necessary, in denominating such a bacterium by a specific name, to give the authority from whom the name is taken. Within recent years doubt has arisen as to whether many of the non-septate spirillary forms, ¢.g., Spirochete pallida, are to be looked on as bacteria at all,—one view being that in, it may be, many cases they represent a stage in the life-history of what are really protozoa. The question is an important. one, as these forms include many pathogenic agents. The ultimate classifica- tion of this group of bacteria must at present be left an open question, and it is convenient to denominate the non-septate spiral rods Spirochete, and those whose vital unit is a single eurved rod Spuriila. II. The Higher Bacteria.—These show advance on the lower in consisting of definite filaments branched or unbranched. In most cases the filaments at more or less regular intervals are 16 GENERAL MORPHOLOGY AND BIOLOGY 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, how- ever, 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. 5). There are various classes under which the species of the higher bacteria are grouped; but our knowledge of them is still somewhat limited, as many of the members have not yet been artificially cultivated. The beggiatoa group consists of free swimming forms, motile by undulating contractions of their protoplasm. For the demonstration of the rod-like elements of the filaments special staining is necessary. The filaments have no special sheath, and the protoplasm contains sulphur granules. The method of reproduction is doubtful. The thiothriz 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. A Jleptothrix group is usually described which closely resembles the thiothrix group, except that the protoplasm does not contain sulphur granules. It cannot, however, be with certainty said whether such organisms can be sufficiently differentiated from the bacilli to warrant their being placed among the higher bacteria. In the cla@othrix group there is the appearance of branching, which, however, is of 4 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 belong 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 FOOD SUPPLY 17 reproduced. Such bodies are often referréd to as spores, but they have not the same staining reactions nor resisting powers of so high a degree as ordinary bacterial 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, or possibly of defensive nature. 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 proto- plasm 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 in the growth of bacteria which must be considered, 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 2 18 GENERAL MORPHOLOGY AND BIOLOGY growth. Such diffusion may be seen when the organism pro- duces pigment, ¢.g., b. pyocyaneus. In supplying artificial food for bacterial growth, the general principle ought to be to imitate as nearly as possible the natural surroundings, though it is found that there exists a considerable adaptability 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 proteid to be present for their proper nourishment, while others can derive their nitrogen from a non-proteid such 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 furnished by other bacteria. Again the influenza bacillus must, outside the animal body, be provided with fresh blood or serum, and the growth of the gonococcus. and the meningococcus is much favoured if serum be a con- stituent of a medium. The opinion has been expressed that vitamines are provided by such media. 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 forms 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 thrée hours’ drying, while the staphylococcus pyogenes aureus will survive ten days’ drying, and the bacillus diphtheriz 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 RELATION TO GASEOUS ENVIRONMENT 19 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 free oxygen is present. To these the title of obligatory aerobes is given. Other bacteria will only grow when no free oxygen is present. These are called obligatory anaerobes. In still other bacteria the presence or absence of oxygen is a matter of indifference ; such organisms are usually denominated facultative anaerobes,—they being pre- ferably aerobic but capable of existing without oxygen. An example of an obligatory aerobe is b. subtilis; of an obligatory anaerobe, b. tetani, while the great majority of pathogenic bacteria are facultative anaerobes. The precise part played by oxygen tension in the growth of anaerobes may require further investigation, as, in certain species, anaerobiosis is a relative property. 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 on 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 seems to have no effect on its vitality or on that of the b. typhosus; in the case of the bacillus pyocyaneus, however, such pressure is said to destroy life. Temperature.—For every species of bacterium there is a temperature at which it grows best. This is called the “optimum temperature.” There is also in each case a maximum temperature abovee 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 putrefac- tion 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 20 GENERAL MORPHOLOGY AND BIOLOGY 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 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 tempera- tures. Thus many pathogenic organisms lose their virulence if grown above their optimum temperature, and some chromogenic forms, most of which prefer rather low temperatures, lose their capacity of producing pigment, ¢.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 sun- light 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 excluded, though light plus heat is more fatal than light alone. In direct sunlight it is chiefly the green, violet, and the ultra-violet rays which are fatal. The last-mentioned rays, however produced, have a powerful bactericidal action. By using a quartz spectrometer with a tungsten arc, Browning and Russ have recently shown that the ultra-violet rays with bactericidal action occupy a position in the spectrum at some distance from the visible rays—from 2960 to nearly 2100 Angstrom units. The exact extent varies somewhat in the case of different organisms, but the area of rays in the spectrum effective against any one organism is comparatively sharply marked off. The bactericidal rays have little penetrating power, being completely absorbed by human skin in a thickness of 10 mm. These observers have also found that those, and only those, rays which are bactericidal to the staphylococcus aureus are absorbed by an emulsion of that organism. Diffuse daylight has also a bad effect upon CONDITIONS AFFECTING BACTERIAL MOTILITY 21 ,bacteria, though it takes a much longer exposure to do serious harm. A powerful electric light is as fatal as sunlight. Here, as with other factors, the results vary very much with the species under observation, and a distinction must be drawn between a mere cessation of growth and the condition of actual death. 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 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 substances, salts of potassium are the most powerfully attracting bodies, and in comparing organic bodies the important factor is the molecular constitution. Further, the filtered products of the growth of many bacteria have been found to have powerful chemiotactic properties. It is evident that all these observa- tions have a most important bearing on the action of bacteria, though we do not yet know their true significance. Correspond- ing chemiotactic phenomena are shown also by certain animal cells, ¢.g., leucocytes, to which reference is made below. 22 GENERAL MORPHOLOGY AND BIOLOGY 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. That the very complicated 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 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, ¢.g., the souring of milk, the becoming rancid of butter, etc. Bacterial action also underlies many processes of economic importance, such as the ripening of cream and of cheese, and the curing of tobacco. A certain comparatively small number of bacteria have been proved to be the causal agents in some disease processes occurring in man, animals, and plants. This means that the fluids and tissues of living bodies are, under certain circumstances, a suit- able pabulum for the bacteria involved. The effects of the action of these bacteria are analogous to those taking place in the action of the same or other bacteria on dead animal or vegetable matter. The complex organic molecules are broken up into simpler products. We shall study these processes more in detail later. Meantime we may note that the disease- producing effects of bacteria form the basis of another biological division of the group. Some bacteria are harmless to animals and plants, and apparently under no circumstances give rise to disease in either. These are known as 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., b. tetani), 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*saprophytie. As to how far such a saprophytic existence of (disease-producing THE METHODS OF BACTERIAL ACTION 23 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 albuminopus bodies which is mostly involved in the wide and varied process of putrefaction can be undertaken by whole groups of different varieties of bacteria. The action of the latter on such substances is analogous to what takes place when albumins are subjected to ordinary gastric and intestinal digestion. In these circumstances, therefore, the production of albumoses, peptones,.etc., similar to those of ordinary digestion, can be recognised in putrefying solutions, though the process of destruction always goes further, and still simpler substances, ¢.g., creatinin, 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 ‘(e.¢., lactic acid) are produced. During bacterial growth there is not infrequently the abundant production of such gases as sulphuretted hydrogen, carbon dioxide, methane, etc. One common result of bacterial action is thus an alteration of the reaction of a medium, sometimes towards the acid sometimes toward the alkaline side. Reduction phenomena are also frequently observed. 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. Many substances 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 up sugars 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 24 GENERAL MORPHOLOGY AND BIOLOGY be retained in the cells where they are formed. In the latter case the bacterial protoplasm often must be thoroughly disintegrated, eg., by grinding, before the ferment is liberated. Sometimes the breaking down of the organic matter appears to take place within, or in the immediate proximity of, the bacteria, some- times wherever the soluble ferments reach the organic substances, And in certain cases the ferments diffusing out into the surround- ing medium probably break down the constituents of the latter to some extent, and prepare them for a further, probably intracellular, disintegration. Thus, in certain putrefactions of fibrin, if the process be allowed to go on naturally, the fibrin dissolves and ultimately great gaseous evolution of carbon dioxide and ammonia takes place, but if the bacteria, shortly after the process has begun, are killed or paralysed by chloro- form, then only a peptonisation of the fibrin occurs, without the further splitting up and gaseous production. That a purely intracellular digestion may take place is illustrated by what has been shown to occur in the case of the micrococcus ure, 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. According to some, the intracellular ferments alone have the capacity of initiating profound changes in material absorbed, while the easily diffusible agents have only a hydrolysing power. In the investigation of the phenomena of the ferment action of bacteria, it has been noted in certain cases that the ferments formed depend on the food supply offered to the bacterium. Thus in one case a bacterium growing in starch forms diastase, but it does not so do when grown on sugar. The disintegration of organic material, which is so prominent an effect of bacterial growth, must be a by-effect in the synthesis of the complex sub- stances of which the bacteria themselves are built up. The most striking example of such synthetic power is presented in the case of the bacteria which in the soil make nitrogen more available for plant nutrition by con- verting 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 VARIABILITY AMONG BACTERIA 25 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 leguminose. 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. he 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, ¢.g., appear as a coccus, a bacillus, or a leptothrix. This is especially the case with certain bacilli, and it may lead to such forms being classed among the higher bacteria. Pleomor- phism 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 observation within twenty-four hours. CHAPTER II. METHODS OF CULTIVATION OF BACTERIA. Introductory.—In order to study the characters of any species of bacterium, it is necessary to have it growing apart from every other species. In the great majority of cases where bacteria occur in nature, this condition is not fulfilled. 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 it growing on a medium which suits it, we are said to have obtained a pure cultwre. 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 bacterio- logical 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 are necessary according as we are dealing with aerobes 26 STERILISATION BY DRY HEAT 27 or anaerobes. Each of these methods will be considered in turn. Tue MetTHops 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 developed. 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 neces- sary 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 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 one hour to a temperature of 160° C., is sufficient to kill all the organisms which usually pollute articles in a bacteriological laboratory, though circumstances" might arise where this would 28 METHODS OF CULTIVATION OF BACTERIA be insufficient. This means of sterilisation is used for the glass flasks, test-tubes, plates, Petri dishes, the use of which will be @ B Fie, 2.—Hot-air steriliser. described. Such pieces of apparatus are thus obtained sterile and dry. It is advisable to put glass vessels into the chamber before heating it, and to allow them to stand in it after sterilisation till the tem- perature falls. Sudden heating or cooling is apt to cause glass to crack. The method is mani- festly unsuitable for food media. B. Sterilisation by Moist Feat. 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 re- quired in various manipulations. To minimise rusting of knives and steel instruments it is well to boil the water for some time before placing them in it. 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 of a tall metal cylinder on legs, provided with a lid, and covered externally by 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. ’ 1G. 3.—Koch’s steam steriliser in section. STERILISATION BY STEAM 29 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 4 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 practicable, as, with long boiling, gelatin tends to lose its physical property of solidification. The method adopted in this case is to steam for twenty minutes 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 is to be sterilised, it is best to put the medium in while the apparatus is cold, in order to make certain that the whole of the food mass reaches the tempera- ture 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. B (3). Sterilisation by Steam at High Pressure.—tThis 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 thermometer. As in Koch’s steriliser, the contents 30 METHODS OF CULTIVATION OF BACTERIA are supported on a perforated diaphragm. The source of heat is a large Bunsen beneath. The temperature employed is usually 115° CG. or 120° C. To boil at 115° C., water requires a pressure of about 23 Ibs. to the square inch (z.e., 8 lbs. plus the 15 lbs. of ordinary atmospheric pressure). To boil at 120°C, a pressure of about 30 lbs. (2.¢., 15 lbs. 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 tem- peratures above 105° C., and is best sterilised by the intermittent method. Certain pre- cautions are necessary in using the autoclave. In all cases it is necessary to allow the apparatus to cool well below 100° C. before opening it or allowing steam to blow off, otherwise there will be a sudden develop- ment of steam when the pressure is removed, and fluid media will be blown out of the Fig. 4.—Autoclave. a. Safety-valve. flasks. Sometimes the instrument is not ma See pipe. 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 accurate 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 superheated, and the pressure on the gauge again does not indicate the tempera- ture 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 temperature above that point. Such a medium is sterilised on Tyndall’s principle 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. PREPARATION OF ORDINARY CULTURE MEDIA 31 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 afterwards be performed at a higher temperature. Tur PREPARATION OF ORDINARY Cuiture MeEpta. The general principle to be observed in the artificial culture of bacteria is that the medium used should approxi- mate 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. -Other media have ‘been found which can support the life of all the pathogenic bacteria isolated. These consist of proteids or carbohydrates iN #yc. 5,—Steriliser for blood a fluid, semi-solid, or solid form, in a serum. transparent 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 become evident. Many organisms, however,. grow best at a tem- perature at which this nutrient gelatin is finid, and therefore 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. 32 METHODS OF CULTIVATION OF BACTERIA 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 ce 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 lattér in the left hand, squeeze out the juice still held back in the contained meat. Finish this expres- sion 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 hemoglobin. It is now boiled Fic. 6.—Meat press, | thoroughly for two hours, by which pro- cess 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. Make up to 1000 c.c. with distilled water. The resulting. fluid ought to be quite transparent, of a yellowish colour without any red tint. If there is any redness, the fluid must be reboiled and filtered till this colour disappears, otherwise in the later stages it will become opalescent. A large quantity of the extract may be made at a time, and what is not immediately required is put into a large flask, the neck plugged with cotton wool, and the whole sterilised by methods B (2) or (3). This extract contains very little alouminous 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. BOUILLON MEDIA 33 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! . F F . 1000 ec. Sodium chloride : F 5 grms. Peptone albumin? . ‘ ‘ LO: 35 Boil till the-ingredients are quite dissolved, and make slightly alkaline to litmus as directed below. After alkalinisation, 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. 28, 29). In this medium the place of the original albumins of the meat is taken by peptone, a soluble protein 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. 191) 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, meningococcus, are very sensitive to the reaction of their surroundings. Adjustment of the Reaction of Media.—The adjustment of the reaction of bacteriological media is a matter of great com- plexity. The method usually adopted with meat extract, which as prepared above is ordinarily slightly acid, is to add saturated sodium carbonate or sodium hydrate solution till the medium is slightly but distinctly alkaline to red litmus paper and’no longer affects blue litmus paper. The occurrence of an amphoteric reaction—i,e., one where red litmus is turned blue, and blue, red—is thus avoided. The test paper must be immersed in the liquid—on no account is the sampling to be done by trans- ferring drops to the paper by means of a glass rod. The disadvantages of this method are that ordinary litmus is not a delicate indicator and, further, no standardisation of the proper tint to be aimed at is possible. The latter difficulty can be 1Some workers, instead of meat extract as made above, use Liebig’s extract of beef, 2 grammes to the litre. While a medium made up in this way suffices for most of the commonly occurring pathogenic bacteria, it is advisable, in the case of the less robust organisms, to use media freshly made with meat. 2 Whatever kind of peptone is used, it ought always to be tested for the absence of sugars and indol before b ed for a medium. Chapetaut’s, and also Savory and Moore’s, peptone vecommended at present, - 3 34 METHODS OF CULTIVATION OF BACTERIA sufficiently got over by making up a solution of disodic phosphate (Na,HPO,,2H,O), 11°876 grm. to the litre; test paper immersed in this assumes a tint just on the alkaline side of what is usually regarded as the optimum reaction for bacterial growth. In applying this method it is preferable to use azolitmin papers’ (made by immersing filter paper in 0:1 per cent. of the dye overnight and drying) or neutral-red papers (made by treating the paper with 0-02 per cent. neutral-red for three minutes). Both of these papers are more sensitive than ordinary litmus paper. Eyres Method.—Several methods have been introduced for adjusting the reaction by titration, and that of Eyre is widely used. It is applicable to any of the media ordinarily employed. 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, a normal solution is obtained. A measured quantity is placed in a porcelain dish, and a few drops of a ‘5 per cent. solution of phenol-phthalein 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. The soda solutions are best stored in bottles with such a cork as is shown in Fig. 38; on the air inlet is placed a little bottle filled with soda lime and fitted with a eee tubed cork. The CO, of the air which passes through is thus removed. 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 alkaline or acid solution necessary to make a litre of the medium neutral to phenol-phthalein. Thus, for example, “reaction = —15,” will mean that the medium is alkaline, and requires 15 c.c. of normal HCl to make a litre neutral. It has been found that when a medium such as bouillon reacts neutral to litmus, its reaction to phenol- phthalein, according 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 ADJUSTMENT OF THE REACTION OF MEDIA 35 degree of alkalinity is the optimum for bacterial growth. It is probable that when a medium has been rendered neutral to phenol-phthalein by the addition of NaOH, the optimum degree is generally attained by the addition of from 10 to 15 cc. of normal HCl per litre, 2.e., the optimum reaction is from + 10 to + 15. According to Fuller, the optimum reaction for bacterial growth lies about midway between the neutral point indicated by phenol-phthalein and the neutral point indicated by litmus. 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 forty- five 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. phenol- phthalein solution. Run in decinormal soda till neutral point is reached, indicated by the first trace of pink colour, the mixture being kept hot.! 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%= amount necessary to neutralise a litre; and 40x - 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. Eyre uses a soda solution of ten times normal strength, which is delivered out of a 1 c.c. pipette divided into hundredths ; this obviates, to a large extent, the error introduced by increasing the bulk of the medium if a weaker neutralising solution be used. In using these strong solutions care must be taken to remove any fluid adhering to the owtsede of the pipette. When the acid or alkali has been added the reaction of the medium must be again taken before sterilisation. The present state of our knowledge of the principles involved in the proper adjustment of the reaction of media is not satisfactory.2 The reaction of a medium depends on the hydrogen-ion concentration. The precise hydrogen-ion content of media as they are ordinarily prepared, either by the colorimetric or titration methods, has, however, not been 1The 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 samples. 2See Clark, Journ. Infect. Dis., Chicago, vol. xvii. (1915), p. 109. For information regarding indicators, see Walpole, Biochemical Journal, vol. vii. (1913), p. 260; vol. viii. (1914), p. 628, 36 METHODS OF CULTIVATION OF BACTERIA determined, and the optimum concentration for bacterial growth is unknown, though probably it. approximates that of the blood serum. A still more serious difficulty is that no simple means is available for estimating the hydrogen-ion concentration of many media, ¢.g., agar. It is nevertheless true that although the methods in use are largely empirical the products resulting are quite satisfactory for all but the more delicate bacteria dealt with. It is therefore probable that many \organisms will tolerate a reaction which may extend from a point some way on the alkaline side of the optimum to a point some way on the acid side. Precise statements such as are frequently made regarding the adjustment of a medium to a certain reaction—say, on Eyre’s scale—do not represent anything else than an expression of the fact that by the technique practised a suitable medium has been produced. It has not been usually recognised, for instance, that hydrogen-ion concentration is a function of the temperature at which the reaction has been adjusted, Thus, a reaction of, say, +10 adjusted at the boiling-point is no longer a +10 reaction at the incubation temperature at which the medium is actually used. 1 (6). Glucose Broth.—To the other constituents of 1 (a) there is added 1 or 2 per cent. of glucose. The steps in the preparation are the same. Glucose being a reducing agent, no free oxygen can exist in a medium containing it, and therefore glucose broth is used as a culture fluid for anaerobic organisms. 1 (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 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 ; : ? . 1000 ce. Sodium chloride . : ‘ ‘ 5 grms. Peptone albumin . ‘ ‘ : 10 ,, Gelatin ‘ , ‘ ‘ 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 ona sand bath, or inthe “ 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 pro- cess, 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 ig great danger of the neck of the flask breaking if it: has to support the funnel and its contents. The filtration may GELATIN MEDIA 37 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 while melting to have settled into layers of different density. Sometimes the first portion of filtrate ‘is turbid. If so, replace it in the unfiltered part: often the subsequent filtrate in such cireum- stances 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 steam thoroughly. 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. 28. If the auto- clave be used the temperature em- ployed 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 pre- paration depends on the temperature at which growth is to take place. Its firmness is its most valuable characteristic, and to maintain this Fic. 7.—Hot-water funnel. in hot 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. For ordinary use in British laboratories 10 per cent. gelatin is a sufficient strength. 2 (6). Glucose Gelatin.—The constituents and mode of pre- paration are the same as 2 (a), with the addition of 1 to 2 per cent. of grape sugar before sterilisation. This medium is used for growing anaerobic organisms at the ordinary temperatures. 3. Agar Media (French, ‘“‘gélose”).—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 38 METHODS OF CULTIVATION OF BACTERIA is a proteid, agar is a carbohydrate. It is derived from the stems of various seaweeds growing in the Chinese seas, com- mercially 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, which is preferable. 3 (a). “ Ordinary” Agar.—This has the following composi- tion :— Meat extract . : : ‘ . 1000 ce. Sodium chloride ‘ : : 5 grms, Peptone albumin. ; , : 10 _,, Agar : ; : : - 3 LB 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 “Koch” 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). Render slightly alkaline with sodium hydrate solution, and if necessary 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 puta glass plate over the filter funnel to prevent condensation water from dropping off the lid of the steriliser into the medium. If a slight degree of turbidity may be tolerated, it is sufficient to filter through a felt bag or jelly strainer. Plug the flask con- taining the filtrate, and sterilise either in autoclave for fifteen minutes or in Koch’s steriliser for one and a half hours. Agar melts just below 100° C., and on cooling solidifies about 39° C. 3 (0). 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 glucose, or, better still, a corresponding amount of a 10 per cent. sterile solution of glucose after filtration. This medium is used for the culture of anaerobic organisms at temperatures above the melting-point of gelatin. For the growth of the tetanus bacillus a specially suitable medium is composed of meat extract with 2 ‘per cent. agar, 2 per cent. peptone, and ‘5 per cent. alkaline sodium phosphate added, and SPECIAL CULTURE MEDIA 39 made faintly alkaline to phenol-phthalein ; 1 per cent. of glucose is added as above. 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. SPECIAL CuLTuRE MepIa. An enormous variety of different media has been brought forward for use in cases either where special difficulty is ex- perienced in getting an organism to grow, or where some special growth characteristic is to be studied. It is impossible to do more than give the chief of these. Peptone Solution. 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 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 bacteria ; and by the addition of one of the sugars to it the fermentative powers of an organism may be tested (p. 79). Litmus may be added to show any change in reaction. Robertson’s Bullock's Heart Medium. This medium was introduced for the cultivation of anaerobes and is made as follows: 8 oz. of bullock’s heart is minced very 40 METHODS OF CULTIVATION OF BACTERIA finely and then ground in a mortar; 8 oz. of tap water are added and the mixture is heated slowly so as to cook the meat thoroughly ; normal sodium hydrate is added until the reaction is alkaline to litmus. The medium is divided into tubes and autoclaved. It can be adapted for growing cultures of anaerobes in the ordinary atmosphere by running a little sterile liquid paraftin on to its surface. \ Media containing an Indicator. Litmus Media.—To any of the ordinary media litmus (French, 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. 50) 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 dis- charged. Veutral-Red Media.—This dye was introduced by Griinbaum and Hume 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 con- taining ‘5 per cent. of the sugar to be tested, to which ‘5 per cent. of a 1 per cent. watery solution of neutral-redis added. The alka- line medium is of a yellowish brown colour which in the presence of acid passes into a deep rose red. Sometimes there subsequently occurs a change to a fluorescent :green, caused apparently by a change in the composition of the dye, as the fluorescence is not discharged by addition of alkali. (See also p. 356.) Blood Serum Media. Solidified Blood Serum.—Koch introduced this medium for the cultivation of the tubercle bacillus and in order to obtain it in a comparatively clear state, adopted the method of inspissation at 65° C. after sterilising by the intermittent method at’ low temperature—B (4) (p. 30). The procedure is somewhat tedious, and for all ordinary purposes opaque coagulated serum, sterilised by the usual methods, can be substituted. A sufficient quantity of serum is placed in a series of sterile test-tubes; these are then placed in a sloped tray, put in the steam steriliser, and steamed for an hour. We have found the following method, however, to give the best results. The serum in test-tubes is first thoroughly inspissated, in the sloped position, at 65° C. BLOOD SERUM MEDIA 41 Sufficient sterile ‘8 per cent. sodium chloride is added to each tube to cover the medium in the upright position. The tubes are then sterilised in this position in the autoclave for a quarter of an hour at 115° C. and thereafter stored. When a tube is to be used the saline is poured off. The advantages of this method are that com- plete sterility is readily obtained and the medium does not dry up. Koch’s Method. — Plug CG the mouth of a tall cylin- iar drical glass vessel (ey of 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 ar is to be killed. When the Areann aoa artery or vein of the animal (eee is opened, allow the first blood which flows, and which may be contaminated from the hair, ete, to 3] sa S escape ; fill the vessel with ov the blood subsequently shed. ¥ io Carry carefully back to the laboratory without shaking, and place for twenty-four hours in a cool place, pre- ferably "an ice chest. The clear serum will separate A from the clotted blood. The serum obtained by such means is frequently con- taminated with bacteria. These can be removed by filtration through an earthenware candle, and this can be rapidly effected oll in by using an arrangement Fic. 8.—Blood serum inspissator. such as that shown in* Fig. 27, the serum during filtration being kept at about 55°C. 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 scrum may, with all precautions, have been contaminated during the manipulations, and must be sterilised. As it will coagulate if heated above 68° C., advantage must be taken of the intcrmittent 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. The medium should be incubated for a day at 37° C. before use, to see that the result ae Foe Oa O 42 METHODS OF CULTIVATION OF BACTERIA is successful. After sterilisation it is ‘‘inspissated,” by which process a clear solid medium is obtained. ‘‘Inspissation” is an initial stage of coagulation, and is effected hy 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 controlled by a gas regulator, and such an apparatus can, by altering the temperature, be used either for sterilisation or inspissation. Loffler’s Blood Serum.—This is the best medium for the growth of the b. diphtherie, 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 as in the case of solidified blood serum (p. 40). Alkaline Blood Serum (Lorrain Smith’s Method).—To each 100 c.c. of the serum obtained as before, add 1 to 1°5 cc. 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. 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-albumen) is thus obtained, and is of value for the growth of the organisms for which Koch’s serum is used, and especially for the growth of the b. diphtheria. 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.—Marmorek succeeded in main- taining the virulence of cultures of pyogenic streptococci 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. Sterile ox serum may be used in a similar way. In the case SERUM MEDIA FOR GONOCOCCUS 43 of these media, sterilisation is effected by method B (4), and they are used fluid. Serum Media for Gonococcus.—The following media will be found suitable :— Wertheim’s Medium consists of one part of sterile human serum and two parts of peptone agar. The agar is melted in tubes and allowed to cool to 45° C. ; the serum warmed to the same temperature is then added, and the mixture is allowed to solidify in the sloped position. Human serum may be conveniently obtained from placental blood or from blood drawn off for the Wassermann reaction. Gurd’s Medium is a 2 per cent. agar with acid reaction +6 to phenol- phthalein (p. 34), with defibrinated human blood added in the proportion of about 5 drops to 5 c.c. of agar ; the blood is added to the melted agar as in Wertheim’s medium. W. B. M. Martin recommends the substi- tution of sodium phosphate (‘5 per cent.) for sodium chloride in the preparation of the agar, and uses fluid human serum sterilised at 57° C. in place of blood. He also finds that the same agar medium allowed to solidify and then smeared on the surface with a drop or two of human serum or blood gives excellent results. Thomson’s Medium.—This is an agar medium with the addition of human plasma. Nutrient agar (2°5 per cent.) is prepared in the usual way from meat juice with 1 per cent. of Witte’s peptone added and its reaction brought to +6. But instead of the usual 0°5 per cent. sodium chloride, the salts of Ringer’s solution are added, namely, sodium chloride 9 grms., calcium heloride 0°25 grm., and potassium chloride 0°42 grm., per litre. Glucose in the proportion of 2°5 per cent. is also added. The medium is then sterilised and tubed—4 c.c. in each tube. To obtain the plasma a tubeful of human blood is drawn off as for a Wassermann reaction. About 10 ¢.c. are poured at once into a centrifuge tube containing 2 c.c. of a 2 per cent. of sodium chloride solution, to prevent clotting, and fitted with a sterile cork. The blood is then centrifuged till the corpuscles are thrown down. The agar medium in tubes is melted and its temperature brought to 50°C. To each tube is then added with a sterile: pipette 1 c.c. of plasma, and the mixture is allowed to solidify in the sloped position. The medium gives a very abundant growth, and is very suitable for the preparation of vaccines. Any of these media may be used for plate cultures, the agar being melted and cooled to 45° C. as for agar plates; the serum or blood is then added, and the mixture is poured out in Petri dishes. Medium for Meningococcus,—T7rypagur.—Gordon has introduced this medium for the cultivation of the meningococcus. It is prepared as follows :— 1. Take 50 grms. of pea flour (ordinary Pearce Duff's) and add to 1 litre of distilled water with 100 grms. of salt. Mix and steam for half an hour, stirring occasionally ; allow to settle, and filter, then sterilise and label ‘‘Saline Pea Extract.” This pea extract should preferably be freshly made for each batch of agar. 2. Take some fresh bullocks’ hearts, free from fat and vessels, mince the meat very finely, and weigh. To each 4 kilo add 1 litre of water, 44 METHODS OF CULTIVATION OF BACTERIA and make faintly alkaline to litmus with 20 per cent. caustic potash solution. Heat this slowly to 75°-80° C. for 5 minutes. Cool to 37° C., and add 1 per cent. of Liquor Trypsine Co. (Allen & Hanbury), and keep it at 37° for 24 to 3 hours. ‘When trypsinising is finished, test for peptone with copper sulphate and caustic potash as below, then render slightly acid with glacial acetic acid, and bring slowly to the boil fora quarter of an hour. Leave overnight in a cool place, and siphon off the clear liquid in the morning. Make this faintly alkaline to litmus, and sterilise in the autoclave at 118° C. for 1 hour on each of two days (if not to be used at once). The result is trypsinised broth. To make Trypagar.—Take 2 measured quantity of the ttypsinised broth, add 2 per cent. of agar fibre (see below for preparation), and -215 grm. of calcium chloride per litre: Autoclave at 118° C. for three- quarters of an hour to dissolve the agar. Mix together in an urn or saucepan ; titrate with’ caustic soda while boiling, using phenol- phthalein as the indicator, and add the necessary amount of normal caustic potash to give an absolutely neutral reaction. Cool to 60° C., add white of egg (2 to a litre) beaten up with the crushed shells, autoclave ‘again at 118° C. for 75 minutes (or in the steamer for 2 hours), Filter, add to the filtrate 5 per cent. of the sterile pea extract, and sterilise in the ordinary way. For use, a small quantity of sterile rabbit’s blood or serum—5 c.c. to 200 c.c. of mediuin—is added to the medium in the melted state at 50°C. before being poured into capsules, or a drop or two of serum may be spread with a glass rod over the surface of the medium after it has solidified, Preparation of Fibre Agar.—Weigh out the required quantity, ent up small with scissors, place in a large flask or enamel pail, and wash twice quickly in water. Drain thoroughly ; add water just to cover, and put in glacial acetic acid, 2°5 ¢.c. per litre of water. Mix thoroughly and leave for a quarter of an hour. Pour off the liquid and wash thoroughly four or five times to make sure that all the acetic acid is washed out. Drain carefully, and use as above. Biwret Reaction for Peptone.—Take 5 c.c. of broth, add 1 c.c. of 5 per cent. solution of vopper sulphate. Mix, and then add 5 c.c. normal caustic potash. A true pink colour indicates that trypsinisation is sufficient ; a bluish-purple shade, that it is incomplete. Blood Media. Blood-Smeared Agar.—This medium was introduced by Pfeiffer for growing the influenza bacillus, and it has been used for the organisms which do not readily grow on the ordinary media, ¢.g., the gonococcus and the pneumococcus. Human blood or the blood of animals may be used. ‘Sloped tubes” (vide p. 54) 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 sub- limate. 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, smear it on the surface of the agar. BLOOD MEDIA 45 The excess of the blood runs down and leaves a film on the surface. Cover the tubes with indiarubber caps, and incubate them for one or two days at 37° 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. Serum-Smeared Agar is prepared in a similar way by smear- ing 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. Blood Agar.—For many purposes (¢.g., the growth of the whooping-cough bacillus, the bacillus of soft sore, the cultivation of trypanosomes and Leishmaniz), the use of agar containing defibrinated blood, especially rabbit blood, is desirable. The blood may be obtained in several ways, preferably by bleeding from the carotid. For this purpose the vessel is exposed and as long a portion as possible is cleaned. This is ligatured high up, and a ligature is loosely applied round the lower part of the vessel in such a way as not to constrict it. The vessel is clamped above this ligature, and with scissors an oblique opening is made in its side. The clamp being removed, the stream of blood is directed by means of the ligature into the mouth of a stout sterile flask, which ought to contain some fragments of broken glass rod. During the bleeding the flask should be gently agitated, and when filled should be shaken in a bath of water just below blood-heat. We have found that sterile blood can be obtained from the ear vein of the rabbit by the method of bleeding to be subsequently described (p. 126). The ear is well washed with lysol, the lysol dried off with sterile wool, absolute alcohol dropped on and allowed to evaporate, and the blood withdrawn. The first c.c. or so is rejected. However the blood is obtained, after defibrination it is warmed to 45° C., and added to agar of the same temperature in the proportion of about one-third of blood and two-thirds of agar. Needless to say, such media must be incubated before use to ensure that bacteria have not gained access during preparation. Bordet and Gengou’s Medium for Bacillus of Whooping-Cough.— An extract of potato is first prepared by adding two parts of water con- taining 4 per cent. of glycerin to one part of potato chips; the mixture is then boiled and the fluid is separated off. An agar medium is then prepared of the following composition : potato extract, 50c.c. ; ‘6 percent. solution of sodium chloride, 150c.c. ; and agar, 5grms. Of this medium, 2-8 c.c, are placed in each of a series of sterile test-tubes, and then to each there is added, by the method described in the preceding paragraph, an equal part of defibrinated rabbit's (or better, human) blood, obtained 46 METHODS OF CULTIVATION OF BACTERIA by aseptic methods. The mixture is then allowed to solidify in the sloped position. This medium is also very suitable for the growth of the gonococcus, meningococcus, and influenza bacillus. Blood-Alkali Agar (Dieudonné).—This medium, introduced for the culture of the cholera spirillum, for which purpose it has been found extremely suitable, has the property of inhibiting the growth of most of the intestinal bacteria; for example, the b. coli does not grow on it, or does so very slightly. A blood-alkali solution is prepared by adding equal parts of defibrinated ox blood and of normal caustic soda solution ; the solution may then be sterilised in the steam steriliser. Of this solution three parts are added to seven parts of ordinary peptone-agar rendered neutral to litmus, and the mixture is disposed in test-tubes. Novy and MacNeal’s Medium for Culture of Trypanosomes, —125 grammes rabbit or ox flesh are treated with 1000 c.c. distilled water, as in making ordinary bouillon, and there are added to the meat extract 20 grms. Witte’s peptone, 5 grms. sodium chloride, 20 grms. 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 at 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. 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. (see p. 78). The development of acid by fermentation is shown by the alteration of the colour and by 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. dysenterie, b. coli, etc. Eace Mepra. Within recent years media containing either the yolk, or both the yolk and the white of egg, have been used for the culture of the tubercle bacillus by Dorset and others. The following will be found very suitable :— Dorset’s Egg Mediwm.—The contents of four fresh eggs are well beaten up, 25 c.c. of water are added and thoroughly mixed, the mixture being passed through muslin to remove air bells. The fluid is then filled into tubes, and these are heated for four hours in the sloped position at 70° C. Another method of solidifying the medium is to place the tubes high up in a Koch’s steriliser for 3-5 minutes. In either case the medium may then be covered with ‘8 per cent. sodium chloride solution and 'sterilised in the autoclave (p. 29). Before the inoculation of a tube, two drops of sterilised water are placed on the surface. The inoculation material is well rubbed over the surface of [the medium, the tubes are sealed with a few EGG MEDIA 47 drops of paraffin on the top of the plug and are incubated in the sloped position. The addition of a sufficient quantity of a solution of basic fuchsin to colour the medium a pale pink is of advantage, as it makes the early growths more easily seen (Cruickshank). Glycerin Egg Medium (Lubenau).—200 c.c. of 5 per cent. glycerin bouillon, 1:5 per cent. acid to phenol-phthalein, are added to ten fresh eggs beaten up, and dre thoroughly mixed. The medium is then treated as above. An equally good medium may be prepared by adding one part of 6 per cent. glycerin, in ‘8 per cent. sodium chloride solution, to three parts of beaten egg. Potatoes as Culture Material. Potatoes are best used as slices in tubes, according to the method introduced by Ehrlich. 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. 9. Two wedges are thus obtained, each of which is placed broad ric. 9,—Cylinder of end downward in a test-tube of special potato cut obliquely. form (see Fig. 10). In the wide part at the bottom of this tube is placed a piece of cotton wool, which catches any condensa- tion 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 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-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. Fic. 10.— The fluid is then poured off and the sterilisation Ebrlich’s continued for another half-hour. tube, con- Potatoes ought not to be prepared long before taining pi ‘i : of petite, being used, as the surface is apt to become dry and 48 METHODS OF CULTIVATION OF BACTERIA 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 steeping a given weight of potato slices for some time in a known quantity of distilled water, and then estimating the reaction of the water by phenol- phthalein. The required degree of acidity or alkalinity is obtained by adding the necessary quantity of HCl or NaOH solution (p. 34), and steeping again. The water is then poured off and the potatoes placed in tubes. Potatoes before being inoculated ought always to be incubated at 37° C. for a night, to make sure that the sterilisation has been successful. Milk as a Culture Medium. This is a convenient medium for observing the effects of bacterial growth, 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, and is steamed for fifteen minutes in the Koch ; it is then set aside in an ice chest or cool place over- night to facilitate further separation of cream. The milk is siphoned off from beneath the cream and placed in sterile test-tubes. A little litmus, sufficient to tint the milk, is often added before final sterilisation to show change in reaction produced by bacterial growth—ltmus milk. 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 torule, moulds, ete. 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. MEDIA FOR SEPARATING BACTERIAL GROUPS 49 Media used for separating the Members of Bacterial Groups. A great number of media have been devised for use in differentiating the members of the coli-typhoid and other bacterial groups. The general feature of these media is that they contain certain substances, often sugars, which tend to bring out the special characters of the organism under in- vestigation. Sometimes also substances are present which inhibit the growth of bacteria other than those belonging to the group. The following are the media which here deserve most attention :— 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. dysenteriz, etc. The characteristic ingredients are bile salts and various sugars. The stock solution is the following: Commercial sodium taurocholate, °5 gramme; Witte’s peptone, 2 grms.; tap water, 100 c.c. (if distilled water be used, ‘03. per cent. of calcium chloride should be added). ‘The solution is steamed for two hours, filtered when hot, allowed to stand for twenty-four hours or till sedimentation has occurred, and filtered again. For a liquid medium there is added to this -25 per cent. of a freshly prepared 1 per cent. solution of neutral red and the sugar, —when glucose, dulcite, or adonite is used, ‘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. It is well to sterilise it in flasks containing 80 c.c., this being an amount sufficient for three large Petri capsules. When this medium is used for examining urine or faces, plates are ‘inoculated as with Drigalski’s medium (infra) ; for its use in water examinations, see p. 152. When growth of a bacterium producing acid and gas occurs in neutral- red fluid media the latter turns a rose colour, and gas appears in the Durham’s tube. Sometimes a fluorescent appearance is also observed, the significance of which will be discussed in the chapter on B. coli. With the neutral-red solid media the colonies of any organism giving rise to acid will be of a rose-red colour. Litmus is often used instead of neutral red. 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 grms. Witte’s peptone, 20 grms. nutrose, 10 grms. sodium chloride; the mixture is then boiled for an hour, 60 grms. 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. (8) 260 c.c. 4 50 METHODS OF CULTIVATION OF BACTERIA Kubel-Tiemann litmus! solution is boiled for ten minutes, 30 grms, lactose (¢hemically 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 4c.c. of a 10 per cent. sterile solution of water-free sodium carbonate 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 feces, a little is rubbed up in from ten to twenty times its volume of sterile bouillon (a properly made emulsion should just be short of being opaque); in the case of urine or water, the fluid is centrifugalised and the deposit or lower portion is used for the inocula- tion procedures. For use the medium is distributed in Petri capsules in a rather thicker layer than is customary in an ordinary plate. This sheet of medium must be transparent, but must not be less than 2 inm. in thickness—in fact, ought to be about 4mm. 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 in- cubation 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 on the agar during its exposure to the air. The plates are usually inoculated by means of a glass spreader made by bending 2 inches of a piece of glass rod at right angles to the rest of the rod. In the case of fmces one or two loopfuls of an emulsion made as described above are placed on the surface of a plate and thoroughly distributed by means of the spreader ; when the material is less rich in bacteria the spreader may be dipped in the infective fluid. In either case two or three further plates are success- ively spread, without any intervening sterilisation of the spreader. 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 1The 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 alight 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. MEDIA FOR SEPARATING BACTERIAL GROUPS 51 the typhoid bacillus,—being often heaped up in the centre,—and the contour of the colony is often double. Faweus’s Picric Acid and Brilliant Green Medium.—This is a modification of Conradi’s medium which has been used with great success at the Royal Army Medical College in the investigation of typhoid carriers. It is made as follows: To 900 c.c. tap water add 5 grms. sodium taurocholate (which is commercially prepared from ox bile), 30 grms. powdered agar, 30 grms. Witte’s peptone, 5 grms. sodium chloride ; steam for three hours, clear with white of egg, filter through | cotton wool, and bring to a reaction of +15 with normal lactic acid or caustic soda, and sterilise. Dissolve 10 grms. lactose in 100 c.c. sterile distilled water, and add to melted agar. Mix and filter through Chardin paper, sterilise carefully, and store in 100 c.c. flasks, For use, add to each 100 c.c. flask 2 c.c. of a 1-1000 watery solution of brilliant green and 2 c.c. of a 1 per cent. watery solution of picric acid. Pour into large Petri dishes, and leave these to stand inverted at 37° C. till the surface hardens. Inoculate as usual. Colonies of b. typhosus of twenty- four hours’ growth are of about 1 mm. in diameter, transparent and re- fracting ; those of b. coli, on the other hand, have a deep green centre, though later typhoid colonies may also present a pale green centre, ' 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 precipitation of the caseip, which is now filtered off. Dilute sodium carbonate 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 belactose. To this, sufficient Kubel-Tiemann solution of litmus is added, the medium is put into tubes and then sterilised. (This is the original method, but it is better, after the casein has been precipitated, to make the medium slightly alkaline with the sodium carbonate and bring to the boiling- point; then filter, neutralise, add the litmus, and sterilise.) 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. : Any one of these media in the hands of a worker accustomed to its use will yield good results. MacConkey’s medium is that most used by British workers, and it has the merit of being easily prepared. ‘As the result of a considerable experience we have found it most useful and reliable. Next to it we would place Faweus’s modification of Conradi’s brilliant green method. Browning’s Brilliant Green Method.—In this method advantage is taken of the fact that brilliant green has a greater inhibitory effect on b. coli generally than on b. typhosus and the paratyphoid group of bacilli. The amount of the dye necessary to bring about the desired result is not a fixed quantity in each case, as it depends on the number of organisms in the faeces and also on the organic matter. A number of dilutions of the dye are therefore used. Tubes of peptone water (Witte’s peptone 2 per cent. and sodium chloride ‘5 per cent.), each containing 52 METHODS OF CULTIVATION OF BACTERIA 10 c.c., are prepared. The brilliant green (Bayer’s Extra Cryst.) is used as a 1:10,000 solution in distilled water. To «a number of tubes of peptone water, say half a dozen, varying amounts of the brilliant green solution—from 0°1 ¢.c. to 0°7 c.c.—are added in series. Each tube is then inoculated with a loopful of an emulsion of feces in distilled water and the tubes are incubated at 37° C. for twenty-four hours. At the end of this time a loopful is taken from each tube and strokes are made on plates of MacConkey’s medium—three strokes with each loopful. Two plates will be sufficient for the strokes from all the dilutions. After incubation for another twenty-four hours the plates are examined for typhoid colonies; often a pure culture is obtained from one of the dilutions. When the bacilli are scanty the results yielded by the method are remarkable. The method is not suitable for the isolation of dysentery bacilli. Whilst b. coli generally is inhibited by the brilliant green, Browning and his co-workers have found that some strains, especially the inosite- fermenters, ¢.g., b. lactis aerogenes, are equally resistant with b. typhosus, but, on the other hand, are much less resistant to telluric acid. They therefore recommend that 0°33 ¢.c. of a 1 : 1000 solution of telluric acid be added to the tubes of peptone water along with the varying amounts of brilliant green. Whilst a number of tubes, as above described, are essential for the best results, a one-tube method may often be used with success. In this case 0'5 e,c. of the 1:10,000 solution of brilliant green is the optimum quantity. This is specially successful with paratyphoid B. Methods have also been devised, on the same principle as the above, for inhibiting the growth of various organisms present along with b. diphtheriz, and thus aiding the isolation of the latter. We give the following :— Conradi and Troch’s Method for isolating the B. Diphtheris#.—This medium is made by mixing 1000 c.c. water, 10 grms. Lemco, 5 grms. sodium chloride, 20grms. Witte’s peptone, and 6 grms. calcium bimalicum, steaming for half an hour and filtering. To this slightly acid fiuid 1 per cent. of glucose is added and one part is mixed with three parts fresh ox serum. To each 100 c.c. of the bouillon-serum medium 2 c.c. of a 1 per cent. solution of potassium telluricum is added. The finished medium is dis- tributed in Petri capsules and coagulated by a quarter of an hour's exposure to 85° C. A tube of ordinary Loffler’s serum is inoculated with the material to be examined for the diphtheria bacillus and incubated for three hours. The surface is then scraped and two plates of the special medium are inoculated, and incubated for twenty hours. Any diphtheria colonies present are a deep black from a reduction of the dioxide of tellurium ; pseudo-diphtheria colonies show yellow-grey or greyish- black. Smith’s Method.—The following medium, containing telluric acid, has been devised by J. F. Smith ; it gives excellent results. It has the composition :— Peptone-water agar (neutral to litmus) . 5 - 100 «ae Sheep’s serum (sterilised at 57° C.) ‘ 5 : 5 4s 1 per cent. telluric acid solution in distilled water 09 ,, The serum is added to the melted agar at a temperature of 50°C. On this MEDIA FOR GROWING TRICHOPHYTA, ETC. 53 medium the diphtheria bacillus forms large white colonies after incubation for twenty-four hours. The growth of many organisms is inhibited. Media for growing Trichophyta, Moulds, ete. 1. Beer Wort Agar.—Take beer wort as obtainable from the brewery and dilute it till it has ans.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 Media.—Sabouraud recommends the following media, the first being that most frequently used :— (1) Pure tap water. i : i A 1000 c.c. Maltose (‘‘brute de Chanut”) . 3 é 40 grms. Peptone (‘‘ granulée de Chassaing”) . z 10.35 Agar ‘ a ‘ z r 13 as (2) Pure tap water. i 2 : 1000 ec. Glucose (‘‘massée de Chanut”) . : , 40 grms. Peptone (‘‘granulée de Chassaing”) . . 10 ,, Agar . , i - i : : 18 ,, In order to secure uniformity of results over as long a series of observa- tions as possible, it is advisable to make up these media in large quantities, say three litres at a time in a five-litre flask. The agar is put to soak in the water for an hour, the other ingredients are added and dissolved by gradually heating to 120°C. in an autoclave. The medium is then thoroughly mixed by stirring and rapidly filtered through papier Chardin (Cogit, 36 Boulevard Saint Miche], Paris). For this purpose, Sabouraud recommends that ten 500 c.c. flasks should be fitted with funnels and filtration simultaneously carried on in the whole series ; whenever in any one of the flasks the filtrate begins to pass only in drops, a new filter paper is substituted. In this way the three litres of medium can be filtered in a few minutes. We have found that the pro- cedure can be simplified without apparently affecting the efficiency of the medium, by dissolving the agar and sugar in one flask, and the peptone in another. The contents of each are filtered and the two filtrates are then mixed ; in this procedure only two or three filter papers are required for the rapid filtration of a large quantity of the agar and sugar moiety. If filtration in a number of flasks is practised, the contents of all are mixed and then distributed in 6 x § inch test-tubes (plugged with non- absorbent cotton) and sterilised by one exposure in the autoclave at 120° C.—the temperature being very gradually raised. These tubes are used for the primary inoculations, and during incubation, which is necessarily prolonged and usually carried out at 22° C., should be placed in a covered glass jar the lid of which is kept slightly raised at one side with a pad of wool to permit the access of a certain amount of air,— by this device undue drying of the medium is at the same time prevented ; the inoculated tubes should not be covered with rubber caps. The study of the characters of the large colonies of trichophyta, etc., is best carried out with media distributed in 250 or even 500 c.c. Erlenmyer flasks in which the requisite surface of medium with a suitably moist atmosphere is obtained. 54 METHODS OF CULTIVATION OF BACTERIA Tue Use or tHe Orpinary Currure MEDIA. The culture of bacteria is usually carried on in test-tubes conveniently 6x in., but for many purposes smaller tubes, 5x4 in, are equally suitable and medium is thus saved. The tubes 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 ex- tremely resisting spores of the b. subtilis. Cotton-wool plugs are universally used for protecting the sterile contents of flasks and tubes from contamination 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 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 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 species is present. The methods of obtaining pure cultures will presently be described. When a fresh tube of medium is inoculated from an already existing THE USE OF THE ORDINARY CULTURE MEDIA 55 culture, the resulting growth is said to be a “sub-culture” of the first. 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.! If platinum wire is Fic. 12.—Tubes of media. : a. eens MpHEn tube. ' Fic. 11.—Apparatus which may b. Sloped tube. be used for filling tubes. The e. “ Deep es forveulturesiof apparatus explains itself. The anncrooes: indiarubber stopper with its tubes ought to be sterilised before use. not available an excellent substitute,—especially for students’ work,—is found in “resistance wire,” No. 25 B.W.G. This is best mounted in an aluminium handle. Every worker should have three wires. Two are 24 inches long, one of these being straight (Fig. 13, a), and the other having a loop turned upon it 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, \ 56 METHODS OF CULTIVATION OF BACTERIA (Fig. 13, 6). The latter is referred to as the platinum “loop” or platinum “eyelet,” and is used for many purposes. ‘ Taking a loopful” is a phrase constantly used. The third wire (Fig. 13, 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-iridium spud. This consists of a piece of platinum-iridium about 1% inch 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 ES CI ee Lie 5 ener 0 Fig. 13.—Platinum wires in glass handles. u. Straight needle for ordinary puncture inoculations. 0. Platinum ‘ loop.” e, Long needle for inoculating ‘‘ deep” tubes. such manipulations the ordinary platinum wire is awkward to work with, as it bends so easily. If a platinum wire heavily charged with bacteria be sterilised in a Bunsen flame it may ‘‘spark”’ and unkilled bacteria may thus fall on the worker’s bench. In working with organisms highly pathogenic to man, ¢.g., glanders, plague, Malta fever, it is well to substitute for plati- num needles glass rods drawn out to capillary diameter, each of which can be destroyed after use. These before use are sterilised by passing through the flame, and when contaminated are dropped into a 1-1000 solution of corrosive sublimate instead of being heated. Cultures on a solid medium are referred to (1) as “ puncture” or “stab” cultures, or (2) as “stroke” or “slant” cultures, according as they are made (1) on tubes solidified in the upright position, or (2) on sloped 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 adhering to the glass. The short, straight platinum wire is then heated to redness from THE USE OF THE ORDINARY CULTURE MEDIA 57 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, 7.e., away from the right palm. Remove plug from culture tube with right forefinger and thumb, and continue to hold it between the same tingers by the part which projected beyond the mouth of the tube. Now touch the culture with the platinum needle, and, withdrawing it, re- place plug. In the same way remove plug from tube to be inoculated, and plunge platinum wire down the centre of — Frc. 14.—Another method of inoculating the gelatin to within solid tubes, half an inch of the bottom. It must on no account touch the glass above the medium. The wire is then immediately sterilised. A variation in detail of this method is to hold the plug of the tube next the thumb between the fore and middle fingers, and the plug of the other between the middle and ring fingers, then to make the inoculation (Fig. 14). 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 inocula- ting tubes, it is always well, on removing the plugs, to make sure that no strands of cotton fibre are adhering to the inside of the necks. As Fic. 15.—Rack for platinum needles. these might be touched with the charged needle and the plug thus be contaminated, they must be removed by heating the inoculating needle red-hot and scorching them off with it. When the platinum wires are not in use they may be laid ina rack made by bending up the ends of a piece of tin, as in L i al Vk Z 58 METHODS OF CULTIVATION OF BACTERIA Fig. 15. 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 or on one of the solid media so that the colonies formed by the individual organisms are sufficiently far apart to allow their being examined separately. For the purpose, circular shallow glass capsules, each fitted with an overlapping glass cover, are almost universally used; these are known as Petri dishes or capsules. The medium, after being melted, is poured into a sterile capsule and allowed to solidify, so as to form a thin layer; in this way the colonies which after- wards grow are readily accessible. In one method the material containing the bacteria is smeared over the surface of the medium after it has solidified in the capsule,— “smear method.” In i another method the organisms are mixed with the medium when in the melted state, and the mixture Bye, 16,—Pena’s bepeats, is then poured into the capsule (Cover shown partially raised.) and allowed to solidify,—‘“ dilu- tion method.” The former gives the best results in the case of most pathogenic organisms. The smear method is the more convenient, and is that used for the separation of typhoid and dysentery bacilli, meningococci, etc. ; it is, in fact, capable of almost universal application. The procedure varies according to the material to be examined. If the organisms are on a swab, say from the naso-pharynx, con- secutive strokes are made all over the surface of the medium, always the same portion of the swab being brought into contact with it. In this way the organisms are gradually wiped off the swab, tillin the later strokes they may be deposited at sufficiently wide intervals to give separate colonies. Sometimes it is advis- able to smear two plates consecutively with the same portion of the swab. If the material to be examined is fluid, eg., an emulsion of faces, the usual method is to place a loopful on the surface of the medium, and then, with a sterile glass-rod bent at a right angle, to smear the whole surface. If the organisms are found, ou microscopic examination, to be very numerous, say in pus, it will be advisable to dilute with sterile saline before SEPARATION OF AEROBIC ORGANISMS 59 making the smears. The characters of the colonies which appear on the plates can be examined with a hand-lens, magnifying about 6 diameters. In some cases examination tinder a low- power of the microscope is an advantage; the plate in the in- verted position can be put on the stage of the microscope for this purpose. For the culture of special organisms, as afterwards detailed, the agar or other medium is smeared with sterile serum or blood according to the growth requirements of the organism, or the serum or agar is added before the medium is poured. The principle just described may be applied also to agar in tubes, but the results generally are not so satisfactory, and the characters of the colonies cannot be so readily studied. In this case two or three agar tubes are taken, a platinum loop is charged with the material to be examined, and a series of undulating strokes is made from below upwards on the surface of the agar, one tube after the other being used without recharging the needle. The tubes after inoculation should be kept in the upright position, so that the water of condensation is not allowed to run over the surface. ; In the second method, which we have called the “dilution method,” the bacteria are added to the medium when liquid, and mixed by careful shaking ; the inoculated medium is then poured out into a capsule and allowed to solidify. As in this case the organisms are diffused throughout the medium, some of the colonies grow on the surface of the medinm—“ snperficial colonies ”—others in its substance—“ deep colonies.” These often show different appearances, which are sometimes used in the systematic description of an organism. As the bacteria may produce far too many colonies to allow separation, means must also be used for making different dilutions, a separate plate being prepared for each. If gelatine is used, the medium in tubes is melted and kept in a beaker of water at about 28° C. If agar is used, the medium is melted thoroughly by boiling in a vessel of water and then allowed to cool to about 43° C., at which temperature the inoculations are made. The following are the details :— The contents of three tubes, marked a, 0b, ¢,1 are liquefied as above described. 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 1 For marking glass vessels it is convenient to use the red, blue, or yellow oil pencils specially made for the purpose. 60 METHODS OF CULTIVATION OF BACTERIA 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 medium from a to b, Shake } and transfer five loops toc. The plugs of the tubes are in each case replaced and the tubes returned to the beaker. The contents of the three tubes are then poured out into three capsules, In doing so the plug of each tube is removed and the mouth of the tube passed two or three times through the Bunsen flame, the tube being meantime rotated round a longitudinal axis. Any organisms on its rim are thus killed. The capsules are labelled and set aside till growth takes place. For accurate work it will be found convenient to carry out the dilutions in definite proportions. The following is the procedure 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. of fluid is transferred from the first tube to the second. By asimilar procedure ‘05 ¢.c. is transferred from the second to the third, and soon. ‘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 medium,—the medium being afterwards plated and the colonies counted when growth occurs. The number of tubcs required will vary according to the number of bacteria in the original mixture, but usually four or five will be sufficient. Enumeration of Colonies.—The dilution method just described supplies the means of counting the number of living bacteria in a fluid, the proviso being always made that they are capable of growth in the medium used. For pathogenic organisms one of the agar media is generally used, whilst in the case of water, gelatine is most suitable. The dilutions are made by the quantitative method, and a given amount, say ‘1 cc., is taken from one of the dilutions and transferred to a tube of melted medium, and, after gentle mixing, the medium is poured in a Petri capsule. It is advisable to take samples in this way from two or even three of the dilutions. To aid the counting of the colonies which develop, various patterns of ruled glass plates have been introduced. If the ruling is in the form of squares of given size, the number of colonies in several squares is counted, and as the area of the Petri dish can. be got by multiplying the square of its radius by 34, the whole number can then be calculated. Petri dishes are rarely flat, and unequal distribution of the colonies has accordingly to be taken into account. The dilution to be selected for taking the sample for plating will depend upon the relative abundance of the organisms in the original fluid. é Separation of Pathogenic Bacteria by Inoculation of Animals.—It is found difficult, and often impossible, to separate THE CULTURE OF ANAEROBIC ORGANISMS 61 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 grow so slowly that the other organisms present may outgrow them, cover the whole plates, and make separation difticult. 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. 140) 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 pheumococcus. Separation by killing Non-spored Forms by Heat.—This is a method which has a limited application. As has been said, the spores of a bacterium resist heat more than the vegetative forms. When a mixture contains spores of one bacterium and vegetative forms of this and other bacteria, then if the mixture be heated for 10 minutes at 80° C. all the vegetative forms may be killed, while the spores will remain alive and will develop subsequently. Several tubes of different media should be inoculated and treated thus, as the success of the method is very variable. 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 gas, such as hydrogen, through the medium (liquefied if necessary). Further, the medium must be kept in an atmosphere of the same gas while growth is going on. (2) Media for anaerobes may be kept in contact with the air, if they contain a reducing agent which does not 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, 38). In this case the medium ought to be of considerable thickness. The Supply of Hydrogen for Anaerobic Cultwres.—The yas is generated in a large Kipp’s apparatus from pure sulphuric acid and pure zinc. It 62 METHODS OF CULTIVATION OF BACTERIA is passed through three wasli-bottles, as in Fig. 17. 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 J in 10 solution of silver nitrate to remove any arseniuretted 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. Commercial hydrogen a8 sold in cylinders may be used, but this must be purified as above. Pyrogallate of Potassium for Anaerobic Cultures. —In arranging for the Frc. 17.—Apparatus for supplying hydrogen for anaerobic cultures. a. Kipp’s apparatus for manufacture of hydrogen. 6. Wash-bottle con- taining 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. (b, ¢, and d are intentionally drawn to a larger scale than a to show details.) absorption of oxygen by this substance the proportions used in Bulloch’s separation method (below) may be employed. Here 109 grms. solid caustic potash are dissolved in 145 c.c. water, and to this 2-4 grms. pyrogallol are added. 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. 18) of a glass plate as base on which a bell-jar can be firmly luted down with unguentum resine. In 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 APPARATUS FOR ANAEROBIC CULTURE 63 the chamber; 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 2 to 4 grms. of dry pyrogallic acid placed in it towards one side. Culture plates, which should be of rather greater thickness than for ordinary aerobic work, 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, open- ing the tap, and giving a few strokes of the pump. 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 py¢, 18, —Bulloch’s apparatus for chamber and spreads over the anaerobic plate cultures. 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, the rubber tube is placed in a little boiled water, and this, passing through the glass tubes, washes out the potash and prevents erosion of the glass. The whole apparatus may be placed in the incubator till growth occurs. M‘Intosh and Fildes Anaerobic Jar.—These authors have designed a jar in which tubes may be incubated under anaerobic conditions, the oxygen being absorbed by spongy palladium. A glass pickle jar, fitted with a metal lid screwing down on a rubber washer, is employed. Into a hole in this lid there is soldered a small stopcock such as is used in model steam-engines. A bracket of sheet brass, carrying a capsule of fine brass or copper gauze, is fixed to the inner end of the stopcock by nieans of the screw-on collar of the latter (Fig. 19). 0°25 gr. fine asbestos wool is placed in a porcelain capsule and soaked in 1°5 ¢.c. 10 per cent. 64 METHODS OF CULTIVATION OF BACTERIA palladium chloride (to which a little hydrochloric acid may be added if necessary), moulded into a flat cake and slowly dried. The palladium chloride is reduced by heating the dried wool pledget in a smoky gas flame till it is coated with carbon and then burning in a blowpipe. The wool is then folded into the gauze capsule, which should be of just sufficient size to enclose it. The lid being thus prepared, the culture tubes are placed in the bottle. The capsule is heated in a Bunsen, and the lid, with the tap closed, is rapidly fixed on the jar. The tap is opened and a piece of pressure tubing leading from a hydrogen generator. is connected with it. The hydrogen is turned on, and combines with the oxygen in the vessel till no free oxygen remains. At the end of the process the internal pressure prevents the further entry of hydrogen. The jar is allowed to cool, the tap shut off and disconnected from the hydrogen supply, and the apparatus can then be incubated. It is absolutely necessary that the lid of the jar should be airtight. This can be tested by placing a few drops of ether in the jar, fixing on the HL Fic. 19.—Lid of M‘Intosh and Fildes anaerobic jar.2 lid, and plunging the vessel in hot water; any leak can thereby be detected. A fitting similar to above can be adapted to any tin vessel with a ‘‘lever-otf” lid. In this case the lid is hited on with plasticine. Lentz’s Method.—The requisites for this are glass plates, discs com- posed of layers of filter paper compressed together and impregnated with pyrogallol, some circular glass dishes of the form of the halves of a Petri capsule. Plate cultures are prepared in the glass dishes in the usual way and the medium is allowed to solidify. A disc is placed on a glass plate and moistened with a potassium hydrate solution ; a dish is then rapidly inverted over it and luted on the glass plate with plasticine. The other dishes are treated in a similar way. M‘Leod has modified this method in the following way. Instead of glass plates he uses shallow circular porcelain capsules? (Fig. 20) which are covered in by a porcelain diaphragm with the exception of a circular 1 For the use of this figure we are indebted to the National Insurance Medical Research Committee, in whose Special Report, No. 12 (1917), the apparatus is described. 2The capsules may be obtained from Messrs. Thomson, Skinner, & Hamilton, Glasgow. APPARATUS FOR ANAEROBIC CULTURE 65 opening in the middle. The interior of each capsule is divided into two halves by a partition which, however, does not extend the whole way up; in one half, solution of pyrogallic acid is placed, in the-other, solution of potassium hydrate. Plasticine is placed round the margin of the upper surface of each capsule. Plate cultures having been made in glass dishes in the usual way, each dish is inverted and placed over a porcelain capsule and carefully fixed in the plasticine. When this has been done, the two fluids in the capsule are mixed by tilting and the oxygen in the JOO Fic. 20.—M‘Leod’s capsule for anaerobic plating, shown in section. interior is rapidly absorbed. Another improvement is that the edges of the glass dishes which rest in the plasticine are turned up so as to prevent the condensation water from running over the plasticine (Fig. 20, 0). Henry’s Method.—In this modification two shallow circular dishes (portions of Petri capsules) are separated by @ tin diaphragm, in the centre of which is an aperture (Fig. 21). The upper dish contains the plate culture, the lower (smaller) contains pyrogallic crystals. Grooves are present in the metal to receive the margins of the dishes, which are fixed in with plasticine. The lower dish is first fixed in position, and | | Fic. 21.—Henry’s apparatus. just before the upper dish is adjusted, 10 c.c. of caustic potash are run into the lower through the opening in the plate. ‘ 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 5 66 METHODS OF CULTIVATION OF BACTERIA 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 (Fig. 12, c). 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 Cultwres.—This may be used with culture tubes containing any of the media suitable for anaerobes and also for surface growths on sloped tubes. There are required a dry tube of the same diameter as the culture tube, a short U-shaped glass tube, and two pieces of rubber tubing 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 1 or 2 grms. of pyrogallic acid are placed, and the powder is packed down with a layer of filter paper. Ten or twenty cubic centimetres of a 10 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. Buchner’s Anaerobic Tube.—This may be used either for maintaining surface growths of anaerobes or for keeping free from oxygen sloped culture media which are being used for separating anaerobes from mixtures. Dry pyrogallol is placed in a cylindrical jar of diameter sufficient to contain the tube or tubes of media. The tubes are then inserted, potassium hydrate solution (p. 62) is poured into the jar, and its mouth quickly stoppered with a rubber or glass stopper. The stopper is made airtight by sealing with paraffin. The pyrogallol absorbs the oxygen in the jar, and thus the cultures are kept in oxygen-free surroundings. CULTURES OF ANAEROBES IN LIQUID MEDIA 67 Cultures of Anaerobes in Liquid Media.—It is necessary to employ such in order to obtain the toxic products of the growth of anaerobes. Glucose broth is usually most convenient. It is placed either (1) in a conical flask with a lateral opening and a perforated indiarubber stopper, through which a bent glass tube passes (as in Fig. 22 a), by which hydrogen may be delivered, or (2) in a conical flask with a rubber stopper furnished with two holes (as in Fig. 22, 5), 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 liquid; the inner end of the lateral nozzle in the one case, and the inner end of the Fia. 22. a. Flask for anaerobes in liquid media. Lateral nozzle and stopper fitted for hydrogen supply. 0b. A stopper arranged for a flask without lateral nozzle. escape tube in the other, must of course be above the surface of the liquid. The single tube in the one case and the two tubes in the other ought to be partially drawn out in a flame to facilitate subsequent complete sealing. The ends of the tubes through which the gas is to pass are previously protected by pieces of cotton wool tied on them. It is well also 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. (In the case of many anaerobes it 68 METHODS OF CULTIVATION OF BACTERIA is advisable to practise a massive inoculation by pouring into medium part of an actively growing bouillon culture.) The flask is then connected with the hydrogen apparatus by means of a short piece of sterile indiarubber tubing, and hydrogen is passed through for half an hour. In the case of flask (1), the lateral nozzle is plugged with melted paraffin and covered with alternate layers of cotton wool and paraffin, the whole being tightly bound on with string. The entrance tube is now completely drawn off in the flame before being disconnected from the hydrogen 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 disconnected from the hydrogen apparatus. It is well in the case of both flasks to run some melted paraffin all over the rubber stopper. Sometimes much gas is evolved by anaerobes, and in dealing with an organism where this will occur, provi- sion must be made for its escape. This is conveniently done by leading down the exit tube, and letting the end just dip into a trough of mercury (Fig. 23), or into mercury in a little bottle tied on to the Fic. 23.—Flask arranged for culture of end of the exit tube. The staan ae denen gas. pressure of gas within causes db is a trough of mercury into which exit tube dips. 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. The Method of Tarozzi.i—This observer has found that if small pieces of fresh sterile organs are added to ordinary bouillon, growth of anaerobes takes place under ordinary atmospheric con- ditions. For this purpose, portions of liver, spleen, or kidney are most suitable. The method has been used in the cultivation | of spirochetes, organism of poliomyelitis, etc. It has been shown by Douglas, Fleming, and Colebrook that the addition of pieces of vegetable (even after being boiled), bran, and even asbestos wool, is effective in making a fluid medium suitable for the growth of anaerobes, the presence of fine interstices in the material being an important factor in aiding the growth. ‘It HANGING-DROP CULTURES 69 has not yet been determined whether this is the whole explana- tion of Tarozzi’s method. When it is desired to grow anaerobes on the surface of a solid medium such as agar, tubes of the form shown in Fig. 24, a and 6, may be used. A stroke culture having been made, the ITE Fic, 24.—Tubes for anaerobic cultures on the surface of solid media. air is replaced by hydrogen as just described, and the tubes are fused at the constrictions. Such a method is of great value when it is required to get the bacteria free from admixture of medium, as in the case of staining flagella. i? MiscELLANEOUS MEruHops. Hanging-drop Cultures.—It is often necessary to observe micro-organisms alive, either to watch the method and rate of their multiplication, or to investigate whether or not they are motile. This is effected by making hanging-drop cultures. The method in the form to be described is only suitable for aerobes. For this special slides are used. ‘Two forms are in use, and are shown in Fig. 25. 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 the bouillon. The cover is then carefully lowered over the cell on the slide, the drop not being allowed to touch 70 METHODS OF CULTIVATION OF BACTERIA 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 incubated and then examined on a warm stage. (2) The sterile cover-glass is placed on a sterile glass plate. 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 yy a r® B EZ CC SILSP¢AA) Ss Az rae TE ZZZID e Fig. 25. A. Hollow-ground slide for hanging-drop cultures, shown in plan and section. B. Another form of slide for similar cultures. the table «. 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. It is sometimes convenient for the observation of the growth of bacterial colonies or of fungi to make hanging-drop cultures with a solid medium. This can be done by substituting a drop of melted gelatin or agar for bouillon and inoculating the surface after solidi- fication. The method of microscopic examination is described on page 89. The Bacteriological Examination of the Blood.—A fairly EXAMINATION OF THE BLOOD 71 large quantity of blood may be obtained by puncture of a vein ; this is the only satisfactory method, and should be that followed whenever practicable. The skin over a vein in the forearm or on the dorsum of ‘the foot having been sterilised by being painted with tincture of iodine, the vein is made turgid by pressure, and the needle of a syringe of 10-15 c.c. capacity, sterilised by boiling, is plunged obliquely through the skin by the side of the vessel into the lumen of which the point can then be passed. (If a bandage has been used to make the vein turgid, pressure should be maintained on the puncture, after the needle has been withdrawn, until the bandage has been removed ; otherwise a hematoma may be formed by leakage from the vessel.) Several cubic centimetres of blood can thus be with- ‘drawn into the syringe. Some of the blood (e.g., 5 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, especially in severe conditions such as ulcera- tive endocarditis, streptococcus infection, etc. Part of the blood may be incubated by itself for twenty-four hours and cultures then made. Needless to say, the inoculations of media must be done at the bedside, as of course the blood quickly coagulates in the syringe. Coagulation can be prevented by drawing up into the syringe before it is used a quantity of 2 per cent. sterile sodium citrate equivalent to the amount of blood it is intended to withdraw. Patients who are seriously ill have often a low blood pressure, and difficulties may be experienced due to the collapsed state of the veins. It is important here that the skin surfaces should be as little as possible exposed to cold, which still further diminishes the volume of the superficial circulation. In such cases it may be necessary to expose the vein by dissection. 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. 92.) Bacteriological Examination of the Cerebro-spinal Fluid —Lumbar Puncture.—This diagnostic procedure, which is often called for in cases of meningitis, can be carried out with 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 72 METHODS OF CULTIVATION OF BACTERIA sterilised with tincture of iodine, and the hands of the operator should be thoroughly purified. The spines of the lumbar vertebra having been counted, the left thumb or forefinger is pressed into the space between the third and fourth spines in the middle line; the needle is then inserted about half an inch to the right of the middle line at this level and pushed through the tissues, its course being directed slightly inwards and upwards, till it enters the subdural space. When this occurs, fluid passes along 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 3 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, as a sudden movement of the patient may snap an ordinary steel needle. The Bacteriological Examination of the Naso-pharynx.—A specimen of pharyngeal mucus may be obtained by means of a swab of cotton wool on the end of a metal wire. The wire ought to be longer than that used in the case of diphtheria and bent near the extremity. A tongue depressor is used, the wire is introduced into the mouth, and passed up behind the soft palate and then brought into,contact with the posterior pharyn- geal wall. Care must be taken not to touch any part of the mucous membrane of the mouth. The best method, however, is by means of a simple apparatus introduced by West. This con- sists of a glass tube shaped like a catheter, in the interior of which is a thin wire bearing the swab, the latter being just with- in the end of the tube. The bend of the tube can be used to depress the tongue, or a depressor may be used, and when the tube is sufficiently introduced it is turned up behind the soft palate and the end of the wire is pressed so as to protrude the swab, which is then brought into contact with the pharyngeal wall. The swab is then drawn back into the tube (as a matter of fact it usually springs back) and the tube is removed from the mouth. We have found the method to be extremely. simple and effective. Plates of “trypagar” or other suitable medium should be inoculated at once (this is essential) and placed in FILTRATION OF CULTURES 73 the incubator as quickly as possible, as cold has an injurious effect on the organisms. If the inoculations have been made at some distance from the laboratory, the plates should be carried in an apparatus with a hot-water jacket—a bag con- taining a hot-water bottle is often sufficient. 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, specimens withdrawn by a sterile catheter into a sterile vessel are pre- ferable, but it is often 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 sterile flasks, the first of which is rejected in case contamination has occurred. In the female, after similar precautions as regards external cleansing, the catheter must be used. The latter must be boiled for half an hour, and anointed with olive oil sterilised by half an hour’s exposure in a plugged flask to a temperature of 102° C. Here, again, it is well to reject the urine first passed. It is often advisable to allow the urine to stand in a cool place for some hours, to then withdraw the lower portion with a sterile pipette, to centrifugalise this, and to use the urine in the lower parts of the centrifuge tubes for microscopic examination or for culture. Filtration of Cultures.—For many purposes it is necessary to filter all the organisms from fiuids in which they may have been growing. This is done especially in obtaining the soluble toxic products of bacteria. The only filter capable of keeping back such minute bodies as bacteria is that consisting of a tube of unglazed earthenware as introduced by Chamberland. 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 Chamberland “B” pattern; the next finest is the Chamberland “F” pattern, which is quite good enough for ordinary work. The Doulton porcelain filter is also very suitable and efficient. There are several types of filters, differing slightly in detail, all possessing the common principle. Sometimes the fluid is forced through the porcelain’ tube. In one form, used for obtaining sterile water, 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 74 METHODS OF CULTIVATION OF BACTERIA considerable pressure is necessary, it is evident it must be put on a pipe leading directly from the main. Sometimes, when fluids to be filtered are very albuminous, they are forced through a porcelain$£cylinder by compressed —_ carbonic acid gas. The filtra- tion of albuminous fluids may sometimes be facilitated by keep- ing them near blood- heat during the pro- cess. For ordinary bacteriological work, filters of various kinds are in the market (such as those of Klein and others), but the most generally -con- venient is that in Fic. 26.—Geissler’s vacuum pump arranged with which the fluid is manometer for filtering cultures. (The tap and pump are intentionally drawn to a larger sucked : through the scale than the manometer board to show porcelain by exhaust- details. ) ing the air in the receptacle into which it is to flow. This is conveniently done by means of a Geissler’s water-exhaust pump (Fig. 26, g), which must be fixed to a tap leading directly from the main. The connection with the tap must be effected by means of a piece of thick-walled rubber-tubing as short as possible, wired on to tap and pump, and firmly lashed externally with many turns of strong tape. Before lash- ing with the tape the tube may be strengthened by fixing round it with rubber solution strips of the tubbered canvas used for mending punctures in the outer case of a Fic. 27,—Chamberland’s candle bicycle tyre. A manometer tube nd fask arranged for ae (6) and a receptacle (c) (the latter to catch any back flow of water from the pump which may accidentally occur) are intercepted between the filter and’ the ie — FILTRATION OF CULTURES 75 pump. ‘These are usually arranged on a board a, as in Fig. 26. Between the tube f 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. 27. The fluid to be filtered is placed jn 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. 27, proceeds to flask 4, and passes through one of the two perforations with which the rubber stopper of the flask is furnished. Through Fia. 28.—Chamberland’s bougie Fic. 29.—Bougie inserted arranged with lamp funnel for through rubber stopper filtering a small quantity of for same purpose as in fluid. Fig. 28. 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 0. 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 indiarubber washer is placed round the bougie ¢ at its glazed end (vide Fig. 28). 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 funnel and the projecting part of the bougie. It is firmly wired to the funnel above and to the 76 METHODS OF CULTIVATION OF BACTERIA bougie below. The extreme point of the latter is left exposed, and the whole apparatus, being supported on a stand, is con- nected by a glass tube with the lateral tube of the flask 4; the tube a is connected with the exhaust-pump. The fluid to be filtered is placed between the funnel and the bougie in the space ¢, and is sucked through into the flask 0. The efficiency of such a, filter, especially when small amounts of fluid are being dealt with, is much increased if when the level of the fluid falls below the upper end of the candle a closely. fitting test-tube is slipped over the latter. By this device the leakage of air through the exposed part of the candle is prevented. There are now in the market candles with glass sheaths cemented into a nickle-plated fitting from the lower part of which a metal tube emerges; the latter can be passed through a rubber stopper into a filter flask. (2) This modifica- tion is shown in Fig. 29. Into the narrow part of the funnel an indiarubber bung is fitted, with a per- foration in it sufficiently y large to receive the candle, which it should grasp tightly. (c) Muencke’s modifica- Se : tion of the Chamberland a, hea filter is seen in Fig. 30. si A pa aeon f 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 4, 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 in the porcelain cylinder, and the whole top covered, as shown at f, with an indiarubber cap with a central perforation ; the tube d is connected with the exhaust-pump, and the tube ¢ plugged with a rubber stopper. For filtering small quantities of fluid the apparatus shown in Fig. 31 may be used. It consists of a small Chamberland bougie fitted by a rubber tube FILTRATION OF CULTURES 17 to a funnel, the stem of which has been passed through a rubber cork; this cork fits into a conical flask with side arm for connection with exhaust. Before any one of the above apparatus is used it ought to be connected up as far as possible and sterilised in the Koch’s steriliser. The ends of any important unconnected parts ought to have pieces of cotton wool tied over them. After use the bougie is to be sterilised in the auto- clave. Much of the material kept back on the filter can now be removed by forcing water through in a direction opposite to that of the flow of the fluid during filtration, Alternatively, the candle, after being dried, should be passed carefully through a Bunsen flame to burn off all organic matter. If the latter is allowed to accumulate, the pores become filled up. The success of filtration must be tested by inoculating tubes of media from the filtrate, and observing if growth takes place, as there may be minute perforations in a candle 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 \_ y are kept thoroughly closed, and if these - vessels be stored in a cool place in the dark. * eel ena A layer of sterile toluol about half an inch _ ties of fiuid. thick ought to be run on to the top of the filtered fluid to protect it 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 (¢.g., C. J. Martin’s turbine centrifuge), and this method is sometimes adopted in practice. 78 METHODS OF CULTIVATION OF BACTERIA 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. The true sugars are aldehydes or ketones, one or more of the carbon atoms of which is united to a hydroxyl group, one being 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 mono- saccharides, disaccharides, and polysaccharides. The members of the last two groups may be looked on as derived from the combination of two or more molecules of a monosaccharide with the elimination of water (eg., 2CsH,.0,=C,.H».0,, + H,0), but their chemical constitution may be more complex than that of the first group. ; Monosaccharides.—These are classified according to the number of C atoms they contain. The pentoses ordinarily used are arabinose (obtained from gum arabic), xylose (from wood), and rhamnose (which is really a methylpentose). Among the hexoses are glucose (dextrose) with dextro-rotatory properties. Glucose is an aldehyde (aldose), but in fruit there is also a ketone (ketose) called fructose, which from its levo- rotatory properties is also known as levulose. Other hexoses are mannose (from the vegetable ivory nut) and galactose (a hydrolytic derivative of lactose). Disaccharides (C,2H»,0,,).—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. 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 poly-saccharides. Usually an independent process resulting in the splitting of the — higher into the lower is preliminary to the alcoholic fermenta- tion. 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, closely allied bodies which are alcohols with large molecules may be broken down by bacterial action, and these have been used for differentiating the properties of allied BACTERIAL FERMENTATION OF SUGARS = 79 bacteria. Among such substances may be mentioned the trihydric alcohol glycerol (glycerin), the tetrahydric erythritol, the pentahydric adonitol, and the hexahydric dulcitol (dulcite), mannitol (mannite), and sorbitol (sorbite). Similarly glucosides (which are combinations of glucose with other substances), 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 bactertum under observation, and frequently a species which will ferment one sugar has no effect on another. The substances finally produced, speaking roughly, may be alcohols, acids, or gaseous bodies (chiefly 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 pro- cesses depend on two kinds of changes, namely, (a) the evolution of gases and () the formation of acids. Generally speaking, we may say that these 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 gas-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 (wide p. 39), Hiss’s serum water medium (p. 46), or a dextrose-free bouillon (vide 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. It is preferable that the addition should be made in the form of a sterile solution in water. 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 being heated in the presence of substances (such as alkalies) which may act deleteriously upon it; in any case sterilisation should not be at a temperature above 100° C. To obtain a ‘‘dextrose-free’” bouillon it is usual to inoculate ordinary bouillon with some organism, such as b. coli, which is known to ferment dextrose, and allow it 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. 80 METHODS OF CULTIVATION OF BACTERIA It is to be noted, however, that a bouillon rendered ‘‘dextrose-free” by b. coli may still contain carbohydrate fermentable, for example, by a streptococcus. For the observation of gasformation either of the following methods may be employed :— (1) Durham’s Tubes (Fig, 32, 6).—The plug of a tube which contains about one-third more than usual of a liquid medium is removed, and a small test-tube is inverted and _ slipped down into the medium. The plug is replaced and the tube sterilised thrice for ten minutes at 100°C. The air remaining Fig. 32.—Tubes for demonstrating gas-formation by bacteria. a, tube with ‘‘shake” culture. b, Durham’s fermentation tube. ce, ordinary form of fermentation tube. in the smaller tube is thereby expelled. The tube is then in- oculated with the bacterium to be tested. Any gas developed collects in the upper part of the inner tube. As some of the sugars now used for fermentation tests are rather expensive, it is well to arrange the Durham apparatus with very small tubes; with these a satisfactory result can be obtained with only 1 c.c. of medium. (2) The Fermentation Tube (Fig. 32, c).—This consists of a tube of the form shown, and the figure also indicates the extent to which it ought to be filled. It is inoculated in the bend with the gas-forming organism, and when growth occurs the gas INDOL-FORMATION BY BACTERIA 81 collects in the upper part of the closed limb, the medium being displaced into the bulb. If the limb be graduated the amount of gas evolved can be measured, and rough chemical tests can be applied, e.g., the presence of carbonic acid gas can be tested for by absorbing it with a solution of caustic soda and that of hydrogen by ignition (see under b. coli). The development of an acid reaction is demonstrated by the addition of an indicator to the medium, litmus or neutral-red being generally used. The details of the 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 along with the bacterial cultures, as changes in reaction sometimes spontaneously occur in media containing unstable sugars. Tests in which sugars are used are best carried out in Jena glass tubes. 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 protein by a bacterium sometimes constitutes an important specific characteristic. To observe indol production the bacterium is grown, preferably at incubation temperature, in a fluid medium containing peptone. The latter may either be sugar-free bouillon or preferably peptone solution (see p. 39). Any medium containing sugars must be avoided, as the presence of these substances may inhibit the production of indol. Two methods are in use for the detection of this body. (1) Lhe Nitroso-indol Method.—Indol is here recognised by- the fact that when it is acted on by nitric acid in the presence of nitrites, a nitroso-indol compound is produced, which has a rosy red colour. Some bacteria (¢.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 Ggnt. solution of potassium nitrite, and testing with pure nitric 6 82 METHODS OF CULTIVATION OF BACTERIA or sulphuric acid. In any case only a drop of the acid need be added to, say, 10 c.c. of medinm. If no result be obtained at once it is well to allow the tube to stand for an hour, as some- times the reaction is very slowly produced. In many instances incubation at 37° C. for several days may be necessary before the presence of indo] 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. This method has for long been felt not to be satisfactory, and the following at present bids fair to replace it :— (2) Ehrlich’s Rosindol Reactton.—The adaptation of this to bacteriological purposes was brought forward by Bohme in 1906. For ease of application and delicacy of effect the reaction possesses great advantages. It depends on the fact that paradimethylamidobenzaldehyde unites with indol to form a rosindol body whose. colour is readily developed, especially in presence of an oxidising substance such as potassium per- sulphate (K,8,0,). Two solutions are required :— (1) Paradimethylamidobenzaldehyde ; 4 grms. Absolute alcohol (96 per cent.) ‘ : 380 c.c. Concentrated hydrochloric acid ; ; 80 cc. (2) Potassium persulphate . Saturated watery solution. ‘ Toa culture of the organism in 5 c.c. of peptone water add 1 c.c. of (1) and then 1 c.c. of (2), and shake well; if indol be present a rose-red colour will appear in a few minutes. Sometimes the rose colour appears on the addition of solution (1), and the addition of a special oxidising agent is unnecessary. The rosindol com- pound can be separated from the culture by shaking the latter up with amyl alcohol, and MacConkey recommends that this should be done in cases of a doubtful reaction, as sometimes when a faint pink colour appears in the culture tube the extracting alcohol remains colourless, showing that no real reaction has occurred. Marshall has pointed out that by means of the reaction a quantitative estimate of the amount of indol formation can be obtained. To do this a large culture, say 100 c.c., is distilled, and the colour obtained by applying the test to the distillate in a Nessler’s tube is matched against that obtained with different amounts of a standard solution of indol THE DRYING OF SUBSTANCES JW VACUO 83 (prepared by dissolving 1 gr. indol in 5 c.c. absolute alcohol, and making up to 500 c.c. with distilled water). There is no doubt that the Ehrlich test is from five to ten times more delicate than the ordinary nitroso-indol reaction, and it is of especial value in dealing with organisms of the coli- typhoid group. With strains of b. coli it can often be obtained in from twenty-four to forty-eight hours, but in the case of a negative result a culture of from six to seven days ought to be used. The reaction is also obtainable with the cholera vibrio, but further investigation is here necessary, as Marshall states that under certain circumstances the nitrites formed by this bacterium may have an inhibitory effect on the production of the rose colour. . 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 im 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. 33. 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 4, 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 a. 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 stand which rests on the brass plate. The edge of the bell-jar is well luted with unguentum resinz 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 Wolff's bottle containing sulphuric acid. This protects the oil of the pump from contamination with 84 METHODS OF CULTIVATION OF BACTERIA water vapour. Whenever the vacuum is produced the rubber- tube should be at once disconnected from 6, the cock a 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 connecting and disconnecting of rubber-tubing of sufficient thickness to withstand collapse when exhausted is difficult. Ordinary stout rubber-tubing can be used if through it there is passed a length of narrow flexible metal-tubing, the ends of which project beyond the rubber-tubing Fic. 33,—Geryk air-pump for drying in vacuo. so as to enter the parts of the apparatus to which the latter is fitted. The Storing and Incubation of Cultures.—Gelatin cultures must be grown at a temperature below their melting-point, 2.c., for 10 per cent. gelatin, below 22°C. They are usually kept in ordinary rooms or in a cool incubator at about 20°C. Agar. and serum media are employed to grow bacteria at a higher temperature, corresponding to that at which the organisms grow best, usually 37° C. in the case of pathogenic organ- isms. For the purpose of maintaining a uniform temperature incubators are used. These vary much in the details of their structure, but all consist of a chamber with double walls between which some fluid (water or glycerin and water) is placed. STORING AND INCUBATION OF CULTURES 85 This, 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 T much in design. Sometimes a mechanism see devised in Koch’s laboratory is affixed, which automatically turns off the gas if the light be accidentally extinguished. Between the tap supplying the gas, and the burner, is interposed a gas regulator. Such regulators vary in design, but, for ordinary chambers which require to be kept at a constant temperature, Reichert’s is as good and simple as any, and is not expensive. It is shown in Fig. 34. The gas enters at a and from b passes to the burner. When the mercury in f expands to cut off the gas at c sufficient passes by the bye-pass ¢ to keep the flame alight. There is an improved form with a large bulb filled with xylol attached at f. Changes in the bulk of the xylol are com- municated to the mercury. This instrument is very B t delicate and will be found to work well. The varieties of incubators are, as we have said, numerous. We have found those of Hearson of London extremely good, and they are fitted with a good 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 of water from which evaporation may take place. With tubes which will require to be long in the incubator, the plugs should be pushed a little way into the tube and a few drops of melted paraffin dropped on the top of the wool, or the plugs should be covered either by indiarubber 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 tube the cotton-wool plug ought to be well singed in a flame. *:,‘‘ Cool” ineubators are often used for incubating gelatin at* 21° to Fic. 34.—Reichert’s gas regulator. 86 METHODS OF CULTIVATION OF BACTERIA 22° ©. An incubator of this kind fitted with a low-tempera- ture Hearson’s regulator is in the market. Fic. 35.—Hearson’s incubator for use at 37° C. 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 the 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 the following :— (1) Thymol water (saturated in cold) . : . 100 cc. Glycerin ; : : "i 20 cc. Acetate of potash . . é 5 grms. Coignet’s (gold label) gelatin . : : 10 grms. 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,fouridrops of strong formalin to the tube, and fill up to MOUNTING OF BACTERIAL CULTURES 87 within a quarter of an inch of the top of the tube with the following fluid :— : (2) Thymol water (saturated in cold) . - * 100 cc. Glycerin é . : ; : 20 c.c. Acetate of potash . ‘i ; ; ‘i 3 5 grms. 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 up 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 the cement is 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 go as completely to seal it. (6) 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 capsule until the colonies of growth have developed. The watch-glass is now removed from capsule, and a layer of the pre- serving gelatin medium (1) (p. 86), 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 super- fluous 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, ete., which have contained bacteria and with which he has finished, ought to be at once plunged (in the 88 METHODS OF CULTIVATION OF BACTERIA 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 steriliser. A white glazed tile on which a bell-jar can be set is very 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. The Microscope.—For ordinary bacteriological work a good microscope is essential. It ought to have a heavy stand, with coarse and fine adjustments, 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 advisable to have three objectives, ordinary low and high powers, and a ;},inch oil immersion, which is essential. It is well to have two eye-pieces. The student must be thoroughly familiar with the focusing 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 coloured picture is desired, the diaphragm ought to be widely opened. The flat side of the mirror ought to be used along with the condenser. When the observer has finished for the time being with 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. Jn the two last cases advantage is always taken of the affinity of bacteria for certain stains. When they are to be examined in fluids a drop of the liquid may be placed on a slide and covered with a cover-glass.! It is more usual, however, to employ hanging-drop preparations. The technique of making 1Jn bacteriological work it is essential that cover-glasses of No. 1 thickness (i.¢2., ‘14 mm. thiek) should be used, as those of greater thickness are not suitable for a 1,-inch lens. 89 90 MICROSCOPIC METHODS these has already been described (p. 70). 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. In determining whether or not a bacterium is motile, great difficulty is often experienced in distinguishing between true motion and Brownian movement, especially if the organism be small. The essential criterion to be fulfilled is that the bacteria shall be moving in all directions, the observation of individuals lying close together starting to move in opposite directions being important. The observation of hanging-drop preparations must be correlated with the results of staining for’the presence of flagella which, so far as is known, are present in all ordinary motile forms. Dark-Ground Illumination.— Within recent years the method of observing living micro-organisms by oblique illumination has been much practised, and a number of substage condensers are in the market, by means of which this is effected. The general principle involved in these instruments is to stop out the rays passing directly towards the tube of the microscope, and to FILM PREPARATIONS 91 arrange for light being thrown obliquely on objects, eg., bacteria, mounted in a drop of fluid between a slide and cover- glass. The bacteria disperse these rays in all directions, and some passing up through the lens can be focused by it. It is also necessary to place a suitable stop within the objective of the microscope to cut off the peripheral rays. The organisms appear as brightly illumined objects on a dark background. The method has been employed for bacteria in general, and especially for the demonstration of spirochzetes in secretions. Generally speaking, the internal structure of the organisms under observation is well brought out, and their movements can be readily studied. 2. Film Preparations.—(a) Dry Method.—This is the most extensively applicable method for 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 or slide. Many methods are recommended for ob- taining 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. The best method is that recommended by Van Ermengem. The cover-glasses or slides are placed for some time in a mixture « of concentrated sulphuric acid 6 parts, potassium bichromate 6 parts, water 100 parts, then washed thoroughly in water and dried. Ifa fluid is to be examined a loopful may be placed on the cover-glass and spread out over the surface with the needle. When a culture on a solid medium is to be examined, a loopful of water is placed on the cover-glass, and a minute particle of growth rubbed up in it and spread over the glass. The great mistake ‘made by beginners is to take too much of the growth. The point of the straight needle should just touch the surface of the culture, and when this is rubbed up in the droplet of water and the film dried, there should be an opaque cloud just visible on the cover-glass. When a 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 glass between the right forefinger and thumb; if the fingers just escape being burned no harm will accrue to the bacteria in the film. In ordinary routine examinations it is more convenient to 92 MICROSCOPIC METHODS make films on clean glass slides. After these are fixed and stained they may be examined without a cover-glass, a drop of cedar-oil being placed on the film. If such a preparation is to be preserved, the oil should be removed by xylol, which is then dried off, and the slide may then be kept in a box free from dust. In making films of a thick fluid such as pus it is best to spread it out with the needle. The result will be a film of irregular thickness, but suffi- Sermo” ciently thin at many parts for 1 .,¢ Proper examination. It is often me: iano Hi pena Miusable to dilute the material by emulsifying with a loop of water, Scrapings of organs may be smeared directly on a cover- glass or slide. In the case of dood, a small drop is placed near one end of a clean slide, the edge of a second 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, and 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 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. A film prepared in this way may be too thick at one edge, but at the other is beautifully thin. Another method is to allow a drop of blood 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. If it is desired to pre- serve the red blood corpuscles in a film it may be fixed by one of the following methods: by being placed (a) in a hot-air chamber at 120° C. for half an hour; (6) 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. 61 shows a film prepared by the last method.) In using the Romanowsky stains no previous fixation is necessary (vide infra). In the case of wrine, 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 EXAMINATION OF BACTERIA IN TISSUES 93 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 gently wash in water and dry. In this way a much clearer picture is obtained when the preparation is stained. Films dried and fixed by the above methods are now ready to be stained by the methods to be described below. (6) Wet Method.—If it is desired to examine the fine histological structure of the cells of a discharge as well ag 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 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. (8) Formol-aleohol—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 grms. in 10 c.c. of aleohol) 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 (¢.g., Cathcart’s or one of the newer instru- 94 MICROSCOPIC METHODS 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 4 inch in thickness, it 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 he 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) Formalin solution.—This may be used as a 10 per cent. solution of commercial formol-aldehyde in water. Small pieces of tissue are fixed in this in twenty-four hours; they are then placed in 50 per cent. spirit’ | for a similar period, and then in pure spirit. (c) 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. (d) 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 4 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): 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. 1Jn Britain ordinary commercial methylated spirit has mineral 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 ata time. Most pathological laboratories are, however, permitted by the Excise to buy ‘‘ industrial spirit,” which contains only one-nineteenth of wood naphtha. THE CUTTING OF SECTIONS 95 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. The Cutting of Sections.—1. By Means of the Freezing Microtome.—Pieces of tissue hardened by any of the above methods must have all the alcohol removed from them by 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. 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 fiuid and prepare for the entrance of the paraffin. The solvents most in use are chloroform, cedar oil, xylol, and turpentine ; of these, chloroform is the most suitable. The more gradually .the tissues are changed from reagent to reagent in the processes to be gone through, the more successful is the result. A necessity of the process is an oven with hot-water jacket, in which the paraffin can be kept at a constant temperature just above its melting- point, a gas regulator being of course necessary. The tissues occurring in pathological work have a tendency to become brittle if overheated, and therefore the best results are obtained by using parafiin melting at a somewhat low temperature. 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— 1 While the method given is sufficient for ordinary purposes, a more elaborate technique is necessary if it is desired that uo changes shall take place in the tissue. Thus after fixation the tissue must be taken up to absolute alcohol 96 MICROSCOPIC METHODS 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 the tissues must be put inside the oven. 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 bea 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. : In the case of very small pieces of tissue the time given for each stage may be much shortened, and where haste is desirable Nos. 2 and 4 may be omitted. Otherwise it is better to carry out the process as described. 6. Cast the tissues in blocks of paraffin as follows: Pairs of L-shaped pieces of metal made for the purpose by instrument makers must be at hand. By laying two of these together on a glass plate, a rectangular trough is formed. This is filled with melted paraffin taken from a stock in a separate dish. In it is immersed the piece of tissue, which is lifted out of its pure paraffin bath with heated forceps. The direction in which it is to be cut must be noted before the paraffin becomes opaque. When the paraffin has begun to set, the glass plate and trough have cold water run over them. When the block is cold, the metal L’s are broken off, and, its edges having been pared, it is stored in a pill-box. The 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 yu in thickness, and ought, if possible, to be about 4 ». For their manipulation [an a | Fic, 37.—Needle with square of paper on end for manipulating paraffin sections. 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. 37), When cut, sections are floated on the surface of a beaker of water kept at a temperature through successive dilutions of spirit, not differing from each other by more than 10 per cent. Again, when alcohol has been replaced by chloroform the latter must be saturated with chips of paraffin, tirst at room temperature, then at 87° C., and must be kept at 55° C. as short a time as possible. DEHYDRATION AND CLEARING 97 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) Fiaation 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- 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. 103) 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 washéd in water, dried and 7 { 98 MICROSCOPIC METHODS either mounted in xylol balsam or, in the case of films on slides, kept in the dry condition ; wet films 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 pressing 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. Paraffin sections can usually be dehydrated and cleared by the mixture of aniline oil and xylol alone. Balsam as ordinarily supplied has often an acid reaction, and preparations stained with aniline dyes are apt to fade when mounted in it. It is accordingly an advantage to use acid-free balsam. Sections stained for bacteria should always be cleared, at least finally, in xylol, as 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 process of decolorisation can be judged of by observing the appearances under a low objective. | Tae Sraininc 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;H,.NH,). 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 THE STAINING OF BACTERIA 99 rosaniline derives its staining action from the rosaniline, It is therefore called a basic aniline dye. On the other hand, ammonium pigrate 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 cytoplasm 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 :— hie? aoe ethyl-violet, R-5R (synonyms: Hoffmann’s violet, i ablia). Gentian-violet (synonyms : benzyl-violet, Pyoktanin). Crystal violet. Blue Stains. —Methylene-blue} (synonym: phenylene-blue). Victoria-blue, Thionin-blue. Red Stains.—Basic fuchsin (synonyms: basic rubin, magenta). , Safranin (synonyms: fuchsia, Giroflé). Brown Stain.—Bismarck-brown (synonyms: vesuvin, phenylene- brown). 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 dif- ferentiation 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 dilute a little with ten times its bulk of distilled water and filter. 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, ¢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 1 This is to be distinguished from methyl-blue, which is a different com- pound, 100 MICROSCOPIC METHODS in solution in water have a great tendency to decompose. Only small quantities should therefore be prepared at a time. The Staining of Films.—Films are made from cultures as described above, and a few drops of the stain are placed on the surface. 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 siphon arrangement as that figured (Fig. 38). The figure explains itself. When the film has been washed the surplus of water is drawn off with a piece of filter-paper, the preparation is carefully dried high over a flame, a drop of xylol balsam is applied, and the cover-glass mounted on a slide. It is sometimes advan- tageous 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 films on slides, a drop of cedar-wood oil is placed on the film directly, and the preparation is then examined. oS Films of fluids from the body (blood, pus, etc.) can be generally stained in the same way, and this is often quite sufficient for diagnostic purposes. The blue dyes are here preferable, as they do not readily 2 overstain. In the case of such fluids, ; : if the: histological elements also claim ne ee 4 eae attention it is best first to stain the used in washing prepara- Cellular protoplasm with 1-2 per cent. tions. 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 (vide p. 111) 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 MORDANTS AND DECOLORISING AGENTS 101 staining, a drop of water is placed at one side of the cover-glass and a, little piece of filter paper at the other side. The result is that the stain is sucked out by the filter-paper. By adding fresh drops of water and using fresh pieces of filter-paper, the specimen is washed without any violent application of water, and the bacteria are not displaced. For the general staining of films a saturated watery solution of methylene-blue will be found to be the best stain to com- mence with; the Gram method (vide infra) is also used, 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 (0) the sub- sequent 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, ¢.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 gections.. More of the acid may, of course, be added if necessary. Hot water also decolorises to a certain extent; over-stained films can often 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. Different organisms take up and retain the stains with various degrees of intensity, and thus duration of staining and decoloris- ing must be modified accordingly. "We sometimes have to deal 102 MICROSCOPIC METHODS with bacteria which show a special tendency to be decolorised. This tendency can be obviated by adding a little of the stain to the alcohol, or aniline oil, employed in dehydration. In the latter case a little of the stain is rubbed down in the oil. The mixture is allowed to stand. After a little time a clear layer forms on the top with stain in solution, and this can be drawn off with a pipette. : When methylene-blue, methyl-violet, or gentian-violet 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 (24 per cent. in water). The Formule of some of the more commonly used Stain Combinations. 1. Léffer’s Methylene-bluc. Saturated solution of methylene-blue in alcohol : F . 800 Solution of potassium hydrate in distilled water (1-10,000) . 100 ,, (This dilute solution may be conveniently made by adding 1 ¢.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 4-1 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 Loffler’s blue by five minutes’ exposure or longer in the cold. They usually do not require decolorisation, as the tissue elements are not overstained. 2. Kiihne’s Methylenc-blue. Methy lene-blue ; : ; 1°5 grm. Absolute alcohol : : 10 ce. Carbolic,acid solution (1-20) . - 100 ,, Stain and decolorise as with Létier’s blue, or decolorise with very weak hydrochloric acid (a few droys in a bow] of water). 8. 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 one volume with three of water, and filter. Stain sections for five minutes or upwards. Wash very thoroughly with water, otlier- wise 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. GRAM’S METHOD AND ITS MODIFICATIONS 103 4, Gentian-violet in Aniline Oil Water.—Two solutions have here to be made up. (a) Aniline oil water. Add about 5 c.c. aniline oil to 100 c.c. distilled water in a flask, and shake violently till as much as possible of the oil has dissolved. Filter and keep in a covered bottle to prevent access of light. (b) Make a saturated solution of gentian- violet in alcohol. When the stain is to be used, 1 part of (6) is added to 10 parts of (a), and the mixture filtered. The mixture should be made not more than twenty-four hours before use. Stain sections for a few minutes; then decolorise with methylated spirit. Sometimes it is advantageous to add to the methylated spirit a little hydrochloric acid (2-3 drops to 100 ¢.c.). This staining solution is not so much used by itself as in Gram’s method, which is presently to be described. 5. Carbol-Gentian-violet.—1 part of saturated alcoholic solution of gentian-violet is mixed with 10 parts of 5 per cent. solution of carbolic acid, the mixture being wellshaken. It is used as No. 4. 6. Carbol-Fuchsin (see p. 105).—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 bacterva. 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—in other words, whether it is Gram-positive or Gram- negative. It must also be mentioned that some tissue elements may retain the stain as firmly as any bacteria, ¢.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 com- position :— lodine ; p : a3 1 part. -Potassium iodide ‘ 2 parts. Distilled water . F 300, The following is the method :— 1. Stain in aniline oil gentian-violet or in carbol-gentian-violet (vide supra) for about five minutes. 104 MICROSCOPIC METHODS 2. Without washing in water, now treat the section or film with repeated doses of Gram’s solution till its colour becomes a purplish black, and allow the solution to act for 1 minute. 3. Again without washing with water, decolorise with absolute alcohol or methylated spirit till the colour has almost entirely disappeared, the tissues having only a faint violet tint. The period of time for which the alcohol is allowed to act varies in different laboratories. The best period is probably about three minutes. 4. For sections dehydrate completely, clear with xylol, and mount. In the case of film preparations of Gram-positive organisms, the specimen is simply washed in water, dried, and mounted. With films of organisms, whose reaction towards the Gram stain is unknown, a contrast stain (vide infra) should be used. In stage (3) the process of decolorisation is more satisfactorily per- formed by using clove oil after sufficient dehydration with spirit, the clove oil being afterwards removed by xylol. As clove oil is a powerful decoloriser care is necessary in its use. As a contrast stain for the tissues, carmalum or lithia carmine is used before staining with gentian-violet (1), As a contrast stain for bacteria which are decolorised by Gram’s method, carbol-fuchsin diluted with twenty volumes of water or a saturated watery solution of Bismarck- brown may be used before stage (4); the former should not be applied for longer than a few seconds. The following modifications of Gram’s method may be given :— 1. Weigert’s Modification.—The contrast staining of the tissues and stages (1) and (2) are performed as above. (8) 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 in the case of sections, but decolorisation by alcohol is preferable in the case of films of pus, etc. 2. Nicolle’s 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), or by the other methods mentioned above. 3. Jensen’s Modification.—For this there are required : (a) a 0°5 per cent. solution of methy]-violet (6 B) in water ; (b) a solution of iodine, 1 gramme ; potassium iodide, 2 grammes ; water, 100 c.c. ; (c)a solution of neutral-red, 1 gramme in 1000 c.c. water, to which are added 2 c.c. of 1 per cent. acetic acid. Thin films are fixed hy heat and allowed to cool ; treat these with the methyl-violet for 1-4 minute ; wash the stain off with the iodine solution, and allow this to act for 3-1 minute; wash off with absolute alcohol, and treat with fresh alcohol till the necessary decolorisation is complete; wash off the alcohol with the neutral-red, and allow the counter-stain to act for 4-} minute; wash with water; dry with filter paper, and mount. There is great variability in the avidity with which organ- isms stained by Gram retain the dye when washed with alcohol, TUBERCLE STAINS 105 and sometimes difficulty is experienced in saying whether an organism -does or does not stain by this method. Most bacteria are either frankly Gram-positive or frankly Gram- negative, but cases occur where an organism, usually Gram- positive or Gram-negative, tends when grown on certain media to show an opposite tendency, and sometimes an organism is met with in which the individuals in a film show slightly different reactions to the Gram stains. The commonest variation is for a Gram-positive organism to become in older cultures Gram-negative. According to Unna, the Gram stain can only be carried out with the pararosanilin group of dyes (e9., victoria blue, methyl violet, crystal violet, or gentian violet, which is a mixture of the last two). Two theories, a chemical and a physical, have been advanced to explain the reaction. According to the former, the iodine combines with the dye and links it to the bacterial protoplasm. According to the physical, the stain is deposited in the protoplasm, and is relatively slowly washed out by alcohol. The iodine penetrates Gram-positive bacteria most readily and causes a more pronounced deposit of the stain. Recent work indicates that the physical explanation is the more probable, and that differences in the capsule of the two classes of bacteria is an important factor in the reaction. Stain for Tubercle and other Acid-fast Bacillii—These bacilli cannot be well stained with a simple watery solution of a basic aniline dye. This fact can easily be tested by attempt- ing to stain a film of a tubercle culture with such a solution ; with the Gram method, however, a partial staining is effected. Such bacteria require a powerful stain containing a mordant, and must be exposed to the stain for a long time, or its action may be aided 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. 285), and a considerable number of other acid-fast bacilli are now known (p. 283).. Any combination of gentian-violet or fuchsin with aniline oil or carbolic acid or other mordant will stain the bacilli named, but the following methods are most commonly used :— Ziehl-Neelsen Carbol-Fuchsin Stain. Basic fuchsin . ; 1 part. Absolute alcohol . : 10 parts. Solution of carbolic acid (1 : 20) . 100 _=,, 106 MICROSCOPIC METHODS 1. Place the specimen in this fluid, and having heated it till steam rises, allow it to remain there for five minutes, or allow it to remain in the cold stain for from twelve to twenty-four hours, (Films and paraffin sections are usually stained with hot stain, loose sections with cold; in hot stain the latter shrink.) 2. Decolorise with 20 per cent. solution of strong sulphuric acid, nitric acid, or hydrochloric et 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 and 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 methylene-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 . : : . 50 parts, Absolute alcohol ’ « BO a Nitric acid 20° 4 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. 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 from penetrating to the proto- plasm. Like the tubercle bacilli, when once stained they retain the colour with considerable tenacity. The following is the simplest method for staining spores :— STAINING OF CAPSULES 107 1. Stain cover-glass films as for tubercle bacilli. 2. Decolorise with 1 per cent. sulphuric acid in water or with methy- lated 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 staining some, methylated spirit is a sufficiently strong decolorising agent to 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 Moller, is much more satisfactory than the previous. Before being stained, the films are placed in chloroform for two minutes, and then ina 5 per cent. solution of chromic acid for 4-2 minutes, the preparation being well washed after each reagent. Thereafter they are stained and decolorised as above. The Staining of Capsules.—The 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) Hiss’s Method. —The staining solution consists of 1 part of a saturated alcoholic solution of fuchsin or gentian-violet and 19 parts of distilled water. A few drops of the stain are placed on a filw, previously dried and fixed by heat, and the preparation is steamed for a few seconds over aflame. The staining solution is washed off with a 20 per cent. solution of copper sulphate, the preparation (without being washed in water) is dried between filter-papers, and when thoroughly dry is mounted in balsam. The capsules of pneumococci in exudates or growing in a fluid serum medium can be readily demonstrated by this method ; in the case of solid cultures, films should be made without any diluent, or a drop of fluid serum should be used. The method is easily applied, and gives excellent results. (c) Richard Muir's Method (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. 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. 4 Drs Saturated solution of potash alum . 5 a 4. Wash well in water. 5. Treat with methylated spirit for about a minute. 108 MICROSCOPIC METHODS 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 certain culture media may be demonstrated by this method. (d) Capsules can also be demonstrated by the Indian-ink method (p. 111). 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 very 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 91. 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 Peete Alum, saturated watery solution . : : ay = SDE Carbol-fuchsin (vide p. 105). z : ay p SDE 35 Mix thoroughly. ) is dropped on the film, heated till steam rises, and allowed to remain for about half a minute. It is again washed in water, THE ROMANOWSKY STAINS 111 solution (c) is dropped on, heated till steam arises, and allowed to remain for another half minute. The preparation is finally washed in water and dried. The spirochetes are of a dark brown or black colour, and are easily found. This is the best method, and is easily carried out. (2) Indian Ink Method.—An emulsion of indian ink of fine quality is sterilised by steaming and allowed to settle for a few days; a drop of the deposit diluted with an equal quantity of distilled water is well rubbed up and spread on a slide with a drop of the material to be examined (exudate from chancre or condyloma, scraping from congenitally affected organ, etc.). The film is dried and examined with an immersion lens without the interposition of acover. Spirochetes, if present, stand out unstained, surrounded by the dark indian ink, and often positive results are rapidly obtained by means of it. The organisms are not so readily recognised by this method as by dark-ground illumination, and negative observations are thus less valuable. (8) Congo-Red Method (Benians).—A small drop of a 2 per cent. aqueous solution of Congo red is placed on a slide, and a very small quantity of the secretion or exudate to be examined is rubbed into it with the platinum wire. The drop is then spread out into a tolerably thick film and allowed to dry. The film is then treated with a 1 per cent. solution of hydrochloric acid in absolute alcohol, and the preparation is dried in the air, or with blotting-paper, though the latter is apt to tear the film. Spirochetes and bacteria show up unstained on the dark back- ground. (4) Giemsa’s Method, see p. 113. 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 aleohol—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 formule 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. The stains in use thus contain a mixture of methylene-blue and its derivatives in combination with eosin ; the differences in these bodies and the different proportions in which they occur in individual stains account for the different effects produced on the various constituents of a cell. The underlying chemical reactions are complicated and as yet not fully understood. Thus it is not certainly known to what 112 MICROSCOPIC METHODS particular new body the reddish hue produced in chromatin ig due, but the active constituent may be methyl-violet or methyl- azure or thionin, all 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 in leucocytes are beautifully brought out), in staining bacteria in tissues or exudates, the malaria parasite, trypanosomes, the pathogenic spirochetes (such as the spirochete pallida), and protozoa generally. The following are the chief formule in use :— 1. Jenner’s 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) a1'2 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. Ofthe powder, ‘5 grm. is 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 tilter-paper and mounted in xylol balsam. 2. Leishman’s Stuin.—The following solutions are prepared: (a) to a1 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); (6) 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 methy! 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 afew 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, the preparation being frequently tilted to prevent precipitation of the stain, 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 THE ROMANOWSKY STAINS 113 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 must be kept covered, and if in such cases a granular deposit 1s 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 film 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. In certain cases, ¢.g., for the staining of old films or of trypanosomes or Leishmanize in sections, Leishman recommends an initial treat- ment of the preparation with serum. This modification is described in Appendix E, 3. J. H. Wright’s Stain,—In this modification 1 per cent. methylene- blue (BX or Ehrlich’s rectified) and 4 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 mix- ture becomes purplish and a finely granular black precipitate appears in suspension (about 500 c.c. eosinto 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 e.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 fiuid, 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 com- plete ; 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 an equal part 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., 0°8 gr. ; glycerin (Merck, chemically pure), 250 gr.; methyl alcohol (Kahlbaum, I.), 250 gr. This stain has been extensively used for demonstrating spirochetes, but it can be used for any other purpose to which the Romanowsky stains are applicable. For spirochetes the following are Giemsa’s directions :— (1) Fix films in absolute alcohol for fifteen to twenty minutes, dry with filter-paper. (2) Dilute stain with distilled water—one drop of 8 114 MICROSCOPIC METHODS 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. water.) (8) Stain for fifteen minutes (a longer period is often desirable, even twenty-four hours). (4) Wash in brisk stream of distilled water. (5) Drain with filter-paper, dry, and mount in Canada balsam. ; Neisser’s Stain.—(a) The following is the original method introduced by Neisser 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. 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; (b) 2 grms, Bismarck-brown (vesuvin) dissolved in a litre of distilled water. Films are stained in (a) for 1-3 seconds or a little longer, washed in water, stained for 3-5 seconds in (b), dried, and mounted. The protoplasm of the diphtheria bacillus is stained a faint brown colour, the granules a blue colour. Cultures should be grown on a‘ serum medium and examined within 24 hours. Satisfactory results are not always obtained in the case of films prepared from membrane, ete. (b) The following is Neisser’s modified cresoidin method :— 1. Stain films for a few seconds in a mixture of solutions A and B, two parts of the former to one of the latter. A. Methylene-blue ee : 1 part. Absolutealcohol . : 50 parts. Glacial acetic acid . ; 50, Distilled water 1000 ,, B. Crystal-violet (Hochst) . : 1 part. _ Absolute alcohol ; , 10 parts. Distilled water ‘ ‘i 300 ,, 2. Wash in water, and 3. Stain in cresoidin solution (1: 300) for a few seconds (the cresoidin should be dissolved in warm water and the solution then filtered). 4. Wash in water, dry, and mount. Instead of cresoidin the following solution of erythrosin may be used with advantage: Saturated alcoholic solution of erythrosin, 20 parts; saturated watery solution of picric acid, 90 parts; add to the mixture precipitated calcium carbonate to excess; allow to stand for a time, shaking at intervals ; filter. Sabouraud’s Method for Staining Trichophyta.—Remiove the fat from the hair or epithelial squames with chloroform. Place in a test-tube with 10 per cent. formol, and warm for two or three minutes till ebullition commences. Wash well in distilled water, and stain for one minute in Sahli’s blue, which is made up as follows :— Distilled water . . Fi . : . 40 parts. Saturated watery methylene-blue. ee De as 5 per cent. solution of borax in water - ar LO a3 Mix the constituents. Allow to stand for a day, and filter. After staining, wash in water, dehydrate with absolute alcohol, clear in xylol, and mount in balsam. CHAPTER IV. METHODS OF EXAMINING THE PROPERTIES OF SERUM—PREPARATION OF VACCINES— GENERAL BACTERIOLOGICAL DIAGNOSIS—IN- OCULATION OF ANIMALS. Tue Trstinac oF AGGLUTINATIVE AND SEDIMENTING PROPERTIES OF SERUM. In studying the properties of serum it is necessary to have the means of measuring and diluting small quantities of fluid. The simplest method is by means of 1 cc. and ‘1 ¢.c. pipettes, which can be got from an instrument-maker. Each pipette should be graduated in tenths, and should deliver to the end. If the original amount of fluid to be used is small, say less than ‘02 c.c., it should be diluted till it has fully this volume. This may be done by drawing up the fluid in a capillary tube (a piece of quill glass-tubing drawn out in the flame being convenient for the purpose) and marking the upper limit of the fluid, the latter then being blown out in a watch-glass. Equal amounts of ‘8 per cent. salt solution can be measured out with the marked tube and added till the fluid has the necessary volume. Thorough mixture is effected by drawing up the diluted serum in the quill tubing and blowing out again, this being repeated several times. Further dilutions can be made by the graduated pipettes. Where such pipettes are not available, Wright’s method may be used. : Wright’s Method of measuring Small Amounts of Fluids.—A Gower’s 5 c.mm. hemocytometer pipette and some pieces of quill glass-tubing are required. 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 still possessing a bore. If the point be broken off this hair, and mercury be run into the tube, the metal will be caught where the tube narrows and will pass no further—in fact, though air will pass, mercury will not. Into the wide end of this tube 5 c.mm. of mercury, measured from a Gower’s pipette, is run down -till it will go 116 116 METHODS OF EXAMINING SERUM uo further. A mark is made on the tube at the proximal end of the mercury, which is now allowed to run ont, and the tube is carefully cut through at the mark. A piece of ordinary quill tubing is drawn out and broken off just below the point where narrowing has begun, the hair end of the capillary tube is slipped through the broken-off end, and the tube is fixed in position with wax 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 when pressure on the nipple is relaxed. Thus other tubes can be very readily calibrated ae by the mercury being expelled into them, and its limits marked on their bores. For measuring equal parts of different qj PP D fluids, the pipette shown in Fig. 40, d, : in connection with agglutination is very ie useful. fi Methods of testing for Simple Ag- eee glutination.—By agglutination is meant the aggregation into clumps of uniformly disposed bacteria in a fluid ; by seddmenta- tion the formation of a deposit composed of such clumps when the fiuid is allowed to stand. Sedimentation is thus the aera naked-eye evidence of agglutination. The blood serum may acquire this clumping : power towards a particular organism under Fic. 39.— Wright's 5 certain conditions,—these being chiefly met c.mm. pipette. A, ~~ : eee : a casing of quill tubing; With when the individual is suffering from B, rubber nipple; C, the disease produced by the organism, or ie a be i has recovered from it, or when a certain c.mm, capacity; D to degree of immunity has been produced E, hair capillary. artificially by injections of the organism. The nature of this property will be dis- cussed 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, corre- sponding 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 METHODS OF TESTING FOR AGGLUTINATION 117 the mixture is placed in an upright thin glass tube ; sedimenta- tion is shown by the formation within a given time (say from two hours at 55° C. to twenty-four hours at room temperature) 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 with the bacterial emulsion alone, and (6) the serum to be tested should never be brought in the undiluted condition into contact with the bacteria. The stages of procedure 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. 41) or into the hollow bulbous portion of a capillary pipette, such as in Fig. 40, a. (These pipettes 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 pipette. ‘The serum is then to be diluted. 2. The serum may be diluted (a) by means of a graduated pipette— either a leucocytometer pipette (Fig. 40, b) or some corresponding form, In this way successive dilutions of 1:10, 1 : 20, 1: 100, ete., can be rapidly made. This is the best method. (+) By means of a capillary ipette with a mark on the tube, the serum is drawn up to the mark and then blown out into a glass capsule; equal quantities of bouillon are successively measured in the same way, and added till the requisite dilution is obtained. (c) By means of a platinum needle with a loop at the end (Delépine’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. Yhe drops are then mixed. A very convenient and rapid method of combining the steps 1 and 2 is to draw a drop of béood 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. 40, c), and centritugalise or simply allow the red corpuscles to separate by standing. (In this method, of course, the dilution is really greater than if pure serum were used, and allowance must therefore be made in comparing results.) The presence of red corpuscles is no drawback in the case of the microscopic method, but when sedimentation tubes are used the corpuscles should be separated first. 3. The bacteria to be tested should be taken from young cultures, preferably not more than twenty-four hours old, incubated at 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 or ‘8 per cent. solution of sodium chloride. 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 uni- formly mixed with the rest of the fluid. The bacterial emulsion ought to have a faint but distinct tarbidity. (When the exact degree of sediment- 118 METHODS OF EXAMINING SERUM ing 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 b d and finally blown carefully capillary pipette) of the | { it a down close to the lower 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. 25) Fic. 40,—Tubes used in testing agglutinating end which is then sealed and sedimenting properties of serum. off.” The sediment collects at the lower extremity. It is often important to observe not merely the fact that agglutination occurs, but also the weakest concentration of the serum with which the reaction can be obtained. Standard Cultures.—Dreyer has introduced a method of standardising agglutinable cultures, the organisms being killed by formalin. The bacillus (b. typhosus, b. paratyphosus, etc.) is grown for twenty- Z will be found very suitable. Z The ultimate dilution of Z the serum will, of course, be IZ double the original dilution. Dp 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 at room temperature, or at 55° C. in a water bath for two hours. One of Wright’s sedimentation tubes is shown in Fig. 40, 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, z which is then drawn up in the same quantity; the diluted serum will then occupy the position ki. The fluids are then drawn several times up into the bulb, and returned to the capillary tube so as to mix, Y MEASUREMENT OF GROUP AGGLUTININS 119 four hours at 37° C. in ordinary veal peptone bouillon in an Erlenmeyer flask. At the end of this time the flask is shaken and there is added to it 0°1 per cent. of commercial formalin ; it is again shaken and placed at once in a cool chamber at about 2° C. in the dark. The shaking is repeated at intervals for 4 to 5 days, the flask always being replaced in the cold chamber. At the end of three or four days the culture will be found to be sterile and will keep practically indefinitely. Such killed cultures are very suitable for sedimentation tests. Each culture is standardised by (a) its opacity being brought, by dilution with normal saline containing 0°1 per cent. formalin, as nearly as possible identical with that ofa ‘‘standard agglutinable culture,” and (6) by measur- ing its agglutinability as compared with that of the standard culture.! Measurement of Group Agglutinins.—In the case of certain groups of allied organisms,—notably the b. coli and its allies,— it has been found that when a serum clumps one member of the group it may also clump some of the allied forms. If the greatest dilution with which agglutination is obtained be esti- mated, the end-points for the different strains affected will be found to differ. The determination of the end-point is important, as the disease condition from which the serum is derived is generally caused by the organism which is clumped in highest dilution. In comparing the effect of a serum on different bacteria, the sedimentation method is usually employed. A series of emulsions of the different bacteria to be tested is prepared by scraping off the growth on an agar tube, and suspending in normal saline. Each of these should contain approximately the same number of bacteria per unit volume. This is attained by using emulsions of equal opacity, as judged of by noting the point at which transparency to some arbitrary standard such as a particular type or set of parallel lines ceases. A given amount of each emulsion is now mixed with different dilutions of the serum to be tested, the mixtures are all made up to the same volume, say 1 c.c., and the tubes placed at 55° C. for two or three hours. The results are then read. Dreyer takes as standard agglutination the highest dilution in which marked agglutination without sedimentation can be detected by the naked eye. It is often, however, an advantage to examine the results with a hand lens. Further details will be given in deal- ing with individual organisms. The Absorption Method of testing Agglutinins.—This method is applied under circumstances similar to those of the last, namely, when several agglutinins acting on allied organisms are present in a serum. The principle is to remove all the agglutinins acting on one organism, and to study the properties 1 Full details as to the use of standard cultures and sera may be had from the Department of Pathology, University of Oxford. 120 METHODS OF EXAMINING SERUM of those which remain. In practice, the method consists in adding to the serum an equal volume of a thick emulsion of the bacterium (the organisms being scraped off an agar slope), allowing the mixture to stand at 37° C. for two or three hours, aud then separating the bacteria with the centrifuge. The supernatant clear fluid is now pipetted off, and its agglutinating properties studied on the other members of the bacterial group either by sedimentation or by the microscopic method. The object of the method is to determine which member of a bacterial group is causally related to the condition from which the serum is obtained, and examples of its application for this purpose will be found in the chapter on Typhoid Fever (p. 393). Here the principle is that, when an unknown strain belonging to such a bacterial group is under investigation, if its capacities for absorbing agglutinins from a serum are the same as those of an already recognised strain, then the two are probably identical. On the other hand, an allied strain to the organism by which the agglutinin has been produced will absorb only part of the agglutinin. Opsonic METHODS. Method of measuring the Phagocytic Capacity of the Leucocytes.—This was first done by Leishman by a very simple method, as follows :— Equal quantities of blood and of a fine emulsion of the bacterium to be tested are mixed together, a small drop of the mixture is placed on a glass slide and covered with a cover-glass ; the preparation is placed in the incubator at 37° C. for fifteen minutes. The cover-glass is then slipped off and the film on the slide stained by Leishman’s method. A control preparation can be made with normal blood in the same way and the two films are stained as one. The number of bacteria present in, say, fifty polymorphonuclear cells successively examined is determined, and an average struck. By this method Leishman showed that in cases of staphylo- coccus 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. Wright subse- quently showed that phagocytosis depended upon certain sub- stances in the serum to which he gave the name opsonins (see Immunity) and elaborated a method by which its degree could be estimated. The 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, and (4) serum from the infected ‘person. OPSONIC METHODS 121 (1) Preparation of Bacterial Emulsion.—In the case of ordinary organ- isms, ¢.g., the pyogenic cocci, a little of a twenty-four-hour living culture on a sloped agar tube is taken and rubbed up in a watch-glass with ‘85 per cent. saline so as to obtain an emulsion consisting of single bacterial cells. With certain organisms, ¢.g., streptococci in chains, a good deal of tritura- tion may be necessary, and often centrifuging must be practised, for the removal of clumps. 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. If too strong an emulsion be used, the leucocytes may take up so many organisms that these cannot be accurately enumerated. When intensely pathogenic organisms are used, ¢.g., b. pestis, m. meli- tensis, Wright recommends that the culture should be first killed by emulsifying in 40 per cent. formalin. The latter is then removed by centrifuging and the deposit washed with saline. In the case of the tubercle bacillus, Wright directs that a 7-10 day culture in glycerin broth should be sterilised by heat, collected on a filter, washed with salt solution, and dried. Ten milligrams of the dry culture should be powdered in a small agate mortar, a drop of 1 per cent. saline added, and the sticky paste triturated for about five minutes ; further saline is added drop by drop till a thick emulsion is obtained of the bulk of about1c.c. This is centrifuged and the supernatant suspension pipetted off and diluted to the necessary degree. (2) Preparation of Leucocytes.—Here the observer uses his own blood cells. . tion ‘frequently appears in fluid serum media, 4 e.g., f the organism be grown in rabbit or human — serum which has been > obtained under aseptic aca precautions and heated Fic. tre tags pneumococcus froma pure for half an hour at 55° C. culture on blood agar of twenty-four hours’ growth, some in pairs, some in short chains. OF On Agar slopes over Stained with weak carbol-fuchsin. x1000. Which a drop of serum has been run. The pneumococcus is non-hemolytie on blood-agar plates (p. 45), and it ferments saccharose, raffinose, and lactose ; a similar fermentative action on inulin is important, as ordinary streptococci do not ferment this sugar. Apparently some samples of inulin are more readily acted on than others. Usually the test is carried out with Hiss’s inulin serum water medium, in which coagulation of the serum results (p. 46), but some investigators have had more success with inulin bouillon, acid production being estimated, by titration against soda with a phenolphthalein indicator. The pneumococcus is soluble in bile. To demonstrate this, fresh ox bile autoclaved for twenty minutes at 120° C, and filtered is added to a fully developed fluid culture (which must be one in simple bouillon) in the proportion of about a fifth of the OCCURRENCE OF PNEUMOCOCCUS 231 culture. Two per cent sodium taurocholate may be similarly used. The facts that in cultures the pneumococcus often grows in chains, and that occasionally streptococci are found to develop capsules, have raised the question of the relationship of the pneumococcus to other streptococci. In determining the true pneumococci, biological as well as morphological characters must be studied, and here the bile solubility of the pneumococcus, its failure to produce hemolysis, and its capacity of ferment- ing inulin are the important characters. It must be stated, however, as bearing on the close relationships of the pneumo- cocci and streptococci, that Rosenau believes he has succeeded in transforming streptococci into capsulated organisms having all these biological features of the pneumococcus. Considerable attention has been directed to a group of cocci originally described by Schottmiiller, isolated from various dis- ease conditions in man (pneumonia, meningitis, suppurations), which besides possessing voluminous capsules have these sur- rounded by a viscous material which gives a slimy consistence to cultures and also to pathological exudates. These are related to the pneumococci on the one hand and to the streptococci on the other. The work of the Rockefeller investigators (v. infra) suggests that these organisms ought to be classified into two groups. (1) The pnewmococcus mucosus. This organism tends to be not so pointed as the ordinary pneumococcus, and its colonies are larger ; it is non-hemolytic on blood agar, soluble in bile, gives rise to acid and clot in Hiss’s inulin serum-water, and is very pathogenic to white mice and rabbits. Anti-sera produced by strains of this coccus, while showing cross agglutina- tion towards members of their own group, do not agglutinate streptococci and usually also not other pneumococci. (2) The streptococcus mucosus. This organism is generally round, occurs in chains, and the colonies are less transparent than those of the pneumococcus; it is usually non-hemolytic, is not soluble in bile, does not ferment inulin, and is les’ pathogenic to mice than the last. Thus while the pneumococcus mucosus is practically a true pneumococcus, the streptococcus mucosus forms a connect- ing link with the true streptococci. The Occurrence of the Pneumococcus in Pneumonia and other Conditions.—The pneumococci occurs in every variety of the disease—in acute croupous pneumonia, in broncho- pheumonia, in septic pneumonia. In a case of croupous pneu- monia the pneumococci are found all through the affected area in the lung, especially in the exudation in the air-cells. They 232 THE ACUTE PNEUMONIAS 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, ¢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. When the inflam- mation is resolving, some of the organisms often stain badly (e.g., tend to lose the Gram-positive reaction) ; such individuals are probably either dead or dying. Sometimes there occur in pneumonic consolidation areas of suppurative softening, which may spread diffusely. In such areas the pneumococci occur with or without ordinary pyogenic organisms, streptococci being the commonest concomitants. In other cases, especially when the condition is secondary to influenza, gangrene may supervene and lead to destruction of large portions of the lung. In these a great variety of bacteria, both aerobes and anaerobes, are to be found. In ordinary broncho-pneumonias also Fraenkel’s pneumo- coccus is usually present, sometimes along with pyogenic 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 bacilli or the b. coli may be alone present or be accompanied by the pneumo- coccus, 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 neighbouring parts, empyema, pericarditis, and lymphatic enlarge- ments 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 (especially in children), joints, kidneys, liver, etc.), in otitis media, ulcerative endocarditis (p. 217), and meningitis. In fact, there is practically no inflam- matory or suppurative condition in the body in which the pneumococcus in pure culture may not be found. These condi- tions 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 vy EXPERIMENTAL INOCULATION 233 pneumococcus. A large number of cases have been investigated by Netter, who gives the following tables of the relative fre- quency of the primary infections by the pneumococcus in man :— (1) In adults— Pneumonia. : : : . 65°95 per cent. Broncho-pneumonia Capillary bronchitis : : » 15°85 ” Meningitis. : ; z - . 13°00 > Empyema : . 7 : . 853 5 Otitis. . : . 244 ,, Endocarditis ‘ ; a aD 35 Liver abscess . 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 pneu- monia, 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 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 pnewmococeus 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 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 septicemia. Thus, if a rabbit or a mouse be injected subcutaneously with pneumonic sputum, or with a scraping from a pneumonic lung, death oceurs in from twenty-four to forty-eight hours. There is some fibrinous infiltration at the point of inoculation, the spleen is often enlarged and firm, and the blood contains capsulated pneumococci in large numbers (Fig. 61). If the seat of 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 pneumococcus on artificial media may lose their virulence. Now, if such a partly attenuated culture be injected subcutaneously into a rabbit, there is greater local reaction; pneumonia, with exudation of lymph 234 THE ACUTE PNEUMONIAS on the surface of the pleura, and a similar condition in the peritoneum, may occur. It may also be said that if a rabbit be immunised with dead or attenuated cultures and then injected with a virulent culture, similar local reactions may occur at the inoculation site. 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 a ig yA se : ee ae \ er Fic. 61.—Capsulated pneumococcus 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. 1000. 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. By intra-tracheal inoculation in dogs Lamar and Meltzer have produced a fibrinous pneumonia pathologically similar to what occurs in man. EXPERIMENTAL INOCULATION 235 The general conclusion to be drawn -from these experiments thus is that in highly susceptiblé animals virulent pneumococci produce a general septicemia ; 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 importance. 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 septicaemic processes which may be produced 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 by Pasteur and others of this organism in the saliva of healthy men. It can certainly be isolated by inoculation of susceptible animals, from the mouths of a considerable proportion of normal men, from their nasal cavities, etc., being probably in any par- ticular individual more numerous at some times (especially, it is stated, during the winter months, ¢.e., a little before the period of the greatest prevalence of pneumonia) than at others, and sometimes being entirely absent. This may indicate the import- ance of predisposing causes in the etiology of the disease, which applies in the case of the diseases caused by pyogenic staphylo- cocci, streptococci, the bacillus coli, etc. By such causes the vitality and power of resistance of the lung may be diminished, and then the pmeumococcus gain an entrance. We can there- fore understand how less definite devitalising agents such as cold, alcoholic excess, ete., 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 must be stated, however, that ac- cording to the Rockefeller investigations (infra) the pneumo- 236 THE ACUTE PNEUMONIAS coccus occurring in the healthy naso-pharynx is usually of Type IV., ze, belongs to the group least pathogenic to man. The more pathogenic types are found almost exclusively in the mouths of convalescents and of contacts and in rooms where pneumonia cases have been nursed. While these types usually rapidly disappear from convalescents and contacts they may per- sist long enough to justify the view that certain persons may act as carriers of the disease. 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 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. We are therefore justified in holding that it is the chief factor in causing croupous pneumonia, and also plays an important part in other forms. Immunisation against the Pneumococcus.—Animals can be immunised against the pneumococcus by inoculation with virulent cultures killed by heating at 55° C., with cultures which have become attenuated by growth on artificial media, or with the naturally attenuated cocci which occur under various circumstances. Sometimes one or two injections, at intervals of several days, are sufficient for immunisation, but the immunity has been observed to be usually of a fleeting character and may not last more than a few weeks ; a process of intensive and rapid immunisation is described below. The serum of such immunised animals mixed im «tro with pneumococci neutralises the action of these in susceptible animals, may also protect against subsequent inoculation with carefully regulated doses of pneu- mococci, and if injected within twenty-four hours after inocula- tion, may prevent death. Such serum also possesses an agglutinating action in low dilutions on the pneumococcus originating the immunity. STRAINS OF PNEUMOCOCCUS 237 Differentiation of Strains of the Pneumococcus by Anti- sera.—The possibility of effecting this is one of the most important consequences of the study of immunity against the pneumococcus. It had been long recognised that strains of the pneumococcus derived from different sources present individual peculiarities, but it was not till the recent exhaustive investiga- tion of the subject in the Rockefeller Institute, New York, that definite results were obtained. In the study of the agglutinating and protecting properties of antisera prepared by inoculating animals against a long series of cultures isolated from cases of acute lobar pneumonia, it was proved that sera derived from certain strains, on the one hand, would almost indiscriminately agglutinate some of these strains, and, on the other, had little: or no effect on other strains. It was further found that the agglutinating and protective qualities of these sera were parallel. In this way it was possible to group the strains under four types. Three of them (I., IL, III.) were definite, and a fourth (IV.) was formed of strains in which an anti- serum usually only agglutinated the strain which originated it, and had little or no capacity of agglutinating the strains of Types I., II., III. The members of Type III. could be recog- nised not only by their originating agglutinating sera specific to the group, but presented cultural features which characterised them as the pneumococcus mucosus (see p. 231). Types I. and II. between them accounted for 60 per cent. of the cases of pneumonia studied and are of relatively high virulence for man, this being specially the case with Type II. Type III., while accounting for only 12 per cent. of cases, is of highest virulence, the mortality with it being 45 per cent. Type IV. was found in 24 per cent. of cases and caused the lowest mortality (16 per cent.) ; the strains occurring in the mouth of healthy individuals probably belong to this type. The fundamental facts of the New York investigation have been confirmed by observers elsewhere, and are obviously of great practical importance for diagnosis and, as we shall see, for treatment. It is probable, however, that in different parts of the world different strains prevail. Thus, in South Africa, Lister has found that, while the New York Types I. and II. are common, nearly a third of all cases of pneumonia are associated with another type which apparently does not occur to any extent in New York. Methods of classifying Pneumococci by Agglutination.—This depends on the observer being furnished with the type sera (I., II.; III.) of the Rockefeller Institute. A white mouse is inoculated intraperitoneally with 0°5 to 1 ec. of a saline emulsion of a bean-sized piece of sputum, 238 THE ACUTE PNEUMONTAS freed of surface contamination by washing in sterile saline. The mouse may die in from five to twenty-four hours, and if the peritoneal exudate contains a strong and fairly pure growth of the pneumococcus the abdo- minal cavity is washed out with 5c.c. saline, cultures being at the same ‘time made in broth and on blood-agar plates. The peritoneal washings are first centrifuged slowly to precipitate gross material, and the supernatant fluid is then centrifuged at a high speed to precipitate the bacteria. The bacterial deposit is emulsified in saline to form a fairly heavy suspension which is used for a macroscopic sedimentation test. If the pneumococci in blood cultures or in other exudates are to be employed, emulsions may be obtained by similar procedures. A bacterial emulsion being prepared, 0°5 c.c. of Serum I. (1-20), 0°5 c.c. of Serum IT. (undiluted), 0°5 cc. of Serum II. (1-20), and 0°5 c.c. of Serum III.1 (1-5) are placed in four tubes, and 0°5 c.c. bacterial emulsion added to each, and in a fifth tube a mixture of 0°1 c.c. sterile ox bile and 0°4 c.c. bacterial emulsion is made up ; the series is placed in a water bath at 37° C. for one hour, and the result read off. Sedimentation in any one of the four tubes indicates that the strain belongs to the type by the serum of which it is agglutinated : if no reaction occurs in any of the tubes, and the organism is soluble in bile, it belongs to Type IV. The Treatment of Pneumonia with Anti-sera.—Many years ago the Klemperers treated a certain number of cases of human pneumonia by serum derived from immune animals, apparently with a certain measure of success, and more recently Romer issued through Merck a polyvalent serum prepared by immunis- ing different species of animals with growths of the pneumococcus on sheep-serum glycerine bouillon and mixing their sera. The results obtained, though in some cases satisfactory, were irregular, and the subject was illuminated by Neufeld and Haendel, who insisted that in the use of any anti-pneumococcic serum means should be taken for ensuring that it had an antagonistic action on the particular strain present in the particular infection treated. Evidence confirming this view has been obtained in the New York investigations on pneumonia, in which the determination of the different types of the pneumococcus was followed by an estimate of the therapeutic capacities of the anti-sera prepared against Types I., II., III. (v. supra). It was found that while the anti-serum to Type I. had a marked curative effect on cases of pneumonia due to the Type I. pneumococcus, the anti-sera to Types IT. and III. had practically no effect on cases attributable to these types ; furthermore the anti-serum to Type I. had little or no effect on cases caused by Types II. and III. These facts throw light on the irregular and generally disappointing result obtained hitherto with the ordinary polyvalent antipneumococcal 1 There is apparently sometimes difficulty in effecting the agglutination of Type III. on account of the consistence of its capsule, and special methods may be necessary ; see Hanes, Journ. Ea:p, Med., 1914, xix. 38, PATHOLOGY OF PNEUMOCOCCUS INFECTION 239 sera. The Rockefeller serum is prepared by immunising horses first with dead cultures ; daily injections are given for six days, followed by an interval of a week, then six further daily injec- tions are given ; it is sometimes necessary to follow these, up ‘by the use of living organisms. Uniformity of strength in successive sera thus prepared is secured by determining the largest amount of an eighteen hours’ culture against which 0-2 ¢.c. of the serum will protect a white mouse,—a comparison with the effects of the same amount of a standard serum being at the same time made. In the therapeutic application of the serum in a case of Type I. pneumonia large quantities must be used, and it is therefore a necessary preliminary to determine whether the patient exhibits hypersensitiveness to horse serum, and to desensitise him if this exists (see chapter on Immunity). If the way be clear, the serum, diluted with an equal amount of sterile saline made with freshly distilled water, is administered by the intravenous method—10-15 ¢.c. being given at the rate of 1 c.c. per minute —changes in the heart’s action and in respiration and the occurrence of urticaria being watched for, and the treatment being suspended for a quarter of an hour if untoward symptoms seem to increase; if this does not occur, the remainder of the first dose may be given during fifteen minutes. The initial dose should be from 90-100 e.c., and the injections ought to be re- peated every eight hours till about 250 c.c. serum have been given. Very soon after commencement of treatment the tem- perature may rise, but this is quickly succeeded by a fall, with improvement in the patient’s general condition, stoppage of ex- tension of the lung lesion, and prevention of invasion of the blood by the pneumococci. The effects of the treatment so far have been satisfactory,—of 107 cases treated in the Rockefeller Institute Hospital up to October 1917 only 7°5 per cent. died, as compared with a mortality of 25 to 30 per cent. in cases of Type I. pneumonia before the serum treatment was introduced. Up to the present no means of treating pneumonia of Types IT. and IIT. by serum methods have been found practicable, and, as has been stated, the pneumococci of Type IV. do not yield a group anti-serum. 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 may play an important part. Pneumonia is a focal disease which presents at the same time the character 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 graye 240 : THE ACUTE PNEUMONTAS 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 having specific effects, but these have been unsuccessful. The general conclusion has been that the toxins at work in pneumonia are intracellular ; as in other cases, we may have to reckon with the distribution in the infected body of poisonous substances consequent on lysis of the infective agent. While the chief multiplication of the pneumococcus in pneumonia occurs in the lung, the organism frequently is found in the blood, and according to some observers its presence in greater numbers than 15 cocci per c.c. is of fatal import. There has been considerable difference of opinion as to the explanations to be given of the facts observed regarding im- munisation against the pneumococcus, and especially regarding the protective and curative properties of immune sera. There is no evidence that such sera possess either antitoxic or bactericidal properties. Within recent times many have accordingly 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 pneumococci when they are lying in the serum of an animal immunised against this bacterium. Wright instanced the pneumococcus as an organism insensible to bactericidal action but very sensitive to opsonins, and Neufeld and Rimpau have described the occurrence of an opsonic—or, as they called it, a bacteriotropic—effect in the action of an anti-pneumococcic serum. In studying further the relationship of the opsonic effect to pneumococcic 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, the essential nature of which is, however, still entirely obscure. 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 may be above normal in consequence of the leucocytosis which usually accompanies a successful re- sistance to this infection. It has been observed, however, that as the crisis approaches in a case which is to recover, the opsonic index rises, and after defervescence gradually falls to normal. Correlated with this, there has been observed about the crisis an increase in the serum of substances capable of PATHOLOGY OF PNEUMOCOCCUS INFECTION 241 protecting animals against pueumococcal infection. These must at ‘present be looked on as the bodies concerned in the curative action of anti-serum. With regard to them Neufeld and Haendel insist that the concentration in the patient’s blood, rather than the amount present in the body, is the important factor. There is experimental evidence that when the concentra- tion is above a certain degree an enormous number of pneumo- cocci can be successfully disposed of, while with a concentration below this limit.a relatively small dose may prove fatal. As bearing on the factors involved 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. It is further stated that avirulent cultures of the pneumococecus can be made to resist phagocytosis if they are treated with the products of the autolysis of virulent strains or with washings from such strains, and that virulent cocci if washed with saline become capable of being readily phagocyted. While it cannot be stated definitely that the opsonic qualities of the serum are the essential factors in resistance to the pneumococcus, it is probable that the activities of the leucocytes play a part in the process. It has long been known that a leucocytosis occurs in the disease, and the degree of this is related to the outlook in the case. Thus, a low leucocyte count when correlated with the clinical symptoms indicates either a mild infection or a grave condition in which resistance is deficient. A count of over 10,000 per c.mm., progressively increasing, is a favourable sign in an uncomplicated pneumonia. The part played by the leucocytes has also been investigated experimentally by rendering the bone-marrow aplastic by means of benzol; under such circumstances the resistance of the animal to infection is diminished (Winternitz and Kline). A substance derived from the infecting pneumococcus some- times appears in the urine during pneumonia. It gives a precipitin reaction with the anti-serum corresponding to the type of pneumococcus causing the infection, and can be detected by mixing equal quantities of clear centrifuged urine with an equal amount of the anti-serum ; this method can, in fact, be used for determining the type of pneumococcus present in the body. The appearance of this substance in the urine is an indication that the case is a severe one, and a progressive increase in amount is a bad prognostic sign. It may be noted here, in conclusion, that in man immunity against pneumonia may be short-lived, as in a- good many cases of pneumonia a history of a previous attack is elicited. 16 242 THE ACUTE PNEUMONIAS The difficulty of interpreting the various serological facts observed in pneumonic conditions has led Lamar to investigate the action of certain chemical bodies, belonging to the soaps, on pneumococci. Welch long ago observed changes in the proto- plasm of pneumococci in pneumonic exudates, pointing to the occurrence of lysis. Lamar has found that pneumococci treated with sodium oleate and especially with potassium soaps of acids having a high iodine value—e.g., linoleic and linolenic acids— undergo morphological changes and become more subject to autolysis and more sensitive to the lytic action of sera, the latter being especially evident when immune sera are employed. The action of the soap is probably exerted on the lipoidal moiety of the bacterial cells, which are thus rendered more pervious to the serum constituents. There is evidence, however, that the protein constituents of sera exercise an inhibitory effect on the lytic action of the soaps, and Lamar has made the interesting observation that this inhibitory action can to a certain extent be neutralised by the use of boric acid. These observations are of the highest importance, and there is some experimental evidence that they may form the basis for a therapeutic treatment of pneumococcic infections. That they have a bearing on the explanation of natural recovery from such infections is indicated by the fact that in inflammatory exudations soaps form a definite constituent. Vaccine therapy in pneumonia.—lt may be stated here that vaccine therapy has been applied in the treatment of pneumonia, 20 to 30 millions of a stock vaccine being administered pending the preparation of an autogenous vaccine from cultures of the infecting strain made from material obtained by puncture of the pneumonic lung. Needless to say, the greatest care and judg- ment are necessary in the use of such vaccines. In certain cases there has been apparently a good result, but in others there is no evidence that the chance of survival has been greater than when ordinary treatment is applied. Something may be said for a combined treatment with serum and vaccine by the use of sensitised dead bacteria on the lines already described in dealing with streptococcic infections. Further, Rosenow has used as a vaccine pneumococci from which certain toxic properties have been removed by treatment with normal saline. Prophylactic Vaccination.—In the South African mines a special situation exists in consequence of the great susceptibility to pneumonia occurring in the native labourers, who are chiefly recruited from sub- tropical regions. As the case incidence may run from 30 to 150 per thousand per annum, and the mortality from 10 to 30 per thousand, the disease is a very serious one. Almroth Wright introduced prophylactic yaccination, and Lister, founding on his investigations (v. supra), pre- OTHER ORGANISMS IN PNEUMONIA 245 pared a vaccine containing the three prevalent types of the pneumococcus. In the latest applications of the method three injections at seven-day intervals of, in all, 7000 million bacterial bodies, killed by an antiseptic, were administered. A very marked diminution in the incidence of the disease has followed. Methods of Examination.—In stained films of sputum, pus, or other exudate containing pneumococci, the outstanding feature is the predominance of diplococcal forms the elements of which may havea lanceolate shape and which are Gram-positive. Often a capsule stain demonstrates the capsule in such material, and it may even appear stained in Gram films. Cultures on blood agar should be made which after 24 hours at 37° C. will, if the pneumococeus be present, show characteristic colonies. Subcultures on serum bouillon or serum-smeared agar will show capsulation. Bile-solubility and reaction with inulin may be tested ; if advisable a white mouse may be inoculated to test the pathogenicity and to afford in blood films corroborative evidence of capsulation. * OccuRRENCE or OTHER ORGANISMS IN PNEUMONIA. As might be expected, seeing that pneumonia is merely an inflammation occurring in a special tissue, or- ganisms other than the pneumococcus have been found associated with the disease, but in not more than about 5 per cent. of cases in all. The chief of these are the streptococcus pyo- genes, b. influenze, Friedlinder’s — pneumo- bacillus, b. coli (rarely) ; mixed infections with these and with the pneumococcus also . occur. Of the organisms Fic. 62,—Friedlinder’s pneumobacillus, show- named the pneumo- ing the variations in length, also capsules. : : . oe Film preparation from exudate in a case of bacillus is of historic in- pneumonia. x 1000. terest, as it was the first os 3 organism described in yneumonia, though there is little doubt that in early days it was often confused with the pneumococcus, 244 OTHER ORGANISMS IN PNEUMONIA Friedlinder’s pneumobacillus.—This organism does not occur alone in more than about one per cent. of cases of pneumonia. In the sputum it may appear as a very short diplobacillus possessing a capsule, but it also frequently is seen in the form of long rods (Fig. 62). It stains by ordinary methods, but Zoses the stain in Gram’s imethod. It can be easily isolated on agar plats, on which it forms large whitish en ee i _ ‘wef AS ‘s ) Oe i era finde Sy hg re Pity wr Ue f = ne gre sr Oh ed wa e* “s Sty ah . \ 4% ty) E . ee he Sek ns OTE ay “Ny oN i ue us . tes fAise 1 yf ye OY 1 ® pe el] . ~ 4f YY Bes ent Shem. TS eee a Tine maltose, and mannite with acid and gas formation ; the amount of acid formed from lactose is often insufficient to clot milk. It usually can form indol from peptone. The pneumobacillus is probably closely related to the b. coli. When injected into mice and guinea-pigs it originates a septicemia and 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. EPIDEMIC CEREBRO-SPINAL MENINGITIS 245 can be seen in the heart blood to possess capsules. It is less pathogenic to rabbits and dogs, but when injected into the trachea in these animals it originates a pneumonia. As stated above, it is the only organism present. in a small number of cases of human pneumonia, and it has also been isolated from conditions of empyema, meningitis, appendicitis, and pyemia ; a bacillus closely related or identical has been found in rhino- scleroma (q.v.). It is a not infrequent inhabitant of the mouth and nose of healthy individuals. From its historical associations an altogether undue importance has been attached to this bacillus. ‘ In septic pneumonias the ordinary pyogenic bacteria, alone or associated with the pneumococcus, are found. EpipemMid CEeREBRO-SPINAL MENINGITIS OR CEREBRO- SPINAL FRVER. As the resuit of observations on this discase in different parts of the world, it has been now established that the causal agent is the diplococcus intra- cellularis menangrtidis, Be ne re ae first described by Weich- eu N selbaum, and now usually ao. ee known as the meningo- coccus, This organism is a small coccus measuring about 1 » in diameter ; it usually occurs in pairs, the adjacent sides be- ing somewhat flattened against each other. In most cases the cocci are chiefly contained within polymorphonuclear leuco- cytes in the exudation ee (Fig. 65); in some cases, yc, 65.—Film preparation of exudation from however, the majority 4 case of meningitis, showing the meningo- may be lying free. It ae leucocytes. See also Plate I., stains readily with basic Stained with carbol-thionin-blue. x 1000. aniline dyes, but loses the stain in Gram’s method. Both in appearance and in its staining reactions it is closely similar to the gonococcus (vide p. 255). The organism can readily be cultivated outside the body, but the conditions of growth are somewhat restricted— “trypagar” (p. 43), agar with an admixture of serum, ascitic fluid, or blood (p. 45) is to be recommended. The optimum reaction isfone neutral to phenol-phthalein. Growth takes place 246 EPIDEMIC CEREBRO-SPINAL MENINGITIS best at the temperature of the body, and practically ceases at 25° C. On ‘these media the colonies are circular discs with a slightly opaque centre fading into a delicate transparent margin (Fig. 66), and they have a smooth, shining surface; they have a slightly mucoid consistence and readily emulsify in water or normal saline. When examined under a low magnification the centre appears somewhat yellowish, and the margins usually are smooth and quite regular; at a later period of growth slight crenation may appear, especially when the medium is somewhat dry. The colonies may be of considerable size, reaching some- times a diameter of 2 to 3 mm. on the second day. A stroke uw b Fic. 66.—a. Two-day colonies of the meningococcus on Martin's medium (p. 43), x9; 6. the same, in which illumination has been arranged to show finely granular centre and transparent margin, x12. Compare with Fig. 69. i From photographs by Dr. W. B. M. Martin. culture gives a broad line of growth of similar character ; the margins tend to be somewhat crenated, and isolated colonies often occur. 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 and dextrose with acid production, a property which distinguishes it from the micrococcus catarrhalis (vide infra); it has no action on saccharose. Fermentation tests can be carried out by means of either fluid or solid media containing 1 per cent. of the sugar to be tested, along with neutral-red or litmus as an indicator (p. 79). In cultures the organism presents the same appearance as in the body, and often shows tetrad formation. There is algo a EPIDEMIC CEREBRO-SPINAL MENINGITIS 247 great tendency to the production of involution forms (Fig. 67), many of the cocci becoming much swollen, staining badly, and afterwards undergoing disintegration. This change, according to Flexner’s observations, would appear to be due to the pro- duction of an autolytic enzyme, 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. On egg medium (p. 46), however, it survives for a considerable time. The organism is readily killed by heat at “a Rie 60° C., and it is also Lire shit eS very sensitive to weak a oe eee &, aa antiseptics ; drying for a ve TA 88. \ period of aday has been >» «4 2 i sear. 5 found to be fatal to it, (2% 70g J:26 8" 3, *%e% The facts established ac- » . a? eae 7 re "2. “ae o| cordingly show it to be oP BE Se a somewhat delicate 2%. 9.8 pe a Pan parasite, a i, 2 ene, aM : As stated above, the v 4, i ol al of organism occurs in the OB eM aS a 8 g's va exudate in the meninges Nig ree el o/ and in the cerebro-spinal Newey Sho Ry fiuid, and it can usually SAE Ea be obtained by lumbar Fic. 67.—Pure culture of diplococcus intra- puncture. In acute cases, cellularis, showing involution forms. especially in the 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. But it should be recognised that at any stage microscopic examination and even cultivation may sometimes give a negative result. In most cases the lesions are practically restricted to the nervous system, but occasionally complications occur, and in these the organism may be present. It has been found, for ‘example, in arthritis, pericarditis, pneumonic patches in the lung, and in other inflammatory conditions associated with the disease, and also occasionally in purpuric patches in the skin, though the ordinary petechial eruption is of toxic origin, Ina small proportion of cases it may be obtained from the blood during life. 248 EPIDEMIC CEREBRO-SPINAL MENINGITIS Experimental inoculation shows that the ordinary laboratory animals are relatively insusceptible to this organism. Aninflammatory condition may be produced in mice and guinea-pigs by intra-peritoneal injection,’ and a fatal result with symptoms of collapse may follow ; in such cases the organism does not seem to undergo very active multiplication, though it may sometimes be cultivated from the blood, and none of the lesions in the nervous system are reproduced. Similar results are produced by the endotoxin in dead cultures, and occasionally the lethal dose of the dead organisms may equal that of the living (Gordon). There is thus evidence that an active endotoxin plays an important part in the pathology of the disease. Flexner and also Stuart M‘Donald have shown that cerebro- spinal meningitis may be produced in monkeys by injections of the organism into the spinal canal, the latter observer finding that exudate containing meningococci was more effective than cultures. 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 distribution and general characters, and also as regards the histological changes, resemble the disease in the human subject. Even these animals, however, are manifestly less susceptible than the human subject. The meningococcus can usually be found in the naso-pharynx of patients suffering from the disease, and there is no doubt that this is the usual channel of infection. In cases where recovery occurs, the organism may persist for a varying period of time,— usually only for a week or two, but sometimes for months. Thére is difference of opinion as to the route by which the organism passes from the naso-pharynx to the meninges. One view is that it passes directly by the lymphatics to the base of the brain, but satisfactory evidence of this is wanting. The other view is that it passes by the blood stream; this is in accordance with what occurs in other infections, and is also supported by the fact that in some very acute cases with purpuric eruption, it has been found in the blood before meningeal symptoms have appeared, and also occasionally in septicemic types without meningitis. For a considerable time it has been known that contacts with cases of cerebro-spinal fever often harbour the meningococcus in the naso-pharynx, that is, are ‘‘carriers,” and during the war this subject has been extensively investigated. In fact, the examination of contacts has become a routine procedure. The percentage of ‘‘ positives ” amongst contacts varies; sometimes it has been found to be twenty or even higher. Non-contacts also have beer examined during epidemics, and amongst them also a considerable propor- tion, though not so great as amongst contacts, have been found to be carriers. In some carriers the organism occurs sparsely amongst other organisms, but in others in fairly large propor- tion, and occasionally in almost pure culture. In the great IDENTIFICATION OF MENINGOCOCCUS = 249 majority of carriers the organism can be found for only a comparatively short time—a few days even, or a week or two— but in a small proportion it persists for months, these being “chronic” carriers. Snch individuals will, of course, act in maintaining the source of the infection, and it appears that the occurrence of an epidemic disease depends upon a dissemination of the organism through the community as evidenced by a high carrier rate, though the conditions which lead to the dissemina- tion are not understood. Unfortunately we have at present no ready means of estimating the relative virulence of meningococci obtained from the naso-pharynx and from elsewhere. With regard to the epidemiology two facts are of importance. One is that direct infection of a healthy individual from a patient suffering from the disease is comparatively uncommon, though it sometimes occurs; the other is that it is rare for a known carrier to develop the disease. On the other hand, there is substantial evidence of persons being infected from carriers. The facts mentioned would seem to show that the organism is spread widely from individual to individual, in most cases without result, but that when the organism reaches a susceptible individual the disease may rapidly develop. No doubt the number of the organisms in the naso-pharynx is a factor of importance, heavy carriers being especially dangerous. It has been stated by some observers that the presence of the meningococcus leads to, or is associated with, pharyngeal catarrh, and that this often precedes meningeal infection. More extended observations, however, have thrown doubt on this, as it is certainly the case at least that the organism may abound in the naso-pharynx without the presence of catarrh or any abnormality. Manifestly the act of coughing, however, will aid in its diffusion when it.is present. Identification of the meningococcus.—In the case of meningitis, this usually presents no difficulty, as the finding of a Gram-negative diplo- coccus in the cerebro-spinal fluid is practically conclusive. In the case of the naso-pharynx, however, the matter is quite different. Means must be taken to distinguish the organism from others resembling it, which occur in the situation. Till recently, the following points taken together have been usually accepted as justifying a positive diagnosis: conformity in the microscopic characters and in the appearance of the colonies with those of the meningococcus, ready emulsification in saline, absence of growth on agar at 23° C., fermentation of glucose and maltose, and non- fermentation of saccharose. Attempts have been made to obtain identifica- tion by means of agglutination, and in this connection the work of Gordon has been of high value. On examining meningococci from various cases of meningitis, he found that a serum prepared by injecting any one strain did not agglutinate all the strains separated. Proceeding further and 250 EPIDEMIC CEREBRO-SPINAL MENINGITIS preparing sera for other strains which were not agglutinated, he arrived finally at the recognition of four ‘‘types” (1.-IV.), according to ag- glutinating tests, cross agglutination between them being little marked. Of these, types I. and II. are the commonest, the latter being rather the more frequent. All the strains separated from cases of meningitis have been found to be agglutinated by one of the four sera. The inference is that diplococci otherwise like meningococci, which are not agglutinated by any of the type sera, are without pathogenic significance, and are not accepted as true meningococci. Gordon’s results have so far received . sound confirmation from the labours of those engaged in the examination of military cases. Manifestly if a strain were isolated from the cerebro- spinal fluid in a case of meningitis which did not conform to any of the types, a new type would have to be added. Preparation of Agglutinating Sera.—Hine has devised the following method in the case of meningococci. A rabbit receives on one day three intravenous injections of five hundred millions of dead meningococci, with an interval of an hour between the injections ; six days afterwards it receives a single dose of three thousand-millions. On the eighth day the serum has usually a titre of over 1:800. Young rabbits of about a kilogramme in weight give the best results. The sera as supplied by the Central Cerebro-spinal Fever Laboratory are used in four dilutions, to each of which equal amounts of emulsion of the organism to be tested are added, the ultimate dilutions of serum being 1:50, 1:100, 1:200, 1: 400. Emulsions of known type organisms are used as controls at the same time. After the mixtures are made they are put iu a chamber at 55°C. for twenty-four hours, and the results are then read. Apart from the epidemic form of the disease, cases of a sporadic nature also occur, in which the lesions are of the same nature, and in which the meningococcus is present. The facts stated would indicate that the origin and spread of the disease in the epidemic form depend on certain unknown conditions which pro- duce an increased virulence of the organism. 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 pro- bably an attenuated variety of the latter. 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 diplo- coccus intracellularis ; and this result points to the two organisms being distinct, though closely allied, species. Serum Reactions.—An agglutination reaction towards the meningo- coccus is given by the serum of patients suffering from the disease, when life is prolonged for a sufficient length of time. It usually appears about the fourth day, when the serum may give a positive reaction in a dilution of 1:50; ata later stage it has been observed in so great a dilution as 1: 1000. Specitic opsonins may appear in the blood about the same time, and though they are not always proportional in amount to the agglutinins, the two classes of substances have pretty much the same SERUM REACTIONS 251 significance, and may occasionally be of use in diagnosis when lumbar puncture fails to give positive results, Although their presence in large amounts may be said to indicate a marked reaction, they do not supply information of much value in relation to prognosis. Immune-bodies, as shown by bactericidal and deviation of complement tests (pp. 122, 127), may also be developed in considerable amount in the course of the disease. Anti-sera for therapeutical purposes have been introduced by various workerx, and of these the one which has been most extensively used is that of Flexner and Jobling. This serum is prepared from the horse by repeated injections in increasing doses of dead cultures, followed by injections of culture autolysate and of living cultures, these two latter being best administered by the subcutaneous method. Several strains of meningococci are mixed together for purposes of injection, and the immunisa- tion is continued over a period of several months. For treat- ment of the disease the serum is injected under the spinal dura, 30 cc. being generally used for an injection in an adult, this being repeated on subsequent days... Some of the spinal fluid is removed and then the serum is injected, undue pressure being avoided. This serum has been used on a large scale in various parts of the world, and there is general agreement as to its favourable effects—the mortality of the disease, which is gener- ally 70 to 80 per cent., having been reduced to about 30 per cent. or even less. By means of its use the tendency to the occurrence of chronic lesions has also been markedly diminished. The action of such anti-sera cannot as yet be fully explained. They certainly contain opsonins, agglutinins, immune-bodies which bind complement, and possibly also anti-endotoxins. After the injection the number of meninggcocci hecomes markedly reduced, probably as a result of increased phago- cytosis; there can scarcely be any direct bactericidal action owing to the absence of complement. Recently, monovalent sera against each of the four types of meningococeus (p. 250) and also a polyvalent serum have been prepared for military eases by Gordon and his co-workers. The standardisation of such anti-sera is a matter of some difficulty ; at first the devia- tion of complement method was used (p. 127), but now the opsonic index is regarded with more favour as an index of the potency of the serum. Gordon has recently pointed out the importance of estimating the anti-endotoxic action, and has described a method for this purpose. Mackenzie and Martin treated cases by the intra-spinal injection of the fresh serum of patients suffering from the disease or who have recovered from it, such serum being in many cases rich in immune-bodies for the ’ 252 EPIDEMIC CEREBRO-SPINAL MENINGITIS meningococcus, and possessing a greatly increased bactericidal action as compared with normal serum. Though the number of cases treated by this method was not large, a distinctly favourable result was obtained. Allied Diplococci.—In the naso-pharynx there occur other Gram-negative diplococci which morphologically have a close re- semblance to the meningococcus. Many of these are chromogenic, eg., m. catarrhalis flavus, and can thus be readily distinguished ; others differ in their fermentative actions. Of these latter the diplococcus or micrococcus catarrhalis has the closest resemblance to the diplococcus intracellularis. In addition to occurring in health this organism has also been found in large numbers in catarrhal conditions of the pharynx and respiratory passages. Its microscopic appearances are practically similar to those de- scribed above, and it also occurs within leucocytes. Its colonies on serum agar, though on the whole they tend to be rather more opaque, closely resemble those of the meningococcus. The organism usually grows on gelatin at 20° C. without lique- fying the medium, and it has none of the fermentative properties described above as belonging to the diplococcus intracellularis. The diplococcus pharyngts stccus (v. Lingelsheim) grows at room temperature, and its colonies are very tough and adhere to the surface of the medium; it can thus readily be distinguished from the meningococcus. It has marked fermentative properties, acting on glucose, maltose, saccharose, and levulose. The diplococeus mucosus has colonies of slimy consistence ; it grows at room temperature, and it forms capsules, which can be demonstrated by the method of Hiss. The points of difference between the meningococcus and the gonococcus are given on p. 258. There are various other Gram-negative species of diplo- cocci, which can be readily distinguished, and which have no pathogenic importance so far as is known. A Gram-positive diplococcus called the diplococcus crassus is also of common occurrence; it is rather larger than the diplococcus intra- cellularis, and especially in sub-cultures may tend to assume staphylococcal forms. Meningitis due to other Organisms.—\Meninyitis may also 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 pnewmococcus. 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 Iesion by means of the blood stream. This organism also infects the meninges not infrequently in lobar pneumonia, and in some MENINGITIS DUE TO OTHER ORGANISMS 253 cases with head symptoms we have found it present where there was merely a condition of congestion. Occasionally epidemics of meningitis have been due to the pneumococcus. The pnewzvo- bacillus also has been found in a few cases. Meningitis is not infrequently produced by streptococei, 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 few instances, but sometimes the pneumococcus is the causal agent. Sporadic cases of meningitis occur associated with organisms which resemble the influenza bacillus morphologically and also in presenting hemophilic culture reactions, but which possess pathogenic properties for rabbits and guinea-pigs. Both in the cerebro-spinal fluid and in cultures, these bacilli frequently show a tendency to produce long filamentous forms and also may show a beading of the protoplasm, which gives them a diph- theroid appearance. The cases from which such bacilli have been isolated have chiefly occurred in children, are extremely fatal, and probably often follow on an otitis media, from which condition similar organisms have been isolated. Sometimes the meningitis is part of a septicemic or pysmic process,—in the latter the joints are often affected. It is impossible at present to say whether the organisms associated with such conditions are true influenza bacilli or are merely allied to them. They certainly tend to be more widely distributed in the body of the infected individual than is the case in the disease known clinically as influenza. On the other hand, influenza appears under several forms, and considerable variations may exist in the virulence of strains responsible for different outbreaks. An invasion of the meninges by the anthrax bucillus occurs, but is a rare condition ; it is attended by diffuse hemorrhage in the sub-arach- noid space. In tubercular meningitis the tubercle bacillus, of course, is present, especially in the nodules along the sheaths of the vessels, In conclusion, it may be stated that maxed infections may occur in meningitis. Thus the pneumococcus has been found associ- ated with the tubercle bacillus and also with the meningo- coccus, sometimes appearing as an additional infection to the latter. Methods of Examination.—During life these involve the microscopic investigation of the centrifuged cerebro-spinal fluid and making cultures therefrom (p. 245). For the former, smears stained by carbol-thionin-blue and by Gram’s method make the recognition of the meningococcus relatively easy, and the 254 EPIDEMIC CEREBRO-SPINAL MENINGITIS presence of Gram-negative cocci, especially within cells, is practically diagnostic of a case of cerebro-spinal fever. Tubes of trypagar, serum agar (pp. 43, 45), or agar containing 25 per cent. of ascitic or ovarian fluid, may then be inoculated. The difficult cases are those where no bacteria can be found microscopically in the Iumbar fluid. Here the character of the exudate may give help. A predominance of polymorpho- nuclear cells is usually manifest in meningococcic, pneumo- coccic, and infiuenzal cases, whereas in tubercular meningitis the exudate is, as a rule, chiefly lymphocytic, though poly- morphs, often degenerated, also occur. In such circumstances, besides other media, a tube of blood-smeared agar should be inoculated in case the pneumococcus or the influenza bacillus is the causal organism. To speak generally, if with a polymorpho- nuclear exudate no growth occurs in the media mentioned, the case is most likely to be due to the meningococcus. The isolation of the organism from the naso-pharynx (p. 72) will give confirmatory, though of course not conclusive, evidence. It must be kept in view, however, that in meningitis high up, produced by any of the organisms mentioned, polymorph leucocytes may be present in the fluid obtained by lumbar puncture before the organisms themselves appear. In tubercular cases it is sometimes impossible to demonstrate the bacilli microscopically in the exudate, though on careful search they may usually be found. For method of examination of the naso-pharynx vide p. 72. CHAPTER IX. GONORRHGA AND SOFT SORE. GoNoRRHG@A. Introductory.—The micrococcus now known to be the cause of gonorrhoea, and called the gonococcus, was first described by Neisser, who in 1879 gave an account of its microscopical char- acters as seen in the pus of gonorrhceal 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 human 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 gonorrhea 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. 68). 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 cells, but when it becomes purulent the large proportion within leucocytes is a very striking feature. In the leucocytes they lic 26 256 GONORRHGA AND SOFT SORE within the protoplasm, especially superficially, and are often so numerous that the leucocytes appear to be filled with them, and their nuclei are obscured. As the disease becomes more chronic, the gonococci gradually become fewer, though even in long-standing cases they may still be found in con- siderable numbers. They are also present in the purulent secretion of gonorrhceal conjunctivitis, also in various parts of the female genital organs when these parts are the seat of true gonorrheal infection, and they have been found in some cases in the secondary infections : : of the joints, as will be Fic. 68.—Portion of film of gonorrheeal pus, described below. showing the characteristic arrangement of Staining.—The gono- the gonococci within leucocytes, See also A . Plate I., Fig. 5. coccus stains readily and Stained with fuchsin. x 1000. 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 conditions of growth are somewhat restricted. The most suitable media are “blood-agar ” and the serum media already described for the purpose (pp. 43, 45). It is advisable to inoculate the media within half-an-hour after obtaining the material from the body, and to place the tubes at once in the incubator. 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 ordinary “sloped tubes” or as a thin layer in a Petri’s capsule. The young colonies are usually visible within forty-eight hours, and often within twenty-four hours; it is important, however, to note that sometimes growth may not appear till the fourth day. They appear around the points of inoculation as small semi-transparent discs of rounded shape. oe AID CULTIVATION OF GONOCOCCUS 257 The colonies vary somewhat in size, and tend to remain more or less separate. Later, the margin tends to be undulated and the a Fra. 69.—Colonies of gonococcus on serum-agar ; (a) three days’ growth ; (6) and (c) five days’ growth. x9, From photographs by Dr. W. B. M. Martin. centre more opaque; a radial marking may be present (Fig. 69). The first cultures die out somewhat quickly, but in sub-cultures, kept at 37° C., the organism remains alive for a considerable time, sometimes three weeks. After about a week more active foci of growth may appear in some of the colonies in the form of heaped-up opaque-points, thus giving an appearance suggestive of contamination. In the early stage of the disease the organism is present in the male urethra in practi- cally purelcondition, 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 inocula- tion be expressed from the deeper part of the urethra, Fic. 70.—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. cultures may often be obtained which are pure from the first. Tn culture, the organisms have similar microscopic characters to those described (Fig. 70), but show a remarkable tendency to 17 258 GONORRHGA AND SOFT SORE 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 com- paratively few normal cocci may be found. The less suitable the medium the more rapidly does degeneration take place. When mixed with other organisms the gonococcus may be separated by serum-agar plates (p. 43). On ordinary agar and on glycerin-agar some growth may take place when the reaction is just alkaline to litmus, but these media are quite unsuitable for ordinary purposes. The organism does not grow on gelatin, potato, ete. Comparison with Meningococcus.—The morphological and cultural characters of the gonococcus and meningococcus are in many respects closely similar ; the following points are of importance in distinguishing them. The conditions of growth of the gonococcus are more restricted than those of the meningococeus. The gonococcus usually does not grow on the ordinary agar media, whereas the meningococeus grows fairly well, at least after the first sub-culture. The colonies of the latter are rather more opaque and have more regular margins than those of the gonococcus. The meningococcus grows well in neutral bouillon, produc- ing a general turbidity, whereas the gonococcus does not grow ; even in serum bouillon the latter organism flourishes feebly, and the scanty growth falls to the bottom leaving the medium clear, whilst the meningococcus produces abundant growth with general turbidity. The fermentative effects have also been studied, and the chief results obtained are that glucose is the only sugar usually employed which is fermented by the gonococcus, whereas the meningococcus ferments maltose also. (For fermentative tests in the case of the gonococcus, solid media, as introduced by v. Lingelsheim, should be used, the serum medium of Martin, with litmus or neutral-red, and the particular sugar added, being specially suitable.) Specific serum reactions — agglutination, opsonic action, bactericidal action, and fixation of complement—have been studied by Torrey, Elser and Huntoon, and Martin, in the case of the two organisms. The general results obtained are that each organism represents a somewhat heterogeneous group showing considerable variations as regards the tests mentioned (vide also p. 249). An anti-gonococcus serum produced by injecting one strain of gonococcus has the maximum effect on that strain, whilst its action on other strains may be much feebler. An anti-gonococcus serum may have some effect, usually slight, on a meningococcus and vice versa ; this indicates that there are some receptors common to the two organisms. Arkwright finds that the complement-fixation test does not supply a satisfactory distinction between gonococci and meningococci. RELATIONS TO THE DISEASE 259 Relations to the Disease.—The gonococcus is invariably present in the urethral discharge in gonorrhea, and also in other parts of the genital tract when these are the seat of true gonorrhceal infection. Its presence in these different positions has been demonstrated not only by microscopical examination but also by culture. From the description of the conditions of growth in culture it will be seen that a life outside the body in natural conditions is practically impossible—a statement which corresponds with the clinical fact that the disease is always transmitted directly by contagion. Inoculations of pure cultures on the urethra of lower animals, and even of apes, is followed by no effect, but a similar statement can be made with regard to inoculations of gonorrhceal pus itself. In fact, hitherto it has been found impossible to reproduce the disease by any means in the lower animals. On a considerable number of occasions inoculations of pure cultures have been made on the human urethra, both on 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 established, and it is interesting to note how the conditions of growth and the pathogenic effects of the organism agree with the cliaracters 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. ‘oxin of the Gonococcus.—De Christmas has cultivated the gonococeus 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 proteins by alcohol, avd 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, 260 GONORRHGA AND SOFT SORE toxin resulted after fiv cessive injections at intervals. In a more recent publication he poir@s’out that the toxin on intracerebral injection 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 disentegration of the organisms. This has, however, been called in question by other investigators. attended with purulent ah He found that no tolerance to the 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 lacune. 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 mucous membrane of the urethra, and also in the prostate, and may thus be capable of producing infection. The prostatic secretion may sometimes be examined by making pressure on the prostate from the rectum when the patient has almost emptied his bladder, the secretion being afterwards discharged along with the remaining urine (Foulerton), In acute gonorrhea there is often a considerable degree of inflammatory affection of the prostate and vesicule 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 deter- mining. A similar statement also applies to the occurrence of orchitis and also of cystitis in the early stage of gonorrhea. 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 gonorrheea, 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, ete., 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 DISTRIBUTION IN THE TISSUES 261 gonorrheea of two years’ standing, a inoculation on the human subject proved it to be still virtent. 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 gonorrhceal 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 membrane 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 un- associated with other organisms. Further, in a large proportion of the cases in which the gonococcus has not been found, no organisms of any kind have been obtained from the pus, and in these cases the gonococci may have been once present and have subsequently died out. Lastly, they may pass to the peritoneum and produce peritonitis, which is usually of a local character. A In gonorrheal conjunctivitis the mode in which the gonococci spread through the epithelium to the subjacent connective tissue is closely analogous to what obtains in the case of the urethra, Their relation to the leucocytes in the purulent secretion is also the same. Microscopic examination of the secretion alone in acute cases often gives positive evidence, and pure cultures may be readily obtained. As the condition becomes more chronic, gonococci are less numerous and a greater proportion of other organisms may be present. Some observers have recently put forward the view that the “chlamy- dozoa” (p. 623) found in trachoma represent a mutation stage of the gonococcus, but there does not appear to be sufficient evidence that this is the case. » Relations to Joint-Affections, etc.—The relations of the gono- coccus to the sequele of gonorrhcea 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 large number of cases of arthritis following gonorrhea pure cultures of the gonococcus may be obtained. A similar 262 GONORRHGA AND SOFT SORE statement applies to inflammation of the sheaths: of tendons following gonorrhea. Secondly, in a considerable proportion of cases no organisms have been found. It is, however, probable that in many 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 pyemic nature, various pyogenic cocci have been found to be present. In the instances in which the gonococcus 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, though sometimes purulent in appearance. In one case Bordoni-Uffreduzzi cultivated the gonococcus from a joint-affection, and afterwards produced gonorrhea 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 gonorrheal endocarditis has been established by recent observations. Cases apparently of this nature occurring in the course of gonorrhcea had been previously described, but the complete bacteriological test has now been satisfied in several instances. In one case Lenhartz produced gonorrhea in the human subject by inoculation with the organisms obtained from the vegetations. That a true gonor- rheeal septicemia may 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). Vaccines.—Both gonorrhea itself and the secondary infections have been treated by means of vaccines, but the results reported vary greatly. On the whole most success has been obtained in the case of joint infections and allied conditions, though even here reports are contradictory. The initial dose employed has been usually about five million cocci, but care ig necessary in starting the treatment, especially in the case of acute gonorrhea. Harrison recommends that the organisms be killed by 0°5 per cent. carbolic acid instead of by heat. Methods of Diagnosis.—For microscopical examination, dried films of the suspected pus, etc., may he stained by any of the simple solutions of the basic aniline stains. We prefer methylene- 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 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 examination alone, we aay say that the presence in a urethral discharge of a large number of SOFT SORE 263 micrococci having the characters, position, and staining reactions de- scribed above, is practically conclusive that the case is one of gonorrhcea. There is no other condition in which this sum-total of 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 gonorrhea with such conditions as a urethral chancre with urethritis, will also be avoided. Even in chronic cases the typical picture is often well maintained, and microscopic examination — alone may give a definite positive result. When other organisms are present, and especially when the gonococci are few in number, it is diffi- cult, 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 gonorrhcea in the female. In the case of the female a drop of secretion should be taken on a platinum loop from the urethra or, with the aid of a speculum, from the cervix uteri, the adjacent parts being cleansed as far as possible by swabbing with sterile cotton wool. 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 impossible, for example, to distinguish by this means the gonococeus from the meningococcus. Cultures alone supply the test, and the points above detailed are to be attended to. Sorr 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 state- ments 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 » in length, and ‘5 uw in thickness (Fig. 71). It is found mixed with other organisms in the purulent discharge from the surface, and is chiefly arranged in small groups or in short chains. When studied in sections through the ulcer, it is found in the superficial part of the floor, but more deeply situated than other organisms, and may be present in a state of purity amongst the leucocytic infiltration. In this position it is usually arranged in chains, which may be of con- siderable length, and which are often seen lying in parallel rows between the cells. The bacilli chiefly occur in the free condition, but occasionally a few may he 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 264 GONORRHGA AND SOFT SORE 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 he- come superadded. This bacillus takes up the basic aniline stains fairly readily, but Joses the colour very rapidly when a decolorising agent is applied. Accordingly, in film preparations when Fic. 71.—Film preparation of pus from soft dshydrat oe ee > ) in length, are met with, both in cultures and in Me the tissues. They are straight or slightly curved, and are of uniform thick- a ae na es : ness, or may show slight ‘1c. 738. —Tubercle bacilli of the human type, : ‘ . from a pure culture on glycerin agar. swelling at their Page Stained with carbol-fuchsin. x1000. tremities. When stained they appear uniformly coloured, or may present small uncoloured spots along their course, with darkly stained parts between. There is no satisfactory evidence that such appearances represent spore-formation, as some have supposed ; and it has been shown that “beaded” bacilli have no higher powers of resistance than those which stain uniformly. The bacillus is seen in the “beaded” form when grown on media containing sperm or olive oil (A. H. Miller). The bacilli in the tissues occur scattered irregularly or in little masses. They are usually single, or two are attached end to end and often form in such a case an obtuse angle. True chains are not formed, but occasionally short filaments are met with. In cultures the bacilli form masses in which the rods are closely applied to one another and arranged in a more or less parallel manner. Tubercle bacilli are quite devoid of motility. es H- ‘THE TUBERCLE BACILLUS | 269 i 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 may occur. These may be in the form of long filaments, sometimes swollen or clubbed at their extremities, may be irregularly beaded, and may even show the appearance of branch- ing. Such forms have been studied by Metchnikoff, Maffucci, Klein, and others. Their significance has been variously interpreted, for while some look upon them as.degenerated or involution forms, others regard them as indicating « special phase in the life-history of the organism, A ee. ‘ \ ° Fic. 74.—Tubercle bacilli in phthisical sputum ; they are longer than - is often the case. See also Plate II., Fig. 7. Film preparation, stained with carbol-fuchsin and methylene-blue. x 1000. 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 been found that under certain circumstances tubercle bacilli in the tissues produce a radiating structure closely similar to that of the actinomyces. Club-like structures may be present at the periphery. 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. 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 streptothricee, the bacillary parasitic form being one stage of the life-history of the organism. This group is often spoken of as the mycobacteria. 270 TUBERCULOSIS Staining Reactions.—The tubercle bacillus takes up the ordinary stains very slowly and faintly, and for successful stain- ing 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. 105). 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. Much’s Method.—Much maintains that in addition to the ordinary acid-fast bacillus, the organism exists in the form of a bacillus which is not acid-fast and also in the form of free granules. These two forms are demonstrable by certain modifications of Gram’s method, of which the following is specially suitable :— Methyl-violet B.N., 10 c.c. of a saturated alcoholic solution in 100 c.c. of a 2 per cent. watery solution of carbolic acid ; stain by boiling over the flame for a few minutes or at 37° C. for 24-48 hours, then treat with Gram’s iodine for 1-5 minutes, 5 per cent. nitric acid for one minute, 3 per cent. hydrochloric acid for 10 seconds, and complete the decolorisation with a mixture of acetone and alcohol in equal parts. There seems to be no doubt that in certain conditions more tubercle bacilli can be demonstrated in the tissues by Much’s method than by the Ziehl-Neelsen method. Chemical Composition.—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 with caustic potash, yielded a body which was probably a chitin, and which was acid-fast when stained for twenty-four hours with carbol-fuchsin. Benians considers that a waxy material in some way encloses the protoplasm and fatty constituent, and confers on the organism the property of resisting the penetration of acid and alcohol. Cultivation.—The medium first used by Koch was inspissated blood serum (vide p. 40). If inoculations are made on this CULTIVATION OF TUBERCLE BACILLUS 271 medium with tubercular material free from other organisms, 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 break 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 comparatively small size and remain separate, becoming confluent only when many occur close to- gether. 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 sur- face of the serum and at the bottom of the tube may grow over the surface of the condensation water on to the glass (Fig. 75, A). The growth is always of a dull appearance, and has a considerable degree of consistence, so that it is difticult to dissociate a portion thoroughly in a drop of water. In older = 4 ee © cultures the growth may Fie. 75.—Cultures of tubercle bacilli on ‘acquire a slightly brown- glycerin agar. ish or buff colour. When AandB. Mammalian tubercle bacilli of human F : type; A is an old culture, B one of a few the small colonies are ex- weeks’ growth. i ‘C. Avian tubercle bacilli. The growth is whiter amined under a low POWEF and smoother on the surface than the others. of the microscope, they are seen to be extending at the periphery in the form of wavy or sinuous streaks which radiate outward, and which have been compared to the flourishes of a pen. The central part shows similar markings closely interwoven. These streaks are com- posed of masses of the bacilli arranged in a more or less parallel manner, On Dorset’s egg medium and especially on glycerin egg 272 TUBERCULOSIS medium the organism grows well, producing an abundant wrinkled layer which has usually a yellowish, buff, or pinkish colour. These media are specially suitable for direct cultivation from the tissues. 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- 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. The organism also flourishes well on glycerin potato, and this medium is suitable for primary cultures from tubercular lesions. 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 superficially as a dull whitish wrinkled pellicle which may reach the walls of the flask ; this mode of growth is specially suitable for the produc- tion of tuberculin (wide infra). The culture has a peculiar fruity and not unpleasant odour. On ordinary agar and on gelatin media no growth takes place. The use of animal tissues in glycerin bouillon as a medium for the growth of the tubercle bacillus has been introduced by Frugoni, and is one which gives excellent results) He recommends that small wedges of rabbit’s lung should be sterilised in the autoclave, and placed in tubes of glycerin bouillon in such a way that their surface is kept moist by the medium, without the fragments being submerged. The growth is probably more rapid and luxuriant than in any other method. The optimum temperature for growth is 37° to 38° C. Growth ceases about 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, ¢9., 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 after two months, and similar results are obtained when the bacilli are kept in distilled water for several weeks. ACTION ON THE TISSUES 273 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 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 Huids 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-poisous 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- 18 274 TUBERCULOSIS cell often shows signs of degeneration, such as hyaline change and vacuolation, or it may be more granular than the rest of the cell. The exact mode of formation of a tubercle follicle varies, however, in different tissues. ‘ 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 proliferation without the protoplasm dividing. These epithelioid cells may sometimes be the lining cells of capillaries, Some consider that the giant-cells result from a fusion of the epithelioid 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 oceur- rence gives rise to an appearance closely resembling a typical giant-cell. 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 amyloid change in the organs is believed by some to be chiefly due to the products of other, especially pyogenic, organisms, secondarily present in the tubercular lesions. 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. 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 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 ACTION ON THE TISSUES 275 still virulent. In subacute lesions, with well-formed tubercle follicles and little caseation, the bacilli are generally scanty. They are most numerous in acute lesions, especially where caseation is rapidly spreading, for example, in such conditions as caseous catarrhal pneumonia (Fig. 76), acute tuberculosis of the spleen in children, which is often attended-with a good deal of rapid caseous change, etc.; in such conditions they often form Fic. 76.——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. large masses which are easily seen under a low power of the microscope. 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 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 276 TUBERCULOSIS manner at the periphery, occasionally also in epithelioid] cells and in leucocytes. WR The above statements, however, apply only to tuberculosis in the human subject, and even in this case there are exceptions, In the ox, on the other hand, the presence of tubercle bacilli within giant-cells is a very common occurrence; and it is also common to find them in considerable numbers scattered Fic. 77.—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. irregularly throughout the cellular connective tissue of the lesions, even when there is little or no caseation present (Fig. 77). 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. In discharges from tubercular lesions which are breaking EXPERIMENTAL INOCULATION 277 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. 105). 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 tubercular meningitis the bacilli can often be found in the cerebro-spinal fluid obtained by lumbar puncture. Tn cases of genito-urinary tuberculosis they are usually present in the urine; but as they are much diluted it is difti- cult to find them unless a deposit 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. 78 In tubercular ulceration of the intestine their pres- ence in the feces may be demonstrated, as was first Fic. 78.—Tubercle bacilli in urine ; showing shown by Koch; but in one of the characteristic clumps, in which 5 en they often occur. this case their discovery Stained with carbol-fuchsin and methylene- is usually of little im- blue. x 1000 portance, as the intestinal lesions, as a rule, occur only in advanced stages when diagnosis is no longer a*matter of doubt. Experimental Inoculation.—Tuberculosis can be artificially produced in animals in a great many different ways—by injection of the bacilli into the subcutaneous tissue, into the peritoneum, into the anterior chamber of the eye, into the veins; by feeding the animals with the bacilli; and, lastly, by making them inhale the bacilli suspended in the air. The exact result, of course, varies in different animals and according to the method of inoculation, but we may state generally that when introduced into the tissues of a susceptible animal, the bacilli produce locally the lesions above described, terminating in caseation ; that there occurs a tubercular affection of the neighbouring lymphatic glands, and that lastly there may be a rapid extension of the bacilli to other organs by the 278 TUBERCULOSIS blood stream and the production of general tuberculosis. Of the animals generally used for the purpose, the guinea-pig is most susceptible: When a guinea-pig is inoculated subcutaneously with tubercle bacilli from a culture, or with material containing them, such as phthisical sputum, a local swelling gradually forms which is usually well marked about the tenth day. This swelling becomes softened and caseous, and may break down, leading to the formation of an irregular ulcerated area with caseous lining. The lymphatic glands in relation to the parts can generally be found to be enlarged and of somewhat firm consistence, about the end of the second or third week. Later, in them also caseous change occurs, and a similar condition may spread to other groups of glands in turn, passing also to those on the other side of the body. During the occurrence of these changes, the animal loses weight, gradually becomes cachectic, and ultimately dies, sometimes within six weeks, sometimes not for two or three months. Post mortem, in addition to the local and glandular changes, an acute tuberculosis is usually present, the spleen being specially affected. This organ ix swollen, and is studded throughout by numerous tubercle nodules, which may be minute _and grey, or larger and of a yellowish tint. If death has been long delayed, calcification may have occurred in some of the nodules. Tubercle nodules, though rather less numerous, are also present in the liver and in the lungs, the nodules in the latter organs being usually of smaller size though occasionally in large numbers. The extent of the general infection varies; sometimes the chronic glandular changes constitute the out- standing feature. Statements as to differences in the pathogenic effects of bacilli from human and bovine sources will be found below (p. 280). : Varieties of Tuberculosis.—1. Human and Bovine Tubercu- losis.—Up till recent years it was generally accepted that all mammalian tuberculosis was due to the same organism, and, in particular, that tuberculosis could be transmitted from the ox to the human subject. The matter became one of special interest owing to Koch’s address at the Tuberculosis Congress in 1901, in which he stated his conclusion that human and bovine tuberculosis are practically distinct, and that if a susceptibility of the human subject to the latter really exists, infection is of very rare occurrence,—so rare that it is not necessary to take aly measures against it. Previously to this, Theobald Smith had pointed out differences between mammalian and bovine tubercle bacilli, the most striking being that the VARIETIES OF TUBERCULOSIS 279 latter possess a much higher virulence to the guinea-pig, rabbit, and other animals, and in particular that human tubercle bacilli, on inoculation into oxen, produce either no disease or only local lesions without any dissemination. Koch’s conclusions were based chiefly on the result of his inoculations of the bovine species with human tubercle bacilli, the result being con- firmatory of Smith’s, and also on the supposition that infection of the human subject through the intestine is of very Tare occurrence, Since the time of Koch’s communication an enormous amount, of work has been done on this subject, and commissions of: inquiry have been appointed in vari- ous countries. We may summarise the chief facts which have been established. Prac- tically all observers are agreed that there are two chief types of tubercle bacilli, which differ both in their cul- tural — characters and in their viru- lence—a_ bovine type and a human type. The bacilli of the bovine type, 2 when cultivated, Fc. 79, Bovine tubercle bacilli in milk. x 1000. are usually shorter and thicker and more regular in size; whilst their growth on various culture media is scantier than that of the human type (Fig. 79). From the latter character the British Royal Commission have applied the term dysgonic to the bovine and eugonic to the human type. For distinguishing the growth characters of the two ~types egg media (p. 46) are especially suitable. On Dorset’s medium the human type produces an abundant, dry and wrinkled or verrucose growth, which has often a yellowish or pinkish tint; while the bovine type forms a thin whitish layer, smooth or somewhat granular, rather moist in appearance, and the growth is-much more easily broken up. The difference between the two types is accentuated by the 280 TUBERCULOSIS addition of glycerin to the medium; this greatly favours the: growth of the human type, while it does not favour, or even inhibits, the growth of the bovine type. In fact, on glycerin- egg medium primary, cultures of the latter often fail. These differences are most marked in the early cultures ; in later sub- cultures they tend to diminish. The vitality of the bovine type is less on artificial media, cultures having sometimes a tendency to die,out. As already stated, there is also a great difference in virulence towards the lower animals, the bacillus from the ox having a much higher virulence. This organism when injected in suitable quantities into the ox pro- duces a _ local tubercular lesion, which is usually fol- lowed by a generalised and fatal tuberculosis; whereas injection of human tubercle bacilli produces no more than a local lesion, which undergoes retrogression. (In certain experiments, ¢.9., those of Delépine, Hamilton, and Young, general tubercu- losis has been produced in the bovine species by tubercle ; bacilli from the human sub- Fre. 80.—Cultures of bovine and human ject, but these results are bacilli 5 weeks old on glycerin egg. : A The central tube is human, the tube exceptional.) Corresponding on each side bovine. The three tubes differences come out in the were inoculated on the same day. case of the rabbit; in fact, intravenous injection of suit- able quantities (¢.g., of 0°-1-0°01 mgrm. of dried bacilli sus- pended in 1 c.c. of saline) in this animal is the readiest method of distinguishing the two types—an acute tuberculosis resulting with the bovine, but not with the human type. In guinea- pigs and monkeys a generalised tuberculosis may result from subcutaneous injection of bacilli of the human type, but in this case also the difference in favour of the greater virulence of the bovine type is made out. With regard to the distribution of the two types of organisms, it may be stated that, so far as we know, the bacillus obtained from bovine tuberculosis is VARIETIES OF TUBERCULOSIS 281 always of the bovine type ; in fact this seems to be the prevalent organism in animal tuberculosis (vide infra). Ina human tuberculosis the bacilli in a large majority of the cases are of the human type; but, on the other hand, in a certain proportion bacilli of the bovine type are present. Pulmonary phthisis is almost invariably caused by bacilli of the human type; a few cases have been recorded in which the bovine type has been present, but these constitute less than 1 per cent. of the cases investigated. The Royal Commission found that the bovine type was present in 50 per cent. of cases of primary abdominal tuberculosis in children—that is, in cases where apparently infection had taken place by alimentation; and more recent observations have shown that glandular tuberculosis in children under ten years of age is produced by bovine bacilli in more than 70 per cent. of the cases. In cases of lupus nearly half of the bacilli obtained were of the bovine type, and it is an inter- esting fact that almost all the viruses, both of the human and bovine types, were markedly attenuated in their virulence for animals. In over two hundred cases of tuberculosis in children, given by W. H. Park, the bovine bacillus was present in more than 25 per cent., the percentage being higher in the earlier than in the later years of childhood ; and Fraser has found that of seventy cases of tuberculosis of bones and joints in children in Edinburgh, this was the type present in more than half. This proportion is higher than that found by Eastwood and F. Griffith and by A. Stanley Griffith, in a large number of cases chiefly in England, namely, a little over 25 per,cent.’ Fraser also found that the proportion of cases in which the bovine type is present is much higher when there is no evidence of infection from other members of the family, than when there is the possibility of such infection. Almost all the tubercular lesions from which the bovine type has been obtained have been in children, the presence of the bovine type of bacillus in adult tubercular lesions, phthisical sputum, etc., being of very rare occurrence. It is therefore justifiable to conclude that tuber- culosis is transmissible from the ox to man, and that the milk of tubercular cows is a common vehicle of transmission. Although most of the bacilli which have been cultivated correspond to one of the two types, as above described, it is also to be noted that intermediate varities are occasionally met with, though some of these on analysis have been found to be really due to a mixture of the two types. According to some observers, it is possible to modify bacilli of the human type by passing them through the bodies of certain animals, ¢.g., guinea- pigs, sheep, and goats, so that they acquire the characters of bovine bacilli, but the more recent results, including those of the Royal 282 TUBERCULOSIS Commission, are that this modification does not take place and that the characters of the type are comparatively stable. The question is still an open one, and it is doubtful whether or not a bovine type aftér long sojourn in the human tissues will assume the characters of the human type; if it does, the proportion of cases actually due to the bovine type will be of course larger than is indicated by the characters of the organism obtained from the lesion. It is quite likely that, although the bovine bacilli are more virulent to the lower animals than the human bacilli are, this does not also hold in the case of the human subject. In fact, the comparative chronicity of the primary abdominal lesions in children, in the first instance, would point rather to a low order of virulence towards the human subject. We may also add that there are cases, notably those of Ravenel, in which accidental inoculation of the human subject with bovine tubercle has resulted in the production of tuberculosis. ‘Some other facts obtained by the Royal Commission may be given: The bovine type of bacillus alone was found in the sheep, goat, and horse, whilst in the pig the bovine type was found in the great majority of cases, though in some the human type, and in others the avian tubercle bacillus, was present. In the case of these two latter the lesions were of a more localised kind. The bovine type was also found in the cat. The human type was found in animals in confinement, e.g., the antelope, gnu, chimpanzee, and macacus rhesus, and also in the parrot. The ‘animals most susceptible to inoculation with the human type are the guinea-pig, rhesus, and chimpanzee; the dog, rat, and mouse are practically immune, while the calf, rabbit, pig, and goat occupy an intermediate position., The parrot also has been found to be susceptible to inoculation with the human type. It was also shown that when cows were inoculated subcutaneously with considerable quantities of bacilli either of the human or bovine type the bacilli were excreted in the milk, and that in these cases the udder appeared normal. There is therefore the presumption that when during the course of the disease the bacilli are present in the blood stream, they may make the milk infective even though there are no lesions in the udder. 2. Avian Tuberculosis.—In the tubercular lesions in birds there are found bacilli which correspond in their staining re- actions and in their morphological characters’ with those in mammals, but differences are observed in cultures, and also on experimental inoculation. On glycerin agar and on serum, the growth of tubercle bacilli from birds is more luxuriant, has a moister appearance (Fig. 75, C), and, moreover, takes place at a higher temperature, 43°5° C., than is the case with mammalian tubercle bacilli. Experimental inoculation brings out even more distinct differences. Tubercle bacilli derived from the human subject or from the ox, for example, when injected into fowls, usually fail to produce tuberculosis, whilst those of avian origin very readily do so (on the other hand, the parrot is susceptible to inoculation with both mammalian types). Fowls are also very susceptible to the disease when fed with portions of the organs containing avian tubercle bacilli, but they can consume enormous quantities of phthisical sputum without becoming tubercular (Strauss, Wurtz, Nocard). The Royal Commission found that rabbits and mice are the only mammals susceptible to inoculation with avian tubercle bacilli, though others may succumb to OTHER ACID-FAST BACILLI 283 toxic effects When large doses are used. In the case ofthe rabbit, intravenous injection results in the formation of greyish-white foci in the spleen, but no frue tubercles are formed ; subcutaneous inoculation leads to a peculiar chronic disease in joints, testes, ete., whilst the liver ae tt are free from lesions—a result not obtained with mammalian acilli. There is, therefore, abundant evidence that the bacilli derived from the two classes of animals show important differences, and, reasoning from analogy, we might infer that probably the human subject also would be little susceptible to infection from avian tuberculosis. The question remains—Are these differences of a permanent character? Nocard found that mammalian bacilli of the human type when kept within closed collodion sacs in the peritoneal cavities of fowls over a long period of time, acquired the characters of avian bacilli, but the Royal Commission as the result of similar experiments obtained no evidence of such transformation. It is accordingly not possible at present to give a definite answer to the question. 3. Tuberculosis in the Fish.—Bataillon, Dubard, and Terre cultivated from a tubercle-like disease in a carp, a bacillus which, in staining reaction and microscopic characters, closely agrees with the tubercle bacillus. The lesion with which it was associated was an abundant growth of granulation tissue in which numerous giant-cells were present. It forms, however, luxuriant growth at the room temperature, the growth being thick and moist like that of avian tubercle bacilli (Fig. 82, c). Growth does not occur at the body temperature, though by gradual acclimatisation a small amount of growth has been obtained up to 36° C. Furthermore, the organism appears to undergo no multiplication when injected into the tissues of mammals, and attempts to modify this characteristic have so far been unsuccessful. Weber and Taute have cultivated this organism from mud, and also from organs of healthy frogs. It is thus probably to be regarded as a saprophyte which is only occasionally associated with clisease in the fish. According to the results of different experimenters, it is possible to modify human tubercle bacilli by allowing them to sojouyn in the tissues of cold-blooded animals, c.g., the frog, blind-worm, ete,, so that they flourish at lower temperatures. These results have, however, been recently called in question, as it has been stated the organisms obtained were not modified tubercle bacilli, but other acid-fast bacilli which may be found in the tissues of normal cold-blooded animals. This question must accordingly be considered still an open one. Other Acid-fast BacilliWithin recent years a number of bacilli presenting the same staining reaction as the tubercle 284 TUBERCULOSIS bacilli have been discovered. Such bacilli have a comparatively wide distribution in nature, as they have been obtained from various species of grass, from butter and milk, from manure, and from the surfaces of animal bodies. Microscopically, they agree more or less closely with tubercle bacilli, though most of them are shorter and plumper; many of them show filamentous and branching forms under certain conditions of culture. More- over, on injection, they produce granulation tissue nodules which may resemble tubercles, although on the whole there is a greater tendency to softening and suppuration, and usually the lesions are localised to the site of inoculation. The most im- portant point of distinc- tion is the fact that their multiplication on arti- ficial media is much more rapid, growth usually being visible within forty- eight hours and often within twenty-four hours at 37° C. Furthermore, in most instances growth occurs at the room tem- perature. The general character of the cultures in this group is a some- what irregular layer, often with wrinkled sur- Fra, 81.—Moeller’s Timothy-grass bacillus. face, dry or moist in From a culture on agar, appearance, and varying Stai ith ie : . e 5 9 e J Stained with carbol-fuchsin, and treated with in tint from white to 20 per cent. sulphuric acid. x 1000." yellow or reddish brown. The number of such or- ganisms is constantly being added to, but the following may be mentioned as examples :— Moeller’s Grass Bacilli I. and I1.—The former was found in infusions of Timothy-grass (Phlewm pratense). It is extremely acid-fast, morpho- logically resembles the tubercle bacillus, and in cultures may show club- formation and branching. The local lesions produced may somewhat re- semble tubercles. The colonies, visible in thirty-six hours, are scale-like and of greyish-white colour (Fig. 82, a). Moeller’s bacillus II. was obtained from the dust of a hay-loft. The colonies at first are moist and somewhat tenacious, but afterwards run together, and are of a dull yellowish colour. The general results of inoculation resemble those of grass bacillus I., but are less marked. Moeller also obtained a similar organism from milk. He also discovered a third acid-fast bacillus, which OTHER ACID-FAST BACILLI 285 he obtained from manure and therefore called the ‘‘ Mistbacillus” (dung bacillus). This organism has analogous characters, though presenting minor differences. It also produces 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. 82, 6) not unlike that of Moeller’s grass‘bacilluis II. Korn has also obtained other two bacilli from butter which he holds to be j distinct from one another and from Rabinowitch’s bacillus. The points of distinction are of a minor character. Other more or less similar bacilli lave been cultivated by Tobler, Coggi, and others.? Another bacillus of consider- able 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 has been found to be comparatively common in Britain, The bacilli occur in large numbers in the lesions, the cells being often packed yg, 82. Cultures of acid-fast bacilli oe and eg cates be grown at room temperature. ‘ound in scrapings fromthe sur- (4) yoetter’s Ti cevaaehaaiaa face. They resemble the tubercle & The Petal Rebinoyhoh meter tecaitie, bacillus in appearance, but are — (¢) Bacillus of fish tuberculosis. distinctly shorter; they are equally acid-fast. The organism has been cultivated by Twort and Ingram on egg medium to which there is added 4-1 per cent. of dried and powdered acid-fast bacilli, the Timothy-grass bacillus being most suitable ; growth is slow, the colonies appearing after about four wecks in the primary cultures. Smegma Bacillus,—This organism is of importance, as in form and staining reaction it somewhat resembles the tubercle bacillus and may be mistaken for it. It occurs often in large numbers in the smegma pre- putiale and in the region of the external genitals, especially where there is an accumulation of fatty matter from the secretions. Morphologically it is a slender, slightly curved organism, like the tubercle bacillus, but usually distinctly shorter (Fig. 83). Like the tubercle bacillus, it stains 1 For further details on this subject, vide Potet, Htudvs sur les bacilles dites acidophiles. Paris, 1902. 286 TUBERCULOSIS 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 has culti- vated it on various media, finds that in culture it shows resistance to decolorisation 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 it 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 - BPO, him to apply the decolorising test. Difficulty will only occur when a few scattered - bacilli retaining the fuchsin / rast =F . are found. Z fi My & Its cultivation, which is ‘ attended with some diffi- | * ’ oF culty, was first effected by . Czaplewski. On serum it ". grows in the form of yellow- ,/ ish-grey, irregularly rounded vw \ \r ‘."/ colonies about 1 mm. in a , diameter, sometimes becon- ‘ a pee ve ‘ing confluent to form a com- \ of bh paratively thick layer. He ’ F found that it also grew Pe : on glycerin agar and in S : bouillon. It is non-patho- me > genic to various animals Fic. 83.—Smegma ae Film preparation which have been tested, un- of smegma. or : Ziehl-Neelen stain. x 1000. ee aay ee a Cowie has found that acid- fast bacilli are of common occurrence in the secretions of the external genitals, mammi, ete., 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) ACTION OF DEAD TUBERCLE BACILLI 287 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 flourish 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 of practically 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. In such cases inoculation may be the only reliable test. Action of dead Tubercle Bacilli.i—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 stcrilised 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 tissuc 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-formed giant-cells, and occa- sionally traces of caseation. The bacilli can be well recognised in the nodules by the ordinary staining method. Similar nodules can be pro- duced by intraperitoneal injection. Subcutaneous injection, on the other hand, produces a local abscess, but in this case no secondary tubercles are found in the internal organs. Further, in many of the animals inoculated by the various methods, a condition of marasmus sets in and gradually leads to a fatal result, there being great emaciation before death. These 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. 8. 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 288 TUBERCULOSIS 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. 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 glycerin 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 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 overestimated. Every phthisical patient ought to be looked upon as a fruitful source of infection to those around, and should only expectorate on pieces of rag which are afterwards to be burnt, or into special receptacles which are then to be sterilised either by boiling or by the addition of a 5 per cent. solution of carbolic acid. Another great source of infection is the milk of cows affected with tuberculosis of the udder, and this is responsible for a considerable proportion of tuberculosis of lymphatic glands, bones, and joints, etc., in young children, as above detailed. In the examination of milk, animal inoculation with centri- fugalised samples is the only reliable means of detecting the presence of tubercle bacilli 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 (vide p. 281). In these cases there may be tubercular ulceration of the intestine, or it may be absent. 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 SPECIFIC REACTIONS OF TUBERCLE BACILLI 289 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 de- stroyed 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, In the former, 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 infect the pharyngeal lymphoid tissue, tonsils, ete., 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 Specific Reactions of Tubercle Bacilli—The tubercle bacillus belongs to the group of organisms which do not to any extent secrete soluble toxins, but which nevertheless produce effects in the body at a distance from the site of actual prolifera- tion. The origin of these effects is obscure, but there is abundant evidence that, while the injection of dead bacilli tends to pro- duce local lesions, the introduction of the disintegrated proto- plasm of the bacillus can produce pathogenic effects of a toxic character. Such disintegrated products (which may be looked on as endotoxins), artificially prepared, were introduced by Koch under the name of tuberculins, and the following are the chief forms in use :— (1) Koch’s Old Tuberculin.—This consists of a six-weeks’-old culture of tubercle bacilli in 5 per cent. glycerin bouillon, evaporated down to a tenth of its original volume, killed by heat, and filtered. It thus contains the products of macerated bacilli, substances (not destroyed by heat) formed from the medium during the growth of the organism or extracted from the bacilli by the glycerin, —and the remains of the medium. (2) Tuberculin-O.—Masses of living bacillary growth from surface cultures on agar are dried im vacuo, ground in an agate mill, treated with distilled water and centrifugalised ; the supernatant clear fluid is the tubereillin. As it gave no cloudiness on the adddition of glycerin, Koch concluded that it contained the glycerin soluble products present in the “old tuberculin ” and which were looked on as responsible for the necrotic effects produced by the latter (vide infra). (3) Tuberculin-R.—The deposit in the preparation of tuberculin-O is 19 290 TUBERCULOSIS again ground up in distilled water, centrifugalised, and the clear fluid set aside ; the process is again and again repeated with the residue until, on centrifuging, none is left. The successive supernatant fluids are mixed and concentrated, and constitute the tuberculin. As this fluid gives a cloudiness with glycerin, Koch considered it contained the glycerin-insoluble constituents of the ‘‘old tuberculin.” (4) Koch’s New Tuberculin (Bazillenemulsion).—A bacillary mass is dried and ground in 50 per cent. glycerin in water till a clear fluid results. This tuberculin is thus equivalent to a mixture of tuberculin-O and tuberculin-R. (5) Tuberculin Béraneck.—This preparation is an extract of tubercle bacilli with 1 per cent. phosphoric acid, the effect of which is supposed to be to destroy some of the more harmful constituents. A number of other tuberculin preparations have been used, but the above are the most important. The original tuberculin was introduced by Koch for the treatment of local tuberculous infections. The supposed rationale was that when the artificially produced toxins were injected into the body their action, added to that of the baciili growing in the focus of infection, caused a sudden exacerbation of the necrotic effect occurring around the bacilli, which resulted in ulceration, whereby the living bacilli were thrown off. It has been found that the injection of the tuberculin directly into the tubercular focus is often not followed by a tuberculin reaction, and although there are other factors to be taken into account this militates against the view that a local concentration of toxin is a sufficient explanation of the phenomenon. The tuberculins are now used for the purposes of diagnosis and to originate immunisation. Their action is extremely complicated and not yet clearly understood, and may be considered under the headings of the production of supersensitiveness, and immunity phenomena. _(1) Phenomena of Supersensitiveness.—(a) The Original Tuberculin Reaction.—This can be manifested with any of the tuberculin preparations. Thus, if ‘25 cc. of “old” tuberculin be hypodermically injected into a healthy individual, there occur in three or four hours malaise, tendency to cough, laboured breathing and moderate pyrexia, all passing off in about twenty- four hours. If, however, only 0-01 ¢.c. be injected into a tuber- cular subject, similar symptoms but in a much more aggravated form (the so-called tuberculin reaction) arise, and if there be present a local tubercular focus—e.g., lupus—there occurs round it a definite inflammatory reaction with, it may be, ulceration. Similar phenomena of “ supersensitiveness” are produced by the injection of almost any foreign proteid into an animal. The subject will be discussed in the chapter on Immunity under the PHENOMENA OF SUPERSENSITIVENESS 291 , heading of Anaphylaxis, and it may be said that anaphylaxis is observed when living or dead tubercle bacilli are injected into healthy animals. The-tuberculin reaction is much used in diagnosis and, in addition to the methods just described, the following special modifications are frequently used for this purpose :— ; (6) The Cutaneous Tuberculin Reaction of von Pirquet and the Ophthalmo-reaction of Calmette.—In recent times the diagnosis of tuberculosis has been considerably aided by the introduction of these two tests. Both are essentially of the same nature, and depend, like the original tuberculin reaction, on the sensitiveness of the tissues of tubercular patients to tuberculin. The cutaneous test is carried out as follows: The skin, usually that of the flexor aspect of the forearm, is well cleansed with ether and then allowed to dry. Two drops of tuberculin are placed on the prepared surface about four inches apart, and then midway between the two drops a small spot is scarified with'a small metal bore constructed for the purpose. This serves as a control, any reaction which follows in it being merely a traumatic one. Similar scarification is effected through the drops of tuberculin, so that the scarified spots are exposed to its action. Small portions of cotton wool are placed over the drops to pre- vent the tuberculin from running off, and the latter is allowed to act for ten minutes. After that time the cotton wool is removed ; no dressing is required. In the process of scarification only the epidermis should be injured and blood should not be drawn. The “old” tuberculin of Koch is that used. In the case of a positive reaction an inflammatory redness and swelling make their appearance round the sites of tuberculin inoculation, generally within a few hours, and at the end of twenty-four hours there is a distinct inflammatory papule about half an inch in diameter, with a somewhat paler centre like a spot of urticaria ; sometimes in the centre there are minute vesicles. The maximum effect usually occurs within forty-eight hours, and after that time the reaction gradually recedes. Such is the typical reaction, but of course slighter, and also more intense reactions are met with. In a negative reaction all three points of scarification show merely a slight traumatic redness which soon passes off. For the ophthalmo-reaction Calmette uses a purified tuberculin. The tuberculin is prepared as in Koch’s original method, and is ‘precipitated with 95 per cent. alcohol; the precipitate is then dissolved in water. This process is repeated other two times, and the final precipitate is made up as a | per cent. solution in distilled water. For use, in the case of an adult, a drop of this 292 TUBERCULOSIS solution is placed in the conjunctival sac and the fluid allowed to spread over the surface ; for children about half this quantity is sufficient. In the case of a positive reaction the ocular con- junctiva is congested, the lids become somewhat swollen and their inner surface presents a bright red colour, there is increased secretion of tears and a varying amount of fibrinous exudation. The reaction usually reaches its maximum in from six to ten hours after the instillation, and commences to pass off in from twenty-four to thirty-six hours,—in children a little sooner. The general results obtained by these two reactions appear to correspond closely. A distinct positive result obtained by either is nearly conclusive as to the presence of a tubercular lesion. In cases of latent tuberculosis the reaction is sometimes obtained, sometimes not. Again, in very advanced cases of tuberculosis, especially a short time before death, a negative result may be got; in some of these cases v. Pirquet has met with a colourless papule or a livid spot without exudation, conditions which he describes as indicating a “cachectic reaction.” The ophthalmo- reaction is the more easily applied, at least in adults, but its use is contra-indicated when there is any abnormal condition of the conjunctiva. Even apart from this, however, inflammatory symptoms of disagreeable severity sometimes supervene. It should also be noted that a second test ought not to be applied to the same eye; as the first may produce a condition of super- sensitiveness (p. 290). Von Pirquet claims for his method that in the case of children it can be satisfactorily carried out with greater ease than the ophthalmic test. It will be recognised that the processes underlying the original tuberculin reaction on the one hand, and the cutaneous and ophthalmic reactions on the other, are analogous. In the former there is the occurrence of local inflammation with metabolic changes and fever ; in the latter, of mild inflammatory effects,— in both cases the phenomena being found only in tubercular subjects. The Use of Old Tuberculin in the Diagnosis of Tuberculosis in Cattle.— In cattle, tuberculosis may be present without giving rise to apparent symptoms. It is thus important from the point of view of human infection that an early diagnosis should be made. The method is applied as follows: The animals are kept twenty-four hours in their stalls, and the temperature is taken every three hours, from four hours before the injection till twenty-four after. The average temperature in cattle is 102°2° F. ; 30 to 40 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 IMMUNITY PHENOMENA IN TUBERCULOSIS 293 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. (2) Immunity Phenomena in Tuberculosis. — Although recovery from tuberculosis is of frequent occurrence in man, we have at present no clear idea of the processes at work. The object of the therapeutic application of the tuberculins introduced by Koch was to increase the hypothetically existing powers of resistance of the infected individual. The underlying principle was thus the same as in immunisation procedures (e.g., against the typhoid bacillus) with the difference that immunisation was proceeding in an already infected, animal. One result has been to stimulate inquiries with a view to observing whether the sera of persons suffering from tuberculosis possess the qualities associated with immunity reactions. (1) Immune-bodies and Precipitins—Evidence for the exist- ence of the former in tuberculosis has been sought by applying the method of complement fixation (see p. 127), eg., the serum of a tubercular animal being mixed with tuberculin, the mixture is tested for its capacity of absorbing complement. Following this line, Wassermann and others have found evidence of the presence of an antituberculin in tubercular foci. Generally speak- ing, such an antituberculin is absent from the blood serum of most tubercular patients. In certain cases it may be present in the serum of patients subjected to repeated tuberculin injections. Another immunity phenomenon which may be observed is the formation of a precipitate when some of the serum of a tuber- culous patient is added to a solution of tuberculin, the mixture being allowed to stand for twenty-four hours (precipitin reaction). There is thus evidence in some tubercular infections of a vital reaction resulting in the formation of antagonistic bodies, which may include both immune-bodies and precipitins. It may be said, however, that the sera of certain animals, e.g., rabbit and ox, when mixed with tuberculin, become capable of deviating complement from a hemolytic combination. (2) Agglutinins.—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, com- mencing with, say, | 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 to leave the mixture for twenty-four hours before observing. As with other agglutin- ative observations, it is difficult to correlate the degree of 294 TUBERCULOSIS agglutinating power of the serum with the degree of immunity possessed by the individual from which.it was derived. The method has been used by some as a means of diagnosis, but its value is doubtful and it is certainly inferior to the methods depending on supersensitiveness. (3) Opsonins.— The serum of most normal men and of several. species of animals contains opsonins to the tubercle bacillus. In tubercular subjects these aré frequently diminished, and to obtain a standard of comparison between infected and healthy subjects samples of serum from a number of persons presenting no signs of tuberculosis are taken and mixed. While the technique of the opsonic method presents great difficulties, it may be taken that with the use of such a standard an opsonic index below °8 indicates a deficiency in opsonins and an index above 1:2 indicates an excess. In strictly localised tuberculosis, indices from *1 to ‘8 are frequently found, while in tuberculosis with general disturbance the index fluctuates greatly from day to day, being sometimes below, sometimes above unity. While there is thus evidence that, when the tubercle bacillus gains entrance into the body, reactions similar in nature to those observed in other infections are developed, the processes underlying recovery from tuberculosis are exceedingly obscure, and certain factors have to be taken into consideration which perhaps play a greater part in this disease than in other infec- tions. One of these is the great chronicity so often observed. It is possible that in many cases this is due to wide variations in individual susceptibility and to differences in susceptibility at different age periods. Thus on the whole the most acute cases of tuberculosis are found in childhood. In view of the widespread opportunities for infection which occur, especially in city life, it is probable that the great mass of the adult population is on the border line between complete resistance and a susceptibility of varying degree. On the other hand, there is some evidence that variations exist in the virulence of different strains of the tubercle bacilli, As has been pointed out, it is probable that the bovine variety is less pathogenic for man than the human, but it is probable that even amongst human strains variations in virulence occur, as has recently been insisted upon by Burnet. It has been supposed by many that a cause of insus- ceptibility mm the adult is found in the fact that infection has previously occurred in childhood whereby an immunity is established. The evidence for this at present is rather of an academic nature, and it is certainly extremely difticult to immunise animals against infection with virulent bacilli. It THERAPEUTIC APPLICATIONS OF TUBERCULINS 295 may be said that the relation of the phenomena of supersensitive- ness to those of the development of immunity is at present very obscure. Therapeutic Applications of the Tuberculins.—As has been indicated, the injection of tuberculins into an infected subject may cause necrosis in a focus of infection, and it was originally supposed by Koch (1890-91) that the origination of such a necrosis might free the body of the invading bacilli. It was soon shown, however, that many single bacilli penetrating the tissues around the focus ‘were left unaffected, and this method of treatment was therefore abandoned. Tuberculin-R was introduced by Koch in 1897 as a toxin having ‘a minimum of necrotic effect, and the object of its use ‘“ was to increase the natural powers of resistance of the tubercular subject. Doses commencing with Jj, to xjy mgrm., gradually increased, were given every second day, the rule laid down for the regulation of the dosage being that no amount should be administered which raised the patient’s temperature more than ‘5° F. In such doses profound local and general effects were, however, still produced and these were sometimes of a harmful character. The difficulty of controlling the effects militated against the general use of this tuberculin as a curative agent, and it was thus not until Wright investigated the effects of extremely minute doses of the agent that it again came into prominence for therapeutic purposes. At the present time the tendency is to abandon the attempt to assign to different elements in a tuber- culin the reactive effects on the one hand and the immunising effects on the other ; thus the bacillary emulsion tuberculin is prob- ably as much used as the tuberculin-R, and the object is now to practice such a dosage as shall give the maximum of good effect. For ordinary cases with little or no evidence of constitutional disturbance, an amount of tuberculin corresponding to from a six- hundredth to a one-thousandth of a milligramme of tubercle powder is a sufficient dose ; for more marked cases with little or no fever, from a two-thousandth to a four-thousandth of a milligramme is given. In febrile cases the greatest care must be exercised, and a twenty-thousandth to a fifty-thousandthof a milligramme probably represents the limits of safe dosage. The injections are also now given less frequently, usually at ten-day intervals. The best results are obtained where the tuberculous infection is localised, e.g., in lupus, tubercular joints and glands, genito-urinary tuberculosis, and, generally speaking, the dosage must be regulated by a study of the clinical effects. Under certain circumstances information as to the effect of the treatment is easily available. Thus, if in a 296 TUBERCULOSIS quiescent lung affection a tuberculin injection causes increased cough, increase of expectoration, or slight rise of temperature, the dose given has been too large, and the same is true if in a tuberculosis about the bladder symptoms of urinary irritation supervene. While undoubtedly in many cases good results have been obtained, every administration must be looked upon as of the nature of an experiment, and the treatment should only be in the hands of those who have had great experience of the subject. The fact that in so many cases tubercular infections tend to disappear under ordinary treatment makes it at present extremely difficult to estimate truly the therapeutic effects of vaccine therapy. Wright holds that the opsonic qualities of the serum constitute the means by which the body frees itself of the invading bacilli. A natural cure results when the absorption into the circulation of the products of the local disintegration of the tubercle bacilli so stimulates some reactive mechanism in the body that sufficient opsonin is produced to cause a phagocytosis of all the tubercle bacilli present. The occurrence of a low opsonic index in chronic local tuberculosis is due to the using up of the opsonins in the focus of infection, and in such a case the general mechanism has not been stimulated to produce a conquering amount of opsonin. The object of a vaccination is thus to supply this deficiency. If it is successful, the focus is flooded with lymph rich in opsonin and the bacilli are consequently phagocyted and destroyed. The reaction of the opsonin-producing mechanism is, however, not a simple one, and as in the introduction of other antigens, the injection of tuberculin is followed by a period, normally lasting a day or two, when the amount of opsonin is actually lower than it previously was (occurrence of negative phase). This, in a successful vaccination, is followed by arise in opsonic content of the serum to a point above the level existent at the time of injection (production of positive phase). In certain cases a positive phase is easily obtained ; in other cases there is a tendency to a prolonged persistence of the negative phase, and if during such an occurrence a fresh tuberculin injection be produced, a still greater fall in opsonic content may occur, usually accompanied b clinical exacerbation of the tubercular symptoms. Immunisation is further complicated by the fact that there is a variable and often uncontrollable absorption into the body from the focus of infec- tion of what is really tuberculin derived from the disintegration of the infecting bacilli. Antitubercular Sera.—From what has been said regarding immunity reactions in tuberculosis it will be gathered that it is questionable whether the use of passive immunity in the treatment of tuberculosis has a rational basis. Several investigators, however, have introduced the sera of animals treated with the products of tubercle bacilli for therapeutic purposes. Amongst these are Maragliano, who has treated dogs, asses, and horses with materials derived from the tubercle bacillus, and administers their serum in doses of 2 ¢.c. every two days in human tuberculosis. An anti- tubercular sera has also been introduced by Marmorek, who grows the bacilli in media unfavourable to their vitality and employs such growths METHODS OF EXAMINATION 297 for immunising animals whose serum he states is suitable for the treat- ment of human cases. Methods of Examination.—(1) Microscopie Examination.—Tuberculosis is one of the comparatively few diseases in which a diagnosis can usually be definitely made by microscopic examination alone. In the case of sputum, one of the yellowish fragments which are often present ought to be selected ; dried films are then prepared in the usual way, and stained by the Ziehl-Neelsen stain (p. 105). In the case of urine or other fluids, a deposit should first be obtained by centrifuging 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 to the experienced observer from this cause. (For points to be attended to, vide p. 286.) The detec- tion of tubercle bacilli by microscopical methods in sputum, pus, feces, and even tissues, has been greatly facilitated by the introduction of a preparation called ‘‘antiformin.” This is a mixture of equal parts of liquor sode chlorinate (B.P.) and of a 15 per cent. solution of caustic soda. It has a remarkable disintegrative and dissolving action on the tissues, etc., so that after it has been allowed to act on sputum, for ex- ample, and the mixture is centrifuged, the resulting deposit is scanty and the tubercle bacilli, if present, are accordingly greatly concentrated. The time necessary may be judged of by the appearance of the mixture, but it will generally be found that the desired result will be obtained after about an hour if one part of sputum be added to two or three parts of 20 per cent. antiformin; the mixture should be shaken from time to time, especially when the sputum is tenacious. (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 theemulsion injected. By this method, material in which no tubercle bacilli can be found microscopically may sometimes be shown to be tubercular. © (8) Culttvation.—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 or egg medium, under strict aseptic precautions, with portions of a tubercular organ, ¢.g., the spleen. The portions of tissue should be fairly large, and should be well rubbed into the broken surface of the medium. Cultures may, however, be obtained from sputum by means of antiformin, as this substance readily kills most of the ordin- ary bacteria and has comparatively slight effect on the tubercle bacillus. Antiformin should be allowed to act on sputum in the proportion and for the time mentioned in paragraph (1), the mixture should then be centri- fuged, the supernatant fluid removed, and the deposit washed with sterile water and again centrifuged, these processes being repeated several times. If, then, inoculations be made from the deposit on blood serum or on Dorset’s egg medium and glycerin egg medium, pure cultures of the tubercle bacillus may, in some instances, be obtained. The method is one which gives good results. 298 TUBERCULOSIS Petroff’s method is also recommended as giving satisfactory results. In this, sputum is shaken with an equal volume of 3 per cent. caustic soda solution, and the mixture is placed for half an hour in the incubator at 37°C. At the end of this time it is made neutral to litmus with hydro- chloric acid and then centrifuged. Some of the deposit thus obtained is then planted on egg medium to which gentian violet has been added in the proportion of 1: 10,000, the dye having an inhibitory action on the ‘growth of various organisms. A pure culture of the tubercle bacillus is often obtained. , Another method is that introduced by Twort ; portions of sputum are exposed to the action of a 2 per cent. solution of ericolin (a glucoside) for an hour at 38° C., and thereafter cultures are made on Dorset’s medium. (4) Reactive phenomena.—The presence of immune-substances in the blood and the tuberculin reaction, along with the methods of applying the respective tests, have been described above (p. 293). CHAPTER XI 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 disease 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. Itis 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 essentially a chronic disease, in which there is a great amount of tissue change, with comparatively little necessary impairment of the general health. In other words, the local 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 anesthetic,—maculo-anesthetic of Hansen and Looft,—the out- standing changes are in the nerves, with consequent anesthesia, 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 299 ‘ 300 LEPROSY 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. 84), 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 Fic, 84.—Sections through leprous skin, showing the masses of cellular granulation tissue in the cutis; the dark points are cells containing bacilli deeply stained. Paraffin section ; Ziehl-Neelsen stain. x 80. 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 all situations the change is of the same nature, consisting in an 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 comparatively large size, and may show vacuolation of their protoplasm and a vesicular type of nucleus. These are often known as “lepra-cells.” Amongst the cellular BACILLUS OF LEPROSY 301 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 a focal 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 follows a remarkable series of trophic disturbances, in which the ‘skin, muscles, and bones are especially involved. The skin often becomes atrophied, parch- ment-like, and anesthetic ; frequently pemphigoid bulla or other skin eruptions occur. Partly, owing to injury to which the feet and hands are liable from their anesthetic 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, 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 confirmed the accuracy of Hansen’s observations. The bacilli, as seen in scrapings of ulcerated leprous nodules, or in sections, have the following characters: They are thin rods of practically the same size as tubercle bacilli, which they also resemble both in appearance and in staining reaction. They are straight or slightly curved, and usually occur singly, or two may be attached end to end; but they do not form chains. When stained they may have a uniform appearance, or the protoplasm may be fragmented, so that they appear like short rows of cocci. They 302 LEPROSY 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 Fic. 85.—Superficial part of leprous skin ; the cells of the granula- tion 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. ‘(p. 105); variations, however, exist in this respect, some bacilli losing the stain more readily than others. The bacilli are also readily stained by Gram’s method. Regarding the presence of spores, practically nothing is known, though some of the un- stained 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 BacilliimThey occur in enormous numbers in the leprous lesions, especially in the tubercular form—in fact, POSITION OF THE BACILLI 303 80 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 (Plate IL, Fig. 8). 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 obscured (Fig. 85). They are often arranged in bundles which contain several bacilli a“ Fic. 86.—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. lying parallel to one another, though the bundles lie in various directions (Fig. 86 and Plate IL, Fig. 9). 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 304 LEPROSY 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 anesthetic 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 oc- Nae be NO, pe Ka EES curs. They are _ eee tae D ae: , said to have been qi eT ace Wis ian found in the blood ae es VY eg Rae or during the pres- : a ey, ence of fever and Nee Neg fos 74 -- the eruption of BP Pa i Nees 5 Set fl "'_@ | fresh nodules, and MA yee td Ba Shy a. i dS of they have also te nie Werte ey been observed in oe PRN a ene + ° the blood vessels f o\ i ~—F7. “ ea ey ee post mortem, ae ie if *" chiefly contained vA “ee f A ep 35 1y a. be ro within leucocytes. rill NOE Ne A eee tee A few may be mary may be given of its ee 5 23s \ action in the body. on \ Locally, the bacillus pro- [yeh aOR : ip duces inflammatory change eS pte with fibrinous exudation,. | Vad but at the same time 4 \ Cos Se cellular necrosis is also tn Bo an outstanding feature. ne Though false membranes have not been produced by the toxins, a necrotic “ action may result when Fic. 118,—Xerosis bacillus from a young these are injected sub- agar culture. 1000. cutaneously. The toxins also act upon the blood vessels, and hence wdema and tendency to hemorrhage are produced; this action on the vessels is also exemplified by the general congestion of organs. The hyaline change in the METHODS OF DIAGNOSIS 417 walls of arterioles and capillaries, so often met with in diphtheria, is another example of the action of the toxin. The toxins have also a pernicious action on highly developed cells and on nerve fibres. Thus in the kidney cloudy swelling occurs, which may be followed by actual necrosis of the secreting cells, and along with these changes albuminuria is present. The action is also well seen in the case of the muscle fibres of the heart, which may undergo a sort of hyaline change, followed by granular disintegra- tion and associated with leucocytic infiltration. These changes are of great importance in relation to heart failure in the disease. Changes of a somewhat similar nature have been 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.—These include: (a) Microscopical Examination.—For microscopical examination it is sufficient to tease out a piece of the membrane with forceps and rub it on a cover-glass; if it be somewhat dry, a small drop of 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 Léffler’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. 114) may also be used with advantage, although it is to be noted that sometimes in a secretion the diphtheria bacillus does not react, typically to this stain. Any secretion from the pharynx or other part is to be treated in the same way. Diagnosis by the microscopic examination is now little used, but it is some- times justified in cases of urgency, though only in the hands of an experienced observer. In some cases the bacilli are present in characteristic form in such numbers as to leave no doubt in the matter. (6) Cultivation.—For this purpose a piece of the membrane should be separated by forceps from the pharynx or other part when that is possible. It should be then washed well in a tube containing sterile water, most of the surface impurities being removed in this way. A fragment is then fixed in a platinum loop by means of sterile forceps, and a series of stroke cultures 27 418 DIPHTHERIA is made on the surface of any of the media mentioned (p. 403), the same portion of the membrane being always brought into contact with the surface. More usually a swab taken from the pharynx is smeared over the medium in a similar manner. ’ The tubes are then incubated at 37° C., and are ready for examina- tion in eighteen to twenty-four hours. A representative sample of the whole growth is obtained by rubbing a platinum loop over the surface; films are made from this, stained, and ex- amined in the usual way, Neisser’s stain being also applied. Any doubtful organism should be tested by growing for two to three days in glucose peptone water, tinted with neutral-red, to which a few drops of sterile serum have been added to aid growth. If no acid is produced the organism may be rejected ; if acid is formed, animal tests should be carried out. For the obtaining of a pure culture the telluric acid medium (p. 52) will be found of great service. (c) Inoculation.—The bacillus in question should be grown in bouillon for two to three days, and then a guinea-pig inoculated with 1 c.c. (This is the most suitable method, though it is not strictly a test of pure virulence, the result being to some extent due to toxin produced in the culture.) If the animal dies with the characteristic lesions, further tests may be made with smaller doses. The intra-cutaneous (p. 407) method may also be used. = For interpretation of results, vide p. 414. CHAPTER XVIL. TETANUS!: CONDITIONS CAUSED BY OTHER ANAEROBIC BACILLI. Introductory.—Tetanus is a disease which in natural conditions affects chiefly man and the horse. Clinically it is characterised by the gradual onset of general stiffness and spasms of the voluntary muscles, commencing in those of: the jaw and the back of the neck, and extending to all the muscles of the body. These spasms are of a tonic nature, and, as the disease advances, succeed each other with only a slight intermission of time. There are often, towards the end of a case, fever and rise of respiration and pulse-rate. The disease is usually associated with a wound received ordinarily from four to fourteen days previously, and which has been defiled by earth or dung. The disease is, in the majority of cases, fatal. 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. Nicolaier (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 inoculation, and in this pus, besides other organisms, there was always present, when tetanus had occurred, a bacillus having certain constant microsc.pic 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 1 This disease is not to be confused with the ‘‘tetany” of infants, which in its essential pathology differs from tetanus (vide Frankl-Hochwart, ‘ Die Tetanie der Erwachsenen,” Vienna, 1907). This remark, of course, does not exclude the occurrence of true tetanus in very young subjects, in whom, in fact, infection frequently takes pices often at the umbilicus. 420 TETANUS 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. 119). 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 p» to 5 » in length and ‘4 » in thickness, with somewhat rounded ends. Besides occurring as shorter 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. 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. 120). These flagella, though they may be of considerable length, are usually curled up close to the body of the bacillus. The formation of flagella can be best studied in preparations made from surface anaerobic cultures (p. 69). 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. 121). At incubation temperature b. tetani readily forms spores, and then presents a very characteristic appearance. The spores are round, and in diameter may be three or four times the thickness of the bacilli. “They are developed at one end of a bacillus, which thus assumes what is usually described as the ‘“‘drumstick ” form (Figs. 119, 122). In a specimen stained with a watery solution of gentian-violet or ISOLATION OF THE BACILLUS 42] methylene-blue, the spores are uncoloured except at the periphery, so that the appearance of a small ring is produced ; if a powerful stain such as carbol-fuchsin be applied for some time, the spores 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. Fic. 119.—Film preparation of discharge from wound lin a case of tetanus, showing several tetanus bacilli of ‘‘drumstick” form. (The thicker bacillus present is not a tetanus bacillus, but a putrefactive anaerobe which was obtained in pure culture from the wound.) Stained with gentian-violet. x 1000. 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 contents of dung-heaps, where it probably leads a saprophytic existence, though its function as a saprophyte is unknown. It also occurs in the dust of houses, on, the skin and in the intestines of many—especially of herbivorous—animals. From such sources and from the pus of wounds in tetanus, 422 TETANUS occurring naturally or experimentally produced, it has been isolated by means of the methods appropriate for anaerobic bacteria. The best methods for dealing with such pus are as follows :-— (1) The principle is to take advantage of the resistance of the spores of the bacillus to heat. A sloped tube of inspis- sated serum or a deep tube of glucose agar is inoculated and incubated anaerobically at 37° C. for forty-eight hours, at the Fic, 120.—Tetanus bacilli, showing flagella. Stained by Rd. Muir’s method. 1000. 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. From such material agar anaerobic plates are prepared by one of the methods de- scribed on pp. 62-65. Kitasato compares the colonies in gelatin plates to those of the b. subtilis. They consist of a thick centre with shoots radiating 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 ISOLATION OF THE BACILLUS 423 bacilli, both in its natural habitats outside the body and in the pus of wounds, other spore - forming obliga- tory and facultative anaerobes occur, which grow faster than the tetanus bacillus, and thus overgrow it. (2) If in any dis- charge the spore-bearing tetanus bacilli be seen on microscopic examina- tion, then a method of isolation based on the same principle as the last may be adopted. Inoculations with differ-’ ent dilutions of the sus- pected material are made in half a dozen deep tubes of glucose’ Fic, 121.—Spiral composed of numerous twisted flagella of the tetanus bacillus. Stained by Rd. Muir’s method. x 1000. bouillon, previously raised to a temperature of 100°C. After fA ; = oe ad [ \ i \ { : ao \ i ae x bs moat oR ‘ “Se id Fic. 122.—Tetanus bacilli; some of which possess spores. agar, incubated for three days at 37° C. also Plate IV., Fig. 20. Stained with carbol-fuchsin. x 1000. From a culture in glucose See “all 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 respectively. 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 the organisms present will have been killed, except the tetanus spores which can develop in pure culture. A _ series of deep glucose agar tubes may also be in- oculated from the series of bouillon tubes. 424 TETANUS (3) Anaerobic plates may be prepared directly from the dis- charge of the wound. The isolation of the tetanus bacillus is in many cases a difficult matter, and several methods should always be tried. Characters of Cultures,—Pure cultures having been obtained, sub-cultures can be made in deep upright glucose gelatin or agar tubes. In deep glucose gelatin (in which growth is often very difficult to obtain) 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. 123). Slow liquefaction of the gelatin takes place, with slight gas forma- tion. 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. On anaerobic agar plates colonies have under a low power a feathery outline (Fig. 124). Growth also occurs in blood serum and also in glucose bouillon and Robertson’s bullock’s heart medium under anaerobic conditions.. There is in it at first a slight turbidity, and later a thin layer of a powdery deposit on the walls of the vessel. All the cultures give out a peculiar burnt odour of rather un- pleasant character. In making sub-cultures Mite Las Pra cal on fluid media a considerable amount of bacillus in glucose the original growth should be used for the gelatin, showing inoculation. GekGae Conditions of Growth, etc.—The b. tetani Natural size. grows best at 37°C. The minimum growth temperature is about 14° C., and below 22° C. growth takes place very slowly. Growth takes place only in the absence of free oxygen, the organism being an anaerobe. Sporulation may commence at the end of twenty-four hours in cultures grown at 37° C.—much later at lower tempera- tures. 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 PATHOGENIC EFFECTS 425 losing their virulence. They have also high powers of resistance to antiseptics. Pathogenic Effects.—The proof that the b. tetani is the cause of tetanus is complete. It. can be isolated in pure culture, and when 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 natwrally.—The disease occurs naturally, chiefly in horses and in man. Other animals may, however, be affected. In different animal species variations in | Fic. 124,—Colonies of the tetanus bacillus on anaerobic agar plates, seven days old. x50. the clinical progress of the disease are observed. In man and in the horse the spasms early affect the extensor muscles of the trunk, while in other animals they may first appear in the muscles neighbouring on the site of infection. There is in most cases a definite 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 a purulent or fetid discharge, though this may be absent. In tetanus following clean operation wounds, catgut ligatures may be the source of infection. Microscopic examina- tion of sections may show at the edges of the infected wound ' necrosed tissue in which the tetanus bacilli may be very numerous. If a scraping from the wound be examined micro- 426 TETANUS scopically, bacilli resembling the tetanus bacillus may be recognised. Care must be taken, however, to distinguish it from other thicker bacilli with oval spores placed at a short distance from their extremities, such forms being common in earth, etc., and also met with in contaminated wounds. 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 possibly be merely the bite of an insect. In many parts of the world infec- tion through the umbilicus originates a high mortality from the disease in newly born infants. The absence in many cases of a definite channel of infection has given rise tothe term “idio- pathic” 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 ; infection of the intestinal mucosa may also occur. During the present war the clinical type of tetanus seen in the wounded has been modified in consequence of the wide application of the prophylactic injection of anti-tetanus serum (vide infra). In the first place there has been a tendency to the prolongation of the incubation period, instances where this has extended to many months being not uncommon. In such cases there has usually been an unhealed septic wound, often contain- ing foreign bodies, and the attack of tetanus may be precipitated by operative procedures ; sometimes the wound has healed and ‘tetanus has followed operation for the removal of foreign bodies in the tissues. Again the disease tends to assume the type seen in some animals, the muscles in the neighbourhood of the wound being first affected ; local hardness and stiffness, pain, and exaggeration of local reflexes have thus often been the first, and sometimes the only, clinical phenomena. Such cases of tetanus are also apparently more amenable to treatment with antitetanus serum. The pathological changes found post mortem are not striking. There may be hemorrhages in the muscles, which have been the subject of the spasms. These are probably due to mechanical causes. It is in the nervous system that we naturally look for, the most important lesions. Here there is ordinarily a general redness of the grey matter, and the most striking feature is the occurrence of irregular patches of slight congestion which are not limited particularly to grey or white matter, or to any tract of the latter. These patches are usually best marked in the grey PATHOGENIC EFFECTS 427 matter of the medulla and pons. Microscopically there is little of a definite nature to be found. There is congestion, and there may be minute hemorrhages 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. (6) The Artificially-produced Disease—The disease can be communicated to animals by any of the usual methods of inocula- tion, 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 the case of intravenous inoculation the spasms begin in the extensor muscles of the trunk, as in the natural disease in man. In intraperitoneal injection spasms in the muscles controlled by the splanchnic system are an outstanding feature. After death there is found slight hyperemia without pus formation, at the seat of inoculation. The bacilli diminish in number, and may be absent at the time of death. The organs generally show little change. Kitasato stated 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 428 TETANUS splinters of wood soaked in cultures in which spores were present,, and subsequently subjected for one hour to a tempera- ture of 80° C. The latter treatment not only killed all the vegetative forms of the organism, but, as we shail 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. In 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. [Sometimes, however, in such circumstances death occurs with- out 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 (wide infra).]| By such experiments, supplemented 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 an attempt was made with regard to tetanus to explain the general symptoms by the soluble poisons of the’bacillus. The earlier results, in which certain bases, tetanin and tetanotoxin, 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 a toxalbumin from tetanus cultures, and this body was independently discovered by Faber in the same year. Brieger and Fraenkel’s body consisted practically of an alcoholic precipitate from filtered 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 ordinary bouillon cultures grown under anaerobic conditions, or of the precipitate produced from the same by ammonium sulphate (of. p. 190). The toxic properties of bacterium-free filtrates 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. He found that guinea-pigs were more susceptible than mice, and rabbits less so. In order that a strongly toxic bouillon be produced, it must ! TOXINS OF THE TETANUS BACILLUS 429 originally have been either neutral or slightly alkaline. Kitasato 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 sunlight. To prepare the toxin, freshly made veal bouillon not too long autoclaved should be used and a massive inoculation, preferably from a fluid culture, practised. Individual strains of the bacillus differ in their capacity for producing toxin. The culture must be incubated under anaerobic conditions and the maximum toxicity is developed in from ten to fifteen days. Behring pointed out that after the filtration of cultures containing toxin, the latter may very rapidly lose its power, and in a few days may only possess ;4,th of its original toxicity. This is due to such factors as temperature and light, and especially to the action of oxygen. Toxins should thus have a layer of toluol floated on the surface and be kept in a cool, dark place. The effect of harm- ful agents on the crude toxin is apparently to cause a degenera- - tion of the true toxin so as to form what it is convenient at present to call 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 may contain different varieties of toxic substances. Ehrlich showed that besides the predominant spasm-producing toxin (called by him tetanospasmin), there often exists in crude toxin a poison capable of producing the solution of certain red blood corpuscles. This hemolytic agent he called 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. 190), Tetanolysin 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 phenomena can be demonstrated similar to those noted by Ehrlich as occurring with diphtheria toxin, and which the latter interpreted as indicating the presence of degenerated toxins (toxoids) in the crude poison, With 430 TETANUS 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 proteins on which the bacillus may be living, though the latter has a digestive action on proteins, There is evidence that peptic digestion and toxin formation are due to different vital processes on the part of the tetanus bacillus. The toxin is 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 inilligramme. Animals differ very much in their suscepti- bilities 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 an incubation period between the introduction of the toxin into an animal's body and the appearance of symptoms. This varies according to the species of animal employed, the path of infection, and the dose given. In the guinea-pig it is from thirteen to eighteen hours, in the horse five days, and the in- cubation 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 symp- toms is usually from two to fourteen days, but this period may be lengthened, and the bacilli may remain a con- siderable time shut up in a wound before producing effects. The longer the incubation period, the more favourable is the prognosis, and in chronic cases spontaneous recovery is not uncommon. With regard to the actzon 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 motor cells in the spinal cord, the nerve storm being often precipitated by peripheral irritation. The motor cells in the pons and medulla are also affected, and to a much greater degree than those in the cerebral cortex. TOXINS OF THE TETANUS BACILLUS 431 When injected subcutaneously the toxin is partly 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. In man under ordinary circumstances the first symptoms appear in the neck. After subcutaneous 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-pig there is little doubt that tetanus toxin has an affinity solely for the nervous system. In other animals, ¢.g., 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. | Tetanus toxin introduced into the stomach or intestine is not absorbed, but to a large extent passes through the intestine unchanged. Evidence that any destruction takes place is wanting. Marie and Morax shed some important light, on the mode of action of tetanus toxin in studying 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 the proximal part did not contain toxin, though no doubt it 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 the presence of 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. The absorption by the nerve was fairly rapid, as one hour after injection the toxin was present in it, and the toxin was shown to be exclusively centripetal in its flow. Further observa- tions were made on this subject by Meyer and Ransom, who found 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, 432 TETANUS and a local tetanus of the muscles supplied by the nerve results, With a larger dose the poison passes across the commissure to 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 the larger animals, where the nerve path is longest, the incubation period is also longest. When intravenous injection is practised, the occurrence of tetanus in a part of the body can be pre- cipitated by the injection of a drop of normal saline into the corresponding part of the cord—sufficient injury being thus caused to allow the toxin in the surrounding lymph to obtain access to the nervous elements. With regard to the action of tetanus toxin, Meyer and Ransom believe that there is a double effect on the nerve cells—first, an exaggeration of the normal tonus, which accounts for the continuous stiffness of the muscles ; and secondly, an jncrease in reflex irritability, which is a promi- nent factor in the recurring spasms. While no absorption of toxin takes place by sensory filaments, they have found evidence of affection of the sensory apparatus in the occurrence of what they call tetanus dolorosus. This is a great hypereesthesia 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, in investigating the action of antitoxin, found that its injection into 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, death 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 into the sciatic nerve was followed by tetanus. This would appear to militate against Ehrlich’s position that IMMUNITY AGAINST TETANUS 433 antitoxin is manufactured in the cells which are sensitive to the toxin (see Immunity). : Roux and Borrel, in injecting tetanus toxin into the brain itself, found that the ordinary type of the disease was not pro- duced, but that what they called ‘cerebral tetanus” occurred. This consisted of general unrest, symptoms of a psychic character (apparent hallucinations, fear, etc.), and epileptiform convul- sions. Death took place 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 dose. In 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 experiments with tetanus cultures in animals, death results not from the multiplication of the bacilli, 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 in- jected into an animal, death does not take place. It was found, however, that such spores can be rendered pathogenic by inject- ing. 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 acci- dentally subjected to conditions such as those enumerated. Kitasato now holds that in the natural infection in man, along with tetanus spores, the presence of foreign material or of other bacteria is necessary. Spores alone or tetanus bacilli without spores die in the tissues, and tetanus does not result. Immunity against Tetanus.—Antitetanic Serum,—The arti- ficial immunisation of animals against tetanus received much attention, especially from Behring and Kitasato in Germany, and Tizzoni and Cattani in Italy. little or no pathogenic pro- J \ Se perties when injected in f animals, a comparatively / , ae A _ large amount of pure cul- /— y y pd Lae ture producing only a local swelling which passes off ; and observations on gunshot _A ; / wounds supply no evidence | ao } that it invades the healthy \ & tissues. It may be regarded WG »® y chiefly as a proteolytic 1 J f / saprophyte which grows on if i dead and dying _ tissues er ; and brings about digestive ewe o. softening and putrefactive es changes. It thus readily a invades the tissues already !* ee a eat ae show- damaged by other organ- stained with carbol-thionin blue. x 1000. isms, ¢.g., the b. welchii. There is also experimental ; evidence that its presence aids the pathogenic effects of other organisms. The b. sporogenes is closely allied to another anaerobe described under the name b. putrificus. a. Bacillus histolyticus.—This is another proteolytic and putrefactive anaerobe separated by Weinberg from cases of gas gangrene. It is 2-6 » in length and rather thinner than the b. welchii; it is often arranged in pairs. It is Gram-positive and forms large oval subterminal spores. The surface growth is in the form of a very thin film, with offshoots at the margin. Its action on milk and coagulated serum is similar to that of the b. sporogenes, but is even more rapid. In cooked meat medium also it produces very rapid digestion, with foul odour, and one feature described by Henry is the separation of white balls of acicular crystals which are probably tyrosin—an appearance which is probably characteristic of this organism. The cultures have a foul odour. A striking evidence of the proteolytic action of this organism is seen when it is injected subcutaneously in a guinea-pig. A rapid ~~ 456 QUARTER-EVIL digestion of the tissues in the vicinity occurs so as sometimes actually to expose the bones. Bacillus putrificus.—This anaerobe was first described by Bienstock, who considered it the chief agent in putrefaction—hence the name. It measures usually 5-6 «4 in length, though shorter and also longer fila- mentous forms are met with, and is relatively slender. It is actively motile and forms oval terminal spores which are large in proportion to the size of the rod. It grows readily under anaerobic conditions and gives a very foul odour in all media. The superficial colonies are trans- pareut discs rounded or irregular in form, while the deep ones are woolly in appearance not unlike those of the b. sporogenes. The organism has marked proteolytic action, rapidly liquefying gelatine and coagulated serum, and the ultimate products are comparatively simple compounds, including various gases. Bienstock found that it did not ferment earbo- hydrates, but strains isolated by other workers have fermented certain of the sugars, To the sugar-fermenting variety Bienstock gave the name b. paraputrificus. Inoculation experiments with the b. putriticus show that it is practically devoid of pathogenic properties. QUARTER-EVIL (GERMAN, RAUSCHBRAND ; FRENUH, CHARKBUN 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 occurs especially in certain localities, affects chiefly sheep, cattle, and goats. Infection takes place by some wound of the surface, and then spreads in the region around, inflammatory swelling attended by bloody cedema and emphysema of the tissues. The part becomes greatly swollen, and of a dark, almost black, colour. Hence the name “ black- quarter” by which the disease is often known. The bacillus which{pro- duces 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 edema. 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 long filaments ; occasionally it occurs in short chains. The spores, which are of oval shape and broader than the bacillus, are usually subterminal, though central-spored clostridium forms occur (Fig. 133). This bacillus is actively motile, and possesses numerous lateral flagella. The characters of the cultures, also, resemble those of the bacillus of malignant cedema, 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). The superficial colonies are small greyish rounded discs with a thicker centre ; the deep colonies show a radiating appearance at the periphery. M‘Intosh finds that the organism belongs to the non-proteolytic class. It produces acid clot in milk in three to four days and ferments glucose, maltose, lactose, and saccharose, but not inulin or dulcite. It does not liquefy coagulated serum. The disease can be readily produced in various animals, ¢.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 o-dema, though FUSIFORM ANAEROBIC BACILLI 457 there is said to be more formation of gas in the tissues. Rabbits are more resistant to this disease, whilst they are ¢éomparatively suscep- tible to malignant wdema. 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 filtration from cultures of bouillon containing 5 per cent. glucose and a thick emulsion of sterile calcium carbonate. 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. It is to be noted as an important fact, that while freshly isolated cultures possess a high degree of viruleuce they may have little capacity for toxin production in vitro. Grassberger and Schattenfroh state that there may be an antagonism between maxi- mum virulence and maxi- mum toxin production. One of the properties of the toxin is said to be a capacity for killing leucocytes. The disease is one against which immunity can be pro- duced in various ways, and methods of preventive in- oculation have been adopted in the case of animals liable to suffer from it. This sub- ject was specially worked out by Arloing, Cornevin, and Thomas, and later by others. Immunity may be produced by injection (especially by the intravenous and intra- ; peritoneal routes) with a Fic. 133.—Bacillus of quarter-evil, showin non-fatal dose of the virus spores. From a culture in glucose agar, (i.e, the cedematous fluid incubated for three days at 37° C found in the tissues of affected Stained with weak carbol-fuchsin. x 1000. animals and which contains the bacilli), or by injection with larger quantities of the virus attenuated by heat, drying, etc. It can be produced also by cultures attenuated by heat and by tke products of the bacilli obtained by filtration 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 (¢/. Anthrax, p. 349). The anti- toxin is said to increase the chemiotactic properties of the leucocytes. Fusirorm ANAEROBIC Bacittt PATHOUENIC TO MAN. Babés in 1884 described organisins of this type in a diphtheria-like affection of the fauces, and since that time the presence of similar organisms has been noted in necrotic inflam- mations, ulcerative stomatitis, noma, and like affections. They have also been found in pulmonary lesions and in abscesses in other parts of the body; in these the pus is very foul-smelling. 458 FUSIFORM ANAEROBIC BACILLI The association of fusiform bacilli with a form of angina has been specially recognised since the work of Vincent (1898-99) ; and this condition often goes now under the name of “ Vincent’s angina.” He recognised two forms of the affection—(a) a diphtheroid type, characterised by the formation of a firm yellowish-white false membrane, very like that of diphtheria, associated with only superficial ulceration ; and (4) an ulcerative type, where the membrane is soft, greyish, and foul-smelling, attended with ulceration and surrounding cedema. In the = ” ae eS is CA ge m., > tia d : - ’ Br Fic. 184.—Film preparation from a case of Vincent’s angina, showing fusiform bacilli and spirochetes. Stained with weak carbol-fuchsin. x 1000. former type fusiform bacilli are present alone; in the latter, which is distinctly the commoner, there are also spirochetes. The fusiform bacilli are thin rods measuring on the average 10 to 14 pw in length, and less than 1 » in thickness; they are straight or slightly curved and are tapered at their extremities. The central portion often stains less deeply than the extremities, and not infrequently shows unstained points and granules (Fig. 134; Plate IL, Fig 4). The organisms are non-motile. They stain fairly deeply with Léffler’s methylene-blue or with weak carbol-fuchsin. They lose the stain in Gram’s method. The spirochetes are long delicate organisms showing several ‘FUSIFORM ANAEROBIC BACILLI 459 irregular curves, and are motile; in appearance they resemble the spirochete refringens and similar organisms found in gan- grenous conditions. They stain less deeply than the bacilli. Sometimes they are numerous, sometimes scanty ; they seem to be similar to spirochetes found in the mouth in a variety of other conditions. In a section through the false membrane, when stained with methylene or thionin blue, there is usually to be seen a darkly stained band, a short distance below the surface, which is due to the presence of large masses of the fusiform bacilli closely packedi together ; neither they nor the spirochetes appear to pass deeply into the tissues. Vincent’s results have been confirmed by others, and there is no doubt that fusiform bacilli, of which there are probably several species, are associated with various spreading necrotic conditions. During the war, cases of Vincent’s angina have been of common occurrence and have been met with in small epidemics. Ulcerative gingivitis and stomatitis have been found to be associated with the presence of the same organisms, and in some cases these lesions precede the infection of the fauces. It would be advisable to apply the term “ Vincent’s disease,” as suggested by Bowman, so as to include all the lesions produced by the organisms in question. In phagedenic lesions of the genitals, fusiform bacilli are usually present, with or without spirochetes, though in our experience they are as a rule of smaller size than those met with in the throat. Cultures of fusiform bacilli have been obtained by Ellermann, by Weaver and Tunnicliffe, and by others. They grow only under anaerobic conditions, and the best media are those consisting of a mixture of serum or blood and agar (1 : 3). The organisms form small rounded colonies of whitish or yellowish colour, somewhat like those of a streptococcus, but rather felted in appearance on the surface. Injections of pure cultures in animals sometimes produce suppuration but never necrosis (Ellermann). Tunnicliffe finds that the spirochetes are only stages in the development of fusiform bacilli, as cultures which at an early stage show only fusiform bacilli, afterwards contain spirochetes, and intermediate forms can be found. There seems to be no doubt that in cultures the bacilli grow out into long filaments which may have an undulated appearance ; but it is doubtful whether these are to be regarded as true spiro- cheetes, and still more whether they are the same spirochetes as those seen in the lesions in assvciation with the bacilli. It is also to be noted that fusiform bacilli are sometimes present in the secretions of the mouth in normal conditions, and may occur in increased numbers in true diphtheria. CHAPTER XVIII. THE CHOLERA SPIRILLUM AND ALLIED ORGANISMS. Introductory.—It is no exaggeration of the facts to say that previously to 1883 practically nothing of value was known regarding the nature of the virus of cholera. In that year, Koch discovered the organism now generally known as the ‘comma bacillus” or the “cholera spirillum.” He 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 1892-98, 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 variations have been found. 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 re garded 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 often by vomiting, there are also symptoms of general systemic disturbance which cannot be accounted for merely by the withdrawal of water and certain substances from the system. Such symptoms include the profound general prostra- tion, cramps in the muscles, extreme cardiac depression, the cold and clammy condition of the surface, the subnormal 460 ' MICROSCOPICAL CHARACTERS 461 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 choleru sicca, general collapse occurs with remarkable suddenness, and is rapidly followed: by a fatal result, whilst there is little or no evacuation from the bowel, though post mortem the intestine is distended with fluid contents. As the characteristic organisms in cholera are present mainly in the intestine, the general disturbances are to be regarded as the result of toxic substances absorbed from the bowel. It is also to be noted that cholera is a disease of which the onset and course are much more rapid than is the case in most in- fective diseases, such as typhoid and diphtheria ; and also that recovery, when it takes place, does so more quickly. The two factors to be correlated to these facts are: (a) a rapid multi- plication of organisms, (6) , the production of Fic. 135.—Cholera spirilla, from a culture on rapidly acting toxins. agar of twenty-four hours’ growth. The Cholera Spiril- Stained with weak carbol-fuchsin. x 1000. lum.— Microscopical Characters.—The cholera spirilla, as found in the intestines in cholera, are small organisms measuring about 1°5 to 2 pm in length, and rather less than ‘5 in thickness. They are distinctly curved in one direction, hence the appearance of a comma (Fig. 135); most occur singly, but some are attached in pairs and curved in opposite directions, so that an S-shape results, Longer forms are rarely seen in the intestine, but in cultures in flyids, as may be well seen in hanging-drop preparations, they may grow into spiral filaments, showing a large number \ of turns. In film preparations made from the intestinal con- \gents in typical cases, these organisms are present in enormous numbers in almost pure culture, most of the spirilla lying with their long axes in the same direction, so as to give the appear- ance which Koch compared to a number of fish in a stream. 462 CHOLERA They possess very active motility, which is most marked in Fic. 136.—Cholera spirilla stained to show the terminal flagella. See also Plate IV., Fig. 19. x 1000. the single forms, and this is due to a single terminal flagellum (Fig. 136). Itis very delicate, and measures four or five times the length of the organism. Cholera spirilla. do not form spores. In old cultures the organisms may present great variety in size and shape. Some are irregularly twisted filaments, sometimes globose, sometimes clubbed at their ex- tremities, and also show- ing irregular swellings along their course; others are short and thick, and may have the appearance of large cocci, often stain- ing faintly. All these changes in appearance are to be classed together as involution forms. (Fig. 137.) Staininy. — Cholera spirilla stain readily with the usual basic aniline stains, though Léffler’s methylene-blue or weak carbol-fuchsin is speci- ally suitable. They are Gram-negative. Distribution within the Body.—The chief fact in this connection is that the spirilla are practically confined to the intestine. Recent observations show that they may be found some- Fra. 187.—Cholera spirilla from an old agar culture, showing irregularities in size and shape, with coccoid bodies—involution forms. Stained with fuchsin, numerous faintly - stained x 1000, times in the internal organs, and especially in the gall-bladder and CULTIVATION 463 biliary passages. Greig found, in a large series of post-mortem examinations, that the cholera organism was present in the gall-bladder in more than a quarter of the cases, and that, in a considerable number of these, distinct pathological changes were present. He has found it also in the urine, lungs, and spleen. Another interesting fact observed by him was that in rabbits inoculated intravenously with the living orgarism for the purpose of obtaining agglutinating sera, infection of the gall- bladder and the formation of gall-stones not infrequently oceurred. The all-important factor in the pathology of the disease, however, is the absorption of toxins from the bowel. 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 fiakes floating in the fluid gonsist chiefly of masses of epithelial cells and mucus, amongst which are numerous spirilla, The spirilla also penetrate the follicles of Lieberkihn, and may be seen lying between the basement membrane and the epithelial lining, which becomes loosened by their action. In some very acute cases there may be relatively little desquamation of epithelium, the intestinal contents being a comparatively clear fluid containing the spirilla in large numbers. In other cases of a more chronic type, the intestine may show more extensive necrosis of the mucosa and a considerable amount of hemorrhage 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. 474.) 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. Abundant growth occurs on media with suftici- ently alkaline reaction to inhibit the growth of many intestinal bacteria, e.g., Dieudonné’s medium, p. 46. 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 464 CHOLERA the following appearance: There is at the surface the bubble- — shaped depression ; below this there is a funnel-shaped area of liquefaction, the fluid being only slightly turbid, but showing at its lower end thick masses of growth of a more or less spiral shape in the thin line of liquefaction (Fig. 138). (This appear- ance is, however, in some varieties not produced till much later, ® especially when the gelatin ix very stiff, een eaT | and, in other varieties which liquefy very slowly, may not be met with at all.) At a later stage liquefaction spreads and may reach the side of the tube. When the organism is sub-cultured over a long period of time, it may lose to a large extent the property of liquefying gelatin. 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. 139, A). 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 appear- ‘ance, whilst the mass of growth in the centre is irregular and often broken up at its margins (Fig. 139, B). On the surface of agar media a thin, Fic. 138,.— Puncture almost transparent, layer forms, which an ee presents no special characters. On solidified gelatin —six days’ blood serwm the growth has at first the growth. Naturalsize. same appearance, but afterwards liquefac- tion 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 lfke that of the glanders bacillus; the appearance, however, varies some- . CULTIVATION 465 what with 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). It usually produces acid, without gas formation, from glucose, saccharose, mannite, and maltose; fermentation of lactose, with acid pro- duction, occurs late, namely, after two to three days. In milk also the organism grows well, and produces no Fic. 1389.—Colonies of the cholera spirillum on a gelatin plate— three days’ growth. A shows the granular surface, liquefaction just commencing ; in B liquefaction is well marked. coagulation nor any change in its appearance, at least for several days. On all the media the growth of the cholera spirillum is a relatively rapid one, and especially is this the case in peptone solution and in bouillon, a circumstance of importance in relation to its separation in cases of cholera (vide p. 475). 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 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 duc 30 466 CHOLERA to the fact that both indol and a nitrite are formed by the spirillum in the medium, and hence, in applying the test for indol, the addition of a nitrite is not necessary. It is essential that the sulphuric acid should be pure, for if traces of nitrites are present the reaction may be given by an organism which has not the power of forming nitrites. Humolytic Test.—This method, introduced by Kraus, is performed by means of agar plates (p. 45), a small quantity of sterile defibrinated blood being added to the agar and thoroughly diffused ;' if any organism has hemolytic properties, a clear zone or areola forms around each colony by the diffusion of hemoglobin. As a rule the cholera organism does not produce hemolysis, but the result after twenty-four hours should be taken, as later a clear zone may appear round a cholera colony (Greig). It has, however, been found by several observers that the hemolytic test is best carried out with a fluid culture. Greig, for example, adds varying amounts, from 1 c.c. downwards, of a three days’ culture in alkaline broth to le.c. of a 5 per cent. suspension of goats’ corpuscles, the whole being made up to 2c.c., and thoroughly mixed. The tubes are placed in the incubator for two hours at 37° C., and then in the ice-chest overnight, the results being read next day. He found after testing more than 300 strains of true cholera spirilla that none of them produced hemolysis, whereas this results with organisms of the El Tor group (vide infra). 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 tempera- ture 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 flourish very rapidly. Though we EXPERIMENTAL INOCULATION 467 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 considerable proportion of organic material, we do not know the exact circumstances under which it can flourish 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. During recent epidemics the cholera organism has been culti- vated from the stools of a considerable number of people suffering from slight intestinal disturbance, and even from the stools of quite healthy individuals ; these may be regarded as “cholera-carriers.” Numerous observations, carried out both on convalescents and on contacts having the spirillum in the stools, show that in the great majority of cases it dies out after two or three weeks and usually earlier ; it has, however, been found as long as twelve months afterwards. Greig has found that the excretion of the organism in the stools of carriers is of an intermittent character ; accordingly several examinations are uecessary before they can be pronounced free. There is no doubt that carriers play an important part in the spread of the disease, and can originate epidemics. Cholera organisms are, as a rule, rapidly killed by being thoroughly dried, 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 contamination of the water supply by choleraic discharges is the chief means by which areas of population are rapidly infected. It has been shown that if flies are fed on material containing cholera organisms, the organisms may be found alive within their bodies twenty- four hours afterwards. And further, Haffkine found that sterilised milk might become contaminated with cholera organ- isms 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. Accordingly, attempts to induce the multiplication of the organism within the intestine of animals by artificially arranging favouring 468 CHOLERA conditions occupied a prominent place in the early ex- perimental 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. These experiments were confirmed by other ‘observers, in- cluding Koch. Thinking that probably the spirillum, when introduced by the mouth, is destroyed by the action of the hydrochloric acid of the gastric secretion, Koch first neutralised this acidity by administering to guinea-pigs 5 c.c. of a5 per cent. solution of carbonate of soda, and some time 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 inject- ing tincture of opium into the peritoneum (1 c.c. per 200 grm. weight), in addition to neutralising as before with the carbonate of sodium solution. The result was remarkable, as thirty out of thirty-five animals treated died with symptoms of general prostration and collapse. Death occurs after a-few hours. Post mortem the smal] intestine is distended, its mucous membrane congested, and it contains a colourless fluid with small flocculi and the cholera organisms in practically pure cultures. 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. Some additional facts with regard to choleraic infection of animals may be mentioned. 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 hemorrhagic peritonitis—the organisms, however, being present also in the blood. And of special interest is the fact, discovered by Metchnikoff, that in the case of young rabbits shortly after birth a large proportion die of choleraic infection when the organisms are simply introduced along with the milk, as may be done by infecting the teats of the mother. Further, from these animals thus infected the disease may be transmitted to others by » natural mode of infection. In this affection of young rabbits many of the symptoms of cholera are present. 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 EXPERIMENTS ON THE HUMAN SUBJECT 469 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 sthaller 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 living organisms at the time of death, the fatal result having taken place from an intoxication (¢f. Diphtheria, p. 407). 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. 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. 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, diarrhcea ‘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 470 CHOLERA 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 ‘and of what has been established with regard to cholera carriers, we may consider that only a certain proportion of people are very susceptible to cholera, and the facts just mentioned are, in our opinion, of the greatest importance in establishing the relation of the organism to the disease. . Toxins.—The general statement may be made that filtered cholera cultures as a rule have little toxic action—that is, com paratively little extracellular toxin is produced by the organism. It was, however, shown by R. Pfeiffer that the dead spirilla were highly toxic, and that, in fact, they produced, on injection into guinea-pigs, the same phenomena as living cultures, profound collapse with subnormal temperature being a prominent feature. Pfeiffer considers that the toxic substances are contained in the bodies of the organisms,—that is, they are endotoxins,—and that they are only set free by the disintegration of the latter. 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, appar- ently due to the liberation of Se bee Nh Sy / ee a . float on the surface, then #5 69@. a striking mode of growth : “ s 8 % may result, to which the ie ed ce: -, term “stalactite” has fh a ge Ne + been applied. This con- Bee wae eS g sists in the growth start- pas .. met f® =| ing from the under surface : e ata za >< _ of the fat globules and ° a Ao Na ee «extending downwards in >"> ae the form of pendulous, / string-like masses. These masses are exceedingly i delicate, and readily break Fic. nea a the Leer of pene off on the slightest shak- ond pe cent. alt ager shoving invelaton ing of the flask ; accord- See also Plate IV., Fig. 17. ingly during their forma- Stained with carbol-thionin-blue. x 1000. tion the culture must be kept absolutely at rest. This manner of growth constitutes an important but not ab- solutely specific character of the organism; unfortunately it is not supplied by all strains of the organism, and varies from time to time with the same strain. 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 ANATOMICAL CHANGES 49] 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. The general result has been to show that the organism does not remain alive in natural conditions for long outside the animal body. 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 hemorrhage, and generally ending in a greater or less degree of necrotic softening if the patient lives long enough. The connective tissue around the glands is similarly affected. 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 areas of hemorrhage 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. 145). In sections of the glands in the earlier stages the bacilli are found to form dense masses in the lymph paths and sinuses (Fig. 149), 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 ; 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 usually attended by much hemorrhage ; the bronchial glands show inflammatory swelling. 492 PLAGUE 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; it is also extremely infective. In the septecemie form proper there is no primary bubo discoverable, though there is almost always slight general enlargement of lymphatic glands; Fic. 149.—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. here also the disease is of specially grave character. A bubonic case may, however, terminate with septicemia; in fact, all intermediate forms occur. An intestinal form with widespread 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, EXPERIMENTAL INOCULATION 493 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 5 c.c. of blood may be withdrawn from a vein and dis- tributed in flasks of bouillon (p. 70). 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, appointed by the Secretary of State for India in 1905, found that in some septiceemic 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 classi- tied 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 fra, 150.—Film preparation of spleen of rat malaise, or there may be after inoculation with the bacillus of { * plague, showing numerous bacilli, most of little more than slight which are somewhat plump. discomfort. Between such Stained with carbol-thionin-blue. x 1000. 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 cedema, which is followed by inflammatory swelling of the corresponding 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 to 3 days, guinea-pigs and rats in 2 to 5 days, and rabbits in 4 to 7 days. Post mortem the chief changes, in addition to the glandular 494 PLAGUE enlargement, are congestion of internal organs, sometimes with hemorrhages, and enlargement of the spleen; the bacilli are numerous in the lymphatic glands and usually in the spleen (Fig. 150), and also, though in somewhat less degree, throughout the blood. Infection can also be produced by smearing the material 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, eg., in sputum along with pneumococci. Rats and mice dan also be infected by feeding either with pure cultures 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. Paths and Mode of Infection.—Plague bacilli may enter 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; in the majority of these the lesions of the skin surface were of trifling nature, and there was no local reaction at the site of inoculation. It may now, however, be regarded as established that the ordinary mode of skin infection is by means of the bites of fleas containing the bacilli. It had previously been shown that when fleas were allowed to, feed on animals suffering from plague, plague bacilli might be found for some time afterwards in the stomach, and some observers, for example Simond, had succeeded in trans- mitting 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 PATHS AND MODE OF INFECTION 495 referred to above,! that the importance of this means of infection was established. By carefully planned experiments, the Com- mittee showed that the disease could be transmitted from af 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 pro- duced by fleas removed from plague rats and transferred 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, compara- tively 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 comparatively 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 sus- ceptible animals by means of fleas taken from rats in 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 com- menced. 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, 1 See Journal of Hygiene, vols, vi.—x. 496 PLAGUE 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, another was left without this protection. The monkey in the former cage remained unaffected, but the other monkey contracted 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 ex- periments the common rat-flea of India—Pulex cheopis (Rotlis- child)—-was used, but it has been shown that this flea also infests and bites the human subject. Recent observations show that not only is plague transferable by means of fleas, but that this is practically the only method obtaining in natural condi- tions, with the exception that rats may become infected by eating the carcases of other animals containing large numbers of plague bacilli. It is improbable from the experiments made that bubonic plague is transmitted by direct contact even when of a close nature ; in fact, it has been shown that plague-infected guinea-pigs may suckle their young without the latter acquiring the disease. The general results show that in the bubonic type direct infection by dust and other material through small lesions of the skin plays a comparatively trifling part in the spread of the disease, fleas apparently being in nearly all cases the carriers of infection. The later work of the Committee supplied information of the highest value with regard to the epidemiology of the disease 5 it showed, in short, that plague in its epidemic form is dependent on the epizootic among rats, and with regard to this some further facts may be given. Plague in Bombay occurs in two chief species of rats, the mus rattus, the black house-rat, and’ mus decumanus, the grey rat of the sewers. The former, owing to its presence in dwelling-houses, is chiefly responsible for the transmission of the disease to man; while the latter, on account of the large number of fleas which infest it, is of special importance in maintaining the disease from season to season. The year may be divided into two portions —an epizootic season, from December to May inclusive, and a non-epizootic, from June to November. During the latter period there are few cases of plague in rats on account of fleas PATHS AND MODE OF INFECTION 497 being scanty; especially is this so in the case of mus rattus. In fact, in certain villages where this species alone is present, the disease may actually die out at the end of the epizootic season, and accordingly when plague reappears in these places this is due to a fresh importation—a fact of great practical importance. A fresh epizootic first affects chiefly mus decumanus, and a little later spreads to mus rattus, while a little later still the disease attacks the human subject in the epidemic form; in each case fleas form the vehicle of transmission, and an interval of from ten to fourteen days intervenes between the outbreak of the epizootic and that of the epidemic. The proportion of cases of plague in mus decumanus is much higher than in mus rattus, for the reason mentioned. It has been further shown that the bacilli flourish in the stomach of the flea and are passed in a virulent condition in the feces, that a large proportion of the fleas removed from plague-infected rats contain plague bacilli, and that the fleas may remain infective for a considerable number of days, sometimes for a fortnight. The subsidence of plague when the mean temperature rises above a certain level (about 80° F.) is probably in part, at least, due to the fact that the bacilli disappear much more rapidly from the alimentary tract of fleas at the higher temperatures ; in accordance with this, experimental transmission of the disease to animals by means of fleas is more frequently successful at lower temperatures. C. J. Martin has shown that infection occurs by regurgitation of infected blood from the stomach of the flea during the act of biting, the proventriculus being some- times blocked by a mass of plague bacilli. The possibility of infection by contamination of the skin by the excrement of fleas containing the bacilli, ‘however, cannot be excluded. As regards the dying out of epidemics, some interesting facts have been brought forward by Liston. He and his co-workers have shown that rats taken from different towns vary greatly in their susceptibility to inoculation with plague bacilli, and that immunity is most marked in the rats from the towns which have suffered most severely from plague. This relative immunity appears to be due to the survival of the more resistant animals, and holds also with regard to their young. The diminution of plague amongst rats, and thus the subsidence of an epidemic, accordingly depends on the killing off of the more susceptible animals, 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 32 498 PLAGUE respiratory passages by inhalation. And accordingly a case of plague pneumonia is of great infectivity in producing other cases of plague pneumonia. Small epidemics of plague pneu- monia break out from time to time, but in 1911 an extensive epidemic occurred in Manchuria leading to 50,000 deaths in six months. In this epidemic, direct infection from patient to patient was clearly shown, and rats were not concerned in the spread, Plague pneumonia appears to occur first of all as a complication in a bubonic case, and there is no evidence that the bacilli differ in virulence in the two conditions. 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, hemorrhage in the mucous membrane of the stomach, areas of necrosis in the liver and at the site of in- oculation, may be present. The toxic substances are compara- tively 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, and, 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, pre- sently 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 practically no effect in the direction of conferring immunity. 1. Preventive Inoculation—Haffkine’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 goat’s 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° ©. 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. TOXINS, IMMUNITY, ETC. 499 It is administered by subcutaneous injection in the dose pre- scribed. 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 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 un- inoculated. 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 in- oculated 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, namely, 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 to 20 c.c. 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 hoth 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 heen recorded. The Indian Com- mission, however, came to the conclusion ‘‘that, on the whole, a certain amount of advantage accrued to the patients in cases both 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 500 MALTA FEVER 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 complicated by the natural tendency of the bacilli to cohere in clumps. For the last reason the macroscopic (sedimentation) method is to be preferred to the microscopic (p. 116). A suspension of plague bacilli is made by breaking up a young agar culture in °75 per cent. sodium chloride solution ; the larger fiocculi of growth are allowed to settle, and the fine, supernatant emulsion is employed in the usual way. According to the results of the Gerinan 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 maybe 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 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 microscopically, and cultures on agar or blood serum should be made by the successive stroke method. The cultural and morphological 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 subcu- taneous inoculation. In many cases a diagnosis may be made by micro- scopic examination alone, as in no known condition other than plague do bacilli with the morphological characters of the plague bacillus occur in large numbers in the lymphatic glands. The organism may be obtained in culture from the blood in a considerable proportion of cases by with- drawing 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 recommended. Here a positive diagnosis should not be attempted by microscopic examination alone, especially in a plague-free district, as bacilli morphologically resembling the plague organism may occur in the sputum in other conditions. Matra Frver. Synonyms—Mediterranean Fever: Rock Fever of Gibraltar : Neapolitan Fever, ete. This disease is of common occurrence along the shores of the Mediterranean and in its islands. Since its bacteriology has MICROCOCCUS MELITENSIS 501 been worked out, it has been found to »ccur also in India, China, South Africa, 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 recognised as a distinct affection, and was known under various names, its precise etiology was unknown till the publication of the researches of Surg.-General Bruce in 1887. From the spleen of patients dead of the disease he cultivated a characteristic organism, now known as the Micrococcus melitensis, and by means of inoculation experiments established its causal relationship to the disease. Wright and Semple applied the agglutination test to the diagnosis of the disease, and in 1904 the mode! of spread of the disease was fully studied by a Com- mission, whose work 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 1s very variable, the fever being of the con- tinued type with irregular remissions. In addition to the usual symptoms of pyrexia, there occur profuse perspiration, 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 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. 151). (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, how- ever, is of a contrary opinion, and has 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 502 MALTA FEVER 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 to 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 sometimes present in large numbers. It has also occasion- ally been obtained from the faces. Cultivation.—This can usually be effected by making stroke cultures on agar tubes from the spleen pulp and incubating 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 to 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 =f have a yellowish tint in Fic. 151.—Micrococcus melitensis, from a the centre, b luish-white two days’ culture on agar at 37° C. at the periphery. A Stained with fuchsin. x 1000. stroke culture shows a layer of growth of similar appearance with somewhat serrated margins. 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 medium. In bouillon there occurs a general turbidity 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; a reaction of +10 (p. 34) has been found very suitable. On potatoes no visible growth takes place even at the body temperature, RELATIONS TO THE DISEASE 503 though the organism multiplies to a certain extent. Outside the body the organism has considerable powers of vitality, as it has been found to survive ina 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- 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, as a rule, insusceptible to inoculation by the ordinary method, though some strains may produce pathogenic effects. Durham, by using the intracerebral method of inoculation, however, succeeded in raising the viru- lence, so that the organism is capable of producing in guinea- pigs on intraperitoneal injection illness with sometimes a fatal result many weeks afterwards. Eyre also, by increasing the’ virulence by intracerebral inoculation, was able to produce infection in various animals, especially on intravenous injection. Mode of Spread of the Disease.—The work of the 1904 Commission 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, it was found when the animals appeared healthy, and there was no physical or chemical change discoverable 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 monkeys 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 504 MALTA FEVER 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 ; cases now are relatively few. 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 good social 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 manner in which the disease spreads from goat to goat has not yet been satis- factorily determined. It has recently been found that the sheep may be the subject of infection and that the micrococcus may be excreted in its milk. It remains to be seen to what extent this obtains. The work of the Commission, so far as it went, excluded other modes of infection than the ingestion of infected milk as being of practical importance; if the disease 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. Al- though numerous patients suffering from the disease come to England, there is no known case of fresh infection arising under natural conditions. There is distinct evidence that the disease may be acquired by inoculation through small lesions in the skin, and this method is probably not infrequent amongst those who handle infected milk. It has been shown that the organism may remain alive in the bodies of mosquitoes for four or five days, and possibly these insects may occasionally be the means of carrying the disease; there is no evidence, however, that this takes place to any extent. Agglutinative Action of Serum.—tThe 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; here also dead cultures may be used. The reaction appears comparatively early, often about the fifth day, and may be present for a con- siderable time after recovery—sometimes for more than a year. METHODS OF DIAGNOSIS 505 Distinct agglutination with a 1: 30 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 : 5 may produce some agglutination, and this property is said to be destroyed at 55° C., whereas the specific agglutinin is not affected. Some observers accordingly recommend that, in applying the test, the serum ought to be first heated to 55° C. As regards relation to prognosis, the observa- tions of Birt and Lamb and of Bassett-Smith have given results analogous to those obtained in typhoid (p. 374). The Commission 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 the dose 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 ae. by the agglutinative test just described (for technique, vide p. 116). Cultures are most easily obtained from the spleen either during life or post mortem. Inoculate a number of agar tubes by successive strokes and incubate at 37° C. Film preparations should also be made from the spleen pulp and stained with carbol-thionin-blue or diluted carbol-fuchsin (1:10). Cultures may sometimes be obtained from the blood by the usual methods. Great care must be exercisel in working with cultures of the m. melitensis, as bacteriologists have become infected with the disease, apparently from such sources, in an unusually high proportion of instances as compared with other affections. CHAPTER XxX. DISEASES DUE TO SPIROCHATES—THE RELAPSING FEVERS, SYPHILIS, AND FRAMBCGSIA. THE diseases produced by spirochetes — spirilloses or spiro- chetoses—fall into two main groups, one represented by the human spirillar fevers and the corresponding affections of various animals, and the second having as its two chief members syphilis and yaws, though to the organisms of these diseases various spirochetes found in ulcerative and gangrenous condi- tions seem to be closely related. The members of the first group are essentially blood infections, and the organisms are in most, if not in all cases, transmitted by, blood-sucking ecto- parasites; in the second group thé organisms are primarily tissue-parasites, blood invasion when it occurs being a later phenomenon, and infection would appear to occur by direct contact. Infective jaundice, recently shown to be due to a spirochaete, occupies a somewhat intermediate position, as the organisms occur in the blood stream but tend to settle and flourish in certain organs. As regards general morphology, staining reactions, conditions of growth and culture, the various spirochetes present certain common characters, and, as already stated, it is still uncertain whether they are to be regarded as bacteria or as protozoa, though the balance of opinion is now distinctly in favour of the latter. Retapsing Fevers aNp AFRican Tick FEvER. At a comparatively early date, namely in 1873, when prac- tically nothing was known with regard to the production of disease by bacteria, a highly characteristic organism was dis- covered by Obermeier in the blood of patients suffering from relapsing fever. This organism is usually known asj the spirillum or spirochiete obermetert, or the spirillum of relapsing fever. He described its microscopical characters, and found 506 CHARACTERS OF THE SPIROCH ATE 507 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. His observations were fully confirmed, and his views as to its causal relationship to the disease have been established as correct. Within recent years relapsing fever has been carefully studied in different parts of the world, and the relationships of the organisms have been the subject of much investigation and discussion. This question will be referred to again below. It has also been shown that the so-called “ tick fever” prevalent in Africa is due to a spirochaete of closely similar character, and results of the highest importance have been established with regard to the part played by ticks in the transmission of the disease. As a matter of convenience, we shall give the chief facts regarding these diseases separately. It has also been shown that spirochetal diseases or “ spirilloses,” as they are called, are widespread amongst vertebrates; they have been described, for example, in geese by Sacharoff, in fowls by Marchoux and Salimbeni, in oxen and sheep by Theiler, and in bats by Nicolle 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. Characters of the Spirochete of Relapsing Fever.—The organisms as seen in the blood during the fever are delicate spiral filaments which have a length of from two to six times the diameter of a red blood corpuscle. They are, however, exceedingly thin, their thickness 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 organisms, and their extremities are finely pointed (Fig. 152). 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. 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. Fantham and Porter find that the sp. obermeieri and sp. duttoni multiply both by longitudinal and by transverse division, the former occurring especially during the onset of the fever. They stain with watery solutions of the basic aniline dyes, though somewhat faintly, and are best coloured by the Romanowsky 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 \ 508 RELAPSING FEVER into short segments. They lose the stain in Gram’s method. There is no evidence that they form spores. Novy found that the spirochzte of American relapsing fever remained alive and virulent in defibrinated rats’ blood for forty days. He also succeeded, by Levaditi’s method, in obtaining cultures in collodion sacs containing rats’ blood which were placed in the peritoneum of rats. Noguchi has succeeded in cultivating the spirochetes of the various relapsing fevers by the following method. A piece of sterile tissue, ¢.g., kidney of rabbit, is placed in a test-tube; a few drops of citrated blood from an infected animal are added and then 15 cc. of sterile ascitic or hydrocele fluid. The presence of a loose fibrin is helpful, and growth occurs under or- dinary anaerobic condi- ‘tions. He finds that all the species multiply by longitudinal and prob- ably also by transverse division. Relations to the Dis- ease,—In relapsing fever, after a period of incuba- tion there occurs a rapid rise of temperature which lasts for about five to Fic. aa a of relapsing fever in seven days. At the end of human blood. Film preparation. (After oe ge or Koch.) See also Plate IV., Fig. 18, this time a crisis occurs, x about 1000. 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 organisms begin to appear in the blood shortly before the onset of the pyrexia, and during the rise of temperature rapidly increase in number. They are very numerous during the fever, a large number being often present in every field of the microscope when the blood is examined at this stage. They begin to disappear shortly before the crisis: after the crisis they are entirely absent from the circulating blood. A similar relation between the presence of the organisms in the blood and the fever is found in the case of the relapses. Miinch in 1876 produced the disease in the RELATIONS OF SPIROCHATE TO THE DISEASE 509 human subject by injecting blood containing the spirochetes, 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 experiments were confirmed by Koch. In such experiments the blood taken from patients and containing the spirochztes was injected subcutaneously. In the disease thus produced there is an incubation period which usually Fic. 153,—Spirochete obermeieri in blood of infected mouse. x 1000. lasts about three days. At the end of that time the organisms rapidly appear in the blood, and shortly afterwards the tempera- ture 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.! White mice and rats are also susceptible to infection. In the former animals the disease is characterised by several relapses ; in the latter there is, however, no relapse. 1 Norris, Pappenheimer, and Flournoy, in their expeninents on monkeys with the organism of American relapsing fever, found that several relapses occurred. 510 RELAPSING FEVER Immunity.—Metchnikoff found that during the fever the spirochsetes were practically never taken up by the leucocytes in the circulating blood, but that. at the time of the crisis, cn dis- appearing from the blood, they accumulated in the spleen and were ingested in large numbers by the microphages or poly- morpho-nuclear 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 confirmed by Soudakewitch, who also found that when the disease was pro- duced in splenectomised monkeys (cercocebus fuliginosus) the spirochetes 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. Later observations, however, indicate 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 (macacus 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 he 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 spirochetes to an end, clumped them, and caused their disintegration ; and further, that in one case when the spirochetes and the immune serum were injected 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 spirochetes. Observations by Sawtschenko and Milkich, Novy and Knapp, and Rabinowitsch, also show that in the course of infection there -are developed anti-substances of the nature of immune-bodies, with protective properties, and agglutinins. Novy and Knapp pro- duced a “ hyper-immunity ” in rats by repeated injections of blood containing the spirochetes, and found that the serum of such animals had a markedly curative effect, and could cut short the disease in rats, mice, and monkeys. The course of events in the human 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 organisms have been destroyed, some still surviving in internal organs or in tissues where they escape the action of the serum or phagocytosis. With the disappearance of the immunity, the organisms appear in the blood, the relapse being, however, of shorter duration and VARIETIES 511 less severe than the first attack. This is repeated till the im- munity lasts long enough to allow all the organisms to be killed. It is possible, however, that the survival of résistant spirochetes, or “mutants,” may play a part in the production of the relapses, Varieties.—As already stated, relapsing fever has been studied in different parts of the world, and, apart from the African tick fever, European, Asiatic, and American types have been dis- tinguished. Differences have been made out with regard to clinical features, pathogenic effects, and immunity reactions. It has been shown, for example, by the work of Novy, Strong, and Mackie, that the American spirochete is probably a distinct species, a8 animals immunised against it are still susceptible to infection by the European and Asiatic organisms, and vice versa. The relationship between the two latter is certainly closer, and no distinct immunity differences have been established. Re- lapsing fever in Asia is evidently a much more severe disease than in Europe; Mackie gives the mortality in Bombay at the comparatively high figure of 38 per cent. But differences in this respect, as well as in pathogenic effects, may simply depend on variations in virulence. At present no definite statement can be made on this point. Sergent aud Foley have described a type of relapsing fever occurring in Algiers, which they consider to be different from the recognised forms, and have given the name sp. berbera to the organism concerned; and Balfour has observed cases in Khartoum which he thinks are probably of the same nature. The fact that tick fever and other spirilloses are conveyed by the bites of insects makes it extremely probable that relapsing fever is transmitted in this way. At first the bed-bug was believed to be the vehicle of transmission, and the experiments of Karlinski and of Tictin, which showed that the spirochetes might remain alive and virulent in the body of this insect for some time after it had sucked the blood of a patient, lent some support to this view. Attempts to transmit the disease by means of the bites of bugs were, however, generally unsuccessful ; Mackie produced the disease in only one out of six monkeys used for this purpose, though large numbers of bugs, which had bitten relapsing fever patients, were used. On investigating an epidemic of the disease, however, he obtained a considerable amount of evidence on epidemiological grounds that the disease was carried by the body louse. He also found that the spiro- chetes in the blood which had been ingested underwent great multiplication about three days afterwards, and formed large tangled masses in the stomach contents, The view that the 512 AFRICAN TICK FEVER louse is the agent of transmission of the human disease is strongly supported by the experiments of Manteufel, who was able to transmit infection from rat to rat in nearly 60 per cent, of the experiments made, whereas he obtained only negative - results by means of bugs. Fehrmann considers that the clothes louse may carry the infection. Further observations are still necessary. African Tick Fever. The disease long known by this name as prevalent in Africa Fic. 154,—Film of human blood containing spirochexte of tick fever. x 1000.1 has also been shown to be caused by a spirochate—sp. duttoni. 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 spirochaete. It is, however, chiefly owing to the work of Dutton and Todd in the Congo Free State, on the one hand, and of Koch in 1 We are indebted to Col. Sir William Leishman, R.A.M.C., for the prepara- tions from which Figs. 153-55 were taken. AFRICAN TICK FEVER 513 German East Africa, on the other, that our knowledge of the etiology of the disease has been obtained. 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 seldom attended with a fatal result unless in patients debilitated by other causes. The organisms in the blood are considerably fewer than in the case|of European Fie. 155.—Spirillum of human tick fever (spirillum duttoni) in blood of infected mouse. 1000. 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 15-35 ». Dutton and Todd showed that it was possible to transmit the disease to certain monkeys (cercopithect) 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 33 514 _ AFRICAN TICK FEVER 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 spirochzetes were not simply carried mechanically by the ticks, but probably underwent some cycle of development in the tissues of the latter. Leishman has since shown that the ticks of the second generation may also be infectious. The species of tick concerned is the ornithodorus moubata. These results were confirmed and extended by Koch. He found that after the ticks had heen allowed to suck the blood containing the organisms, these could be found for a day or two in the stomach of the insect. 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. He also traced the presence of the spirochetes in the eggs laid by the infected ticks, and in the young embryos hatched from them. On the other hand, Leish- man has failed to find any evidence of spirochetes in the tissues of ticks later than ten days after ingestion of blood containing them, or in the ova laid by the ticks, or in the young ticks when hatched, though these were proved by experiment to be infective. After ingestion of the blood by the ticks, he found that morpho- logical changes occurred in the spirochetes, resulting in the formation of minute chromatin granules which traverse the walls of the intestine and are taken up by the cells of the Malpighian tubules; they also penetrate the ovaries and may be found in large numbers within the ova. Similar granules are to be seen in the Malpighian tubules of the embryo ticks, where they are also found in the subsequent stages of their life. He has proved that infection of animals may be produced by inocu- lation with crushed material containing the granules but no spirochetes. He accordingly considers that the granules in question represent a phase in the life-history of the parasite, and that infection occurs by inoculation of the skin with the chrontatin granules voided in the Malpighian secretion and not by unaltered spirochetes from the salivary glands. A similar view is taken by Hindle, who has found that when infected ticks, in which the spirochetes have disappeared, are heated to a temperature of 35° C., the spirochetes reappear in the organs and ccelomic fluid. It is also interesting to note that Balfour has found similar granules in ticks (argas persicus) infected with spirochete gallinarum, and he has also observed the formation of granules from spirochetes in the blood of Sudanese fowls treated with salvarsan, SYPHILIS 515 Koch also made extensive observations on the ticks in Ger- man East Africa, and found that of over six hundred examined along the main caravan routes 11 per cent. contained spirochetes, and in some localities almost half of the ticks were infected. In 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 spirochetes although the inhabitants do not suffer from tick fever, a circumstance which is probably due to their having acquired immunity against the disease. It is now generally believed that the sp. duttoni is a species distinct from, though closely allied to, the organisms of the relapsing fevers described above. We have mentioned some differences in the clinical characters of the diseases, and there are also differences in the pathogenic effects of the organisms on inoculation. The sp. duttoni, for example, produces a much more severe disease in monkeys, and is pathogenic :to more species of the laboratory animals than the sp. obermeieri. The most important differences are, however, brought out by immunity reactions. It was shown by Brein! that the immunity produced by the sp. obermeieri did not protect against the sp. duttoni, and that the converse also held good; and it has since been established that a similar difference obtains between the gp. duttoni and the organisms of the Asiatic and American varieties of relapsing fever. Corresponding results are obtained on testing the various serum reactions in vitro. As already stated, Noguchi has cultivated the sp. duttoni outside the body, and from a study of its characters agrees that it is a distinct species. SYPHILIS. The cause of syphilis is the organism discovered by Schaudinn and Hoffmann in 1905 and called by them the spirochete pallida, now often known as the treponema pallidum. They described its characters and its occurrence in syphilitic lesions, and their observations have been fully confirmed. Its recognition, at first somewhat difficult, has been rendered comparatively easy by the introduction of new methods. Spirochete pallida.—This is a minute spiral-shaped organism, showing usually from eight to twelve curves, though longer forms are met with ; the curves are small (each measuring a little over 1 »), comparatively sharp, and regular (Figs. 156, 157, 158). It may be said to measure 4 to 14 p in length, while it is extremely 516 . SYPHILIS thin, its thickness being only ‘25 ». 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, and movements of flexion of the whole body; there is little actual locomotion, and a specimen will often remain in the same field for along time. The ends are pointed and tapering, and, as was first shown by Schaudinn, a flagellum is present at each end. Both in fresh specimens and in dried films (Figs. 156-158) the regu- larity of the spirals is well maintained, though in the latter there is sometimes distortion or drawing out of a spiral. The use of dark-ground illumination (p. 90) is of great service in searching for the organism. Z 4 ™— ‘ Figs. 156 and 157.—Film preparations from juice of hard chancre showing spirochete pallida. Giemsa’s stain. 1000. (From pre- parations by Dr. A. MacLennan.) In ulcerated syphilitic lesions, and also in non-syphilitic lesions of the genitals, other organisms are, of course, present, and not infrequently various other spirochetes. Of these several species have been described, e.g., sp. refringens, sp. balanitidis, sp. gracilis, but there are others which have not yet been differentiated. The first mentioned is a comparatively coarse organism, more highly refractile, while its curves vary during the movements ; in film preparations the curves appear irregular or are lost to a large extent. Some of the other species are of smaller size, but they differ from the sp. pallida in their appear- ance and in the character of their movements. We believe that in the case of genital lesions there is little difficulty to the experienced observer in recognising the sp. pallida, but any difficulty will be removed if the superficial organisms are re- moved and the lymph is taken from the lesion for examination. SPIROCHATE PALLIDA 517 These organisms generally stain deeply with Giemsa’s stain and are of a bluish tint ; the sp. pallida is coloured a faint pink. In lesions of the mouth and probably in some others, e.g., foetid ulcerations, etc., there occur, however, spirochetes which are indistinguishable morphologically from the sp. pallida, ¢.g., the sp. Mmicrodentia and sp. mucosa, found in carious teeth and pyorrheea alveolaris. Both of these organisms have been culti- vated by Noguchi; they have been proved to be devoid of pathogenic properties, and the cultures,-moreover, have a foul odour. The sp. pertenuis of yaws (p. 524) has also the same Fic. 158.—Film preparation from juice of hard chancre showing spirochete pallida. Giemsa’s stain. 2000. (From a preparation by Dr. Haswell Wilson.) microscopical appearances. In the microscopical’ diagnosis of the organism of syphilis, just as in the case of the tubercle bacillus (p. 286), an all-important point is, accordingly, the source of the organism; and we may say that if we except the case of yaws, which does not occur in this country, an organism with the characters described above can be identified with certainty as the sp. pallida provided that it is obtained from the substance of the tissue lesion. The spirochete pallida by the Giemsa stain is coloured somewhat faintly, and of reddish tint, whilst the regular spiral twistings are preserved ; the sp. refringens shows flatter, wave-like bends (Fig. 160), and, like other organisms, is stained of a bluish tint. 518 SYPHILIS Noguchi, on studying different strains of the sp. pallida in cultures, found that they varied in thickness, and he was able to ‘distinguish thick, thin, and intermediate types. He also found that they differed in their pathogenic action, the thick forms on injection into the testicle of a rabbit causing nodular lesions of cartilaginous hardness, the thin forms producing a diffuse in- durative lesion. These observations are suggestive as possibly throwing some light on the variations in the effects in the human subject. The number of publications with regard to the distribution of Fic. 159.—Section of spleen from a case of congenital syphilis, shoring several examples of spirochete pallida. Levaditi’s method. x ce the spirochzete pallida is now 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 eruptions, in condylomata and mucous patches—in fact, one may say generally, in all the primary and secondary lesions. Schaudinn in his last series of cases, numbering over seventy, found it in all, and on a few occasions detected it in the blood during life in secondary syphilis. It has also been obtained from the spleen during life. In the congenital form of the disease the organism may be present in SPIROCHATE PALLIDA 519 large numbers (Plate IL., Fig. 6), as was first shown by Buschke and Fischer, and by Levaditi. In the pemphigoid bulle, in the blood, in the internal 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. 159). It is also preserit in syphilitic placente, though not usually in large numbers. It has been generally supposed that tertiary syphilitic lesions are non-infective, and the results of the earlier observa- tions on the spirochete pallida were apparently in accordance with this view, as they gave negative results. More prolonged search has, however, shown that the organism may occur in tertiary lesions also. It has been found to be present in the peripheral parts of gummata, especially at an early stage of their formation ; and the observations of Schmorl, Benda, J. H. Wright, and others show that it is often y i 3 $2> 7 Ab? fi a) ‘ cate to be found in syphilitic disease Pes ae Shao of arteries, sometimes occurring in considerable numbers in the @ ae | thickened patches in the aorta. | ad ga That the spirochete may persist bs $ ape) in the body for a very long time me ; / after infection, has been abun- : y dantly shown by different ob- sas id servers; in one case, for example, SR ‘ 28 Ms De ee demonstrated Fic. 160.—Spirochete refringens sixteen years after the primary iy film preparation from a case lesion. It can readily be demon- of balanitis. 1000. strated in sections of syphilitic lesions by the method described on page 109. Recently Noguchi and Moore have announced the discovery of the spirochete in the brain in general paralysis of the insane in a certain proportion of cases. The organism was seen in all the layers of the cerebral cortex, with the exception of the outer- mogt, and the cases in which it was found had run a relatively rapid course. Infection has also been transmitted to the rabbit (wide infra) by inoculation with the brain tissue of general paralytics. In preparations from the organs in congenital syphilis large numbers of spirochetes, 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. They also abound sometimes on mucous surfaces, e.g., of the bladder and intestine in cases of congenital syphilis. ‘The enormous numbers of the Ne 520 SYPHILIS organism which may be present in a well-preserved condition in macerated foetuses render it probable that the organism may multiply in the dead tissues under anaerobic conditions. Cultivation. — Although Miihlens and Hoffmann had previously obtained pure cultures of an organism morphologically identical with the spirochete pallida, it is chiefly to Noguchi that we owe the methods of cultivation. We shall accordingly state his results, which in certain respects differ from those of the other two observers. In the first instance his cultures were made from syphilitic lesions in the rabbit, but later directly from the lesions of the human disease. As a culture medium he used a mixture of two parts of 2 per cent. agar and one part of ascitic or hydrocele fluid, to which a small portion of sterile rabbit’s kidney or other organ was added, the medium being placed in deep tubes and covered with a thick layer of paraffin oil. The medium was inoculated through the oil, the maintenance of strict anaerobiosis being essential. When contaminating bacteria were present these formed a thick growth along the line of inoculation, whilst the spirochetes grew as a diffuse haze into the surround- ing medium. By making sub-cultures from parts apparently free from bacterial growth he succeeded in obtaining the organism in the pure condition. At first the organisms were small, but after several days they had the usual length of the spirochete pallida and all its characteristics. An important point is that he found clear evidence that the organism multiplies by longitudinal division. On inoculating monkeys (macacus and cercopithecus) by scarification, in some cases indurated syphilitic papules developed and the blood of the animals gave a positive Wassermann reaction. The etiological relation of the organ- ism has thus been completely established. Transmission of the Disease to Animals.—Although various experiments had previously been made from time to time 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 carried on a large series of observations, and showed that the disease can be transmitted to various species of monkey. Of those 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. The number of experiments on these animals is now very great, and the general result is that the disease has been transmitted by material from all the kinds of syphilitic lesions in which spiro- chetes have been demonstrated, including tertiary: lesions and TRANSMISSION OF THE DISEASE TO ANIMALS 521 even the blood in secondary syphilis. Inoculation is usually made by scarification on the eyebrows or genitals; the sub- cutaneous and other methods of inoculation, with the exception of intratesticular, give negative results. 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 are marked enlargement and induration of the corresponding lymphatic glands. The primary lesion appears on an average about thirty days after inoculation, and secondary symptoms develop in rather more than half of the cases after a further period of rather longer duration. These are of the nature of squamous papules on the skin, mucous patches in the mouth, and sometimes palmar psoriasis. As a rule, the secondary manifestations are of a somewhat mild degree, and in no instance has any tertiary lesion been observed, though this may be due to the animals not having lived long enough. By re-inoculation from the lesions, the disease may be transferred to other animals. The disease may also be produced in baboons and macaques, but these animals are less susceptible, and secondary manifestations do not appear. The severity of the affection amongst apes would in fact appear to be in pro- portion to the nearness of the relationship of the animal to the human subject. The blood of the infected animals comes to give a positive Wassermann reaction. As shown first by Hansell, and afterwards by Bertarelli, the eye of the rabbit is susceptible to inoculation from syphilitic lesions.. The material used is introduced in a finely divided state either into the tissue of the cornea or into the anterior chamber, and syphilitic keratitis or iritis, or both, may result, there being a period of’ incubation of at least two weeks. Levaditi and Yamanouchi have studied the stages in detail, and find that the spirochzetes remain in the inoculated material un- changed for a time ; then organisation occurs and the spirochetes multiply, and later still there is a more rapid multiplication and invasion by them of the tissues of the eye. The period of incu- bation is thus not due to the organism passing through some cycle of development, but simply to its requiring certain con- ditions for multiplying, which are not supplied for some time. The testis of this animal is also a convenient site of inoculation, a syphilitic orchitis being set up, and the disease has been maintained by this method through several generations of animals. The intratesticular method has proved of great value in testing the infectivity of suspected material, and by this means it has been shown that spirochetes from gummata are 522 SYPHILIS not attenuated in virulence. Uhlenhuth and Mulzer produced generalised syphilitic lesions in young rabbits by intracardiac inoculation with syphilitic material. They have also found that the organism can pass through the placenta of the rabbit and infect the foetus. It has long been held that a person suffering from syphilitic disease is not susceptible to a fresh infection, and this has been shown by experimental methods to hold in the artificially pro- duced disease in the ape, the possibility of re-inoculation thus indicating freedom from infection. A considerable number of cases in the human subject have been observed where after treatment with salvarsan a second attack of the disease has been contracted, the inference being that the first attack had been completely cured. In the case of the rabbit, however, it has been found possible to produce a fresh syphilitic lesion when another was still in existence on the cornea. Apparently in this animal the effects of this local lesion do not become general in the same way as in man. The experimental production of the disease has supplied us with some further facts regarding the nature of the virus. It has been shown repeatedly that the passage of fluid contain- ing the virus through a Berkefeld filter deprives it completely of its infectivity; in other words, it 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 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. The presence of the spirochete does not lead to the formation of anti-substances to any marked extent. 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. Luetin.—Noguchi has prepared an extract from pure cultures of the spirochete pallida, which he calls dwetin, and he finds that this gives a characteristic cutaneous reaction in syphilitics. , This reaction is analogous to the tuberculin reaction in tuberculosis, and, like it, appears to depend on a condition of super-sensitive- ness or allergy (p. 595). In a normal individual the intradermic inoculation of luetin produces a local erythema which may SERUM DIAGNOSIS 523 sometimes {go on to the formation of a slight papule on the second day; thereafter the reaction recedes. In the case of syphilitics Noguchi distinguishes three types of positive re- action—(a) papular form, in which a large indurated, reddish papule, 5-10 mm. in diameter, forms and increases for three or four days, the colour becoming dark bluish red; (4) pustwlar form, in which the inflammatory change is more severe, the papule changing into a vesicle and then into a pustule; and (c) torprd form, in which, after a latent period of about ten days, reaction appears and goes on to the formation of a small pustule. Noguchi’s claims as to the clinical value of the reaction are supported by other observers. The results obtained so far show that a positive result is yot when the disease is ‘*** latent and often when the Wassermann reaction is negative. It is often absent in secondary syphilis, but may appear after anti- syphilitic treatment has been carried on for some time. Further elucidation of the nature of the reaction is still required. Serum Diagnosis—Wassermann Reaction.—The method of applying this test has already been given (p. 127); we have now to consider the results of its application. On com- paring the results obtained it will not be an overestimate to say that a positive result may be obtained in at least 90 per cent. of cases where there is evidence of active general infection. The reaction génerally appears first on the fifteenth to thirtieth day after appearance of the sore, and then gradually becomes more marked ; during the period of secondary manifestations it is practically always present; in the tertiary stage with active manifestations a positive result is only a little less frequent. As the disease becomes inactive or is cured the reaction may disappear, but it is to be noted that disappearance of the reaction after being present does not necessarily imply cure of the disease. It may only have become latent, and on its becoming once more active the reaction may reappear ; in fact, its presence would appear to be definitely related to the activity of the syphilitic lesions. A positive reaction is practically always present in general paralysis and in the large majority of cases of tabes, and may be given by the cerebro-spinal fluid as well as by the blood serum in these diseases. As regards other diseases, a positive reaction has been recorded as occurring in leprosy (p. 308) and sleeping-sickness and also in yaws, and occasionally in malaria ; butfapart from these diseases it is practically never met with. At present little can be said in explanation of the Wassermann reaction, It seems to depend on the interaction of lipoidal substances in the extract with proteins in the serum, which 524 FRAMBCSIA OR YAWS are apparently contained in the globulin fraction; but we know nothing as to why this peculiar modification of the serum, should be present in syphilis. It is now generally: accepted that it does not depend on the presence of an anti- substance (immune-body), which in association with the antigen. — (the spirochete) fixes complement. Methods of Examination.—As already said, in the examination of an ulcerated chancre or other lesion it is advisable to get rid of the surface organisms. The surface should be cleaned with saline and dried. A piece of cotton-wool soaked in absolute alcohol or spirit is then applied for about a minute; the alcohol is then washed off with saline, and the surface is again dried. After a short time there is usually a free flow of watery lymph, which is practically free from other organisms, and often contains the spirochaete in large numbers; a small drop of this is placed on a slide, a cover-glass is applied, and the specimen is examined by dark-ground illumination. It is advisable to put a thin ring of vaseline on the slide to support the cover-glass. Dried films also may be made and treated by any of the methods above described (p. 110), of which Fontana’s is to be recommended. Others prefer to scarify the margin of the sore and examine the lymph which exudes, the flow of which may be aided by squeezing, or a small incision may be made with a very sharp knife, and then after bleeding has completely stopped to take the small drop of serum which gathers at the site. In- all cases admixture of blood is to be avoided, as it interferes with the examination by the dark-ground method. In the case of a lymphatic gland or non- ulcerated lesion it is best to puncture with a hypodermic needle, the point of which should be moved about in the tissue. After it is withdrawn a little saline may be placed in the syringe and pressed through the needle, the first small drop which passes, and which washes out the contents, being taken for examination ; here also dark-ground illumination gives the best results. For methods of cultivation, vide p. 520. FRAMB@SIA OR YAWS. Frambeesia is a disease of the tropics, occurring in the west coast of Africa, Ceylon, the West Indies, and other parts. It is characterised by a peculiar cutaneous eruption, and it is markedly contagious. Its resemblance in many respects to syphilis has been noted, and the relation of the two diseases has been the subject of much controversy. It is accordingly a matter of great interest that an organism of closely similar characters to the spirocheete pallida has been found in the lesions of frambeesia. This organism was discovered by Castellani, who gave to it the name spirocheete pertenwis or pallidula. Morpho- logically, it is practically identical with the spirochete pallida ; when ulceration has occurred other spirochetes of less regular form may be present as contaminations. In the skin lesions it has been shown by Levaditi’s method to be present in con- SPIROCH.ATAL OR INFECTIVE JAUNDICE 525 siderable numbers, especially in the epidermis and also amongst the leucocytic infiltration, which comprises more polymorpho- nuclear leucocytes than are seen in the case of syphilis. Castellani showed that the disease could be transferred to monkeys (semno- pithecus and macacus being used for this purpose), and that the organism could be demonstrated in the unbroken skin lesions. The lesions are as a rule confined to the site of inoculation, but the infection is general, as is shown by the presence of spirochetes in the lymphatic glands and the spleen. These results with regard to the presence of spirochete pertenuis in the lesions and the inoculation of apes have been confirmed by other workers, and the etiological relationship of the organism to the disease may now be regarded as established. Nichols has shown that a frambeesia lesion can be produced in the testicle of the rabbit of similar character to the syphilitic lesion, though the period of incubation is shorter. He finds that the best means of dis- tinguishing the two diseases is afforded by inoculating the skin of the monkey. In the case of syyhilis the resulting lesion is flat, dry, and very scaly ; in the case of frambeesia it is elevated, slightly scaly, and very cedematous; here also the period of incubation is shorter in the case of frambeesia. The immunity reactions in monkeys infected with syphilis and frambeesia, as experimentally studied by Castellani and by Neisser, Baermann, and Halberstddter, go to show that the two diseases are distinct. Nichols obtained a corresponding result in the case of the rabbit, as he found that this animal, when cured of a syphilitic lesion of the testicle by means of salvarsan, was susceptible to frambeesia but not to syphilis. On the other hand, Levaditi and Nattan- Larrier found that, although monkeys infected with syphilis were refractory to frambeesia (/’r. pian), monkeys infected with frambeesia were susceptible to syphilis: they therefore concluded that frambeesia is a modified or mild form of syphilis. We may add that patients suffering from frambeesia generally give a positive Wassermann reaction ; they are also very amenable to treatment with salvarsan (Alston and others). The exact relationship of the two diseases cannot be yet accurately defined, but they are probably distinct, though undoubtedly closely related. SPIROCHETAL OR INFECTIVE JAUNDICE. This affection, often known as Weil’s disease, was proved in 1915 by Inada and other Japanese workers to be due to a spirochzte, to which ‘they gave the name spirochete tctero- hemorrhagie, and already the pathology of the condition 526 SPIROCHATAL OR INFECTIVE JAUNDICE has been worked out fairly fully. The disease is characterised by irregular pyrexia, often severe jaundice, which usually appears about the fourth day of illness and may become very marked, a tendency to hemorrhage from mucous surfaces and into the tissues, hemorrhagic herpes, etc., albuminuria, and various other symptoms. Its occurrence in small epidemics had been previously noted, members of the same family or groups of soldiers in barracks being not infre- quently affected; in Japan it was found to occur amongst workers in the same part of a mine. It has occurred during the war amongst the troops in France, and the results of the Fic. 161.—Specimens of spirochete ictero-hamorrhagie, as seen in sections of a suprarenal of an infected guinea-pig. Stained by Levaditi’s method. (From a preparation by Major J. W. M‘Nee, R.A.M.C.) x 1000. Japanese workers have been confirmed by bacteriologists in both the British and French armies. It has also been found on the Italian front and amongst the German troops. The infection has thus had a wide distribution during the war, but the mortality has been much lower than that met with in Japan. Morphology of the Spirochete.—The organism in the blood and tissues measures 6-9 yw in length, but both shorter and longer forms occur, and about °25 yw in thickness; that is, it is a slender organism of about the thickness of the sp. pallida. In cultures it may grow into much longer threads. It is somewhat thicker in the middle and tapers towards the ends, which may be pointed, but there are no terminal flagella; its substance may appear somewhat granular. It presents usually several CULTIVATION 527 spirals, but these are somewhat irregular and often poorly marked (Fig. 161); not infrequently the ends form small hooks. It is motile, the movements being rotatory, undulatory, and also to and fro. It can be studied by all the microscopic methods already described in the case of the sp. pallida (p. 524). Cultivation.—The organism was first successfully cultivated in Noguchi’s medium for sp. pallida, in which the initial growth survives for three to six weeks. The medium remains clear and does not yield any odour. Later it was grown on solid media, blood agar and blood gelatine, the latter being the more suitable. The limits of growth are wide, namely, 15-37° C., the optimum temperature being 22-25°C. Noguchi recommends the follow- ing media as suitable ; he finds that the addition of sterile tissue does not improve the growth :— (a) Rabbit serum, two parts; Ringer’s solution or 0°9 per cent. sodium chloride solution, six parts ; citrated rabbit plasma, one part. (b) The same with the addition of one to two parts of neutral or slightly alkaline agar (2 per cent.), which should be liquefied and added when quite hot (60-65° C.) in order to get a uniform mixture of the agar. Both media are covered with a layer of sterile liquid paraffin, and inocu- lation is made through the paraffin. In these media growth produces slight turbidity. Relations to the Disease.—The organism occurs both in the blood and in the organs. In the former it is found in the first four or five days of the disease ; thereafter it gradually disap- pears, and in the second week, when jaundice is most marked, it cannot be detected. The best method of demonstrating its presence is to draw off some blood, say, 3 c.c., and inject it into the peritoneal cavity of the guinea-pig, in which animal it pro- duces an infection and can easily be found (wide infra). It is rarely present in the blood in the human subject in numbers sufficient to allow its detection by microscopic examination. Of the internal organs the liver contains the organisms in largest quantities ; they may be also found in the suprarenals, and, especially at a later stage, in the kidneys. In all the organs in the human subject the spirochetes are scanty, they are often somewhat irregular and degenerated in appearance, and often in the interior of the special cells. These facts have been explained as being the result of the formation of anti- substances, which drive them from the blood and interstitial tissues. Their late occurrence and persistence for some time in the kidneys are comparable with what occurs in the natural infection of the rat without the occurrence of disease symptoms (vide infra). The spirochete is also excreted in the urine. -528 SPIROCHATAL OR INFECTIVE JAUNDICE This does not occur in the earliest stage of the disease, but from about the tenth day onwards positive results are obtained in in- creasing numbers, till about the twentieth day it may be found in practically all cases. Thereafter it gradually disappears, and is rarely found after the fortieth day. The best method is to examine by dark-ground illumination the deposit thrown down from the urine by a high-speed centrifuge. The gradual development of anti-substances in the blood has been shown to occur during the disease. These appear towards the end of the first week, and seem to be related to the disappearance of the organism from the blood; they become specially marked during the second week. Their presence can be demonstrated by injecting some of the patient’s serum along with the spirochetes into a guinea-pig, death being thus prevented, or at least the onset of the illness being postponed. Destruction of the organisms under the influence of the anti- serum may be observed in the peritoneal cavity of the animal, that is, spirochztolysis occurs, corresponding to Pfeiffer’s pheno- menon in the case of bacteria, Experimental Inoculation.—The injection of blood or of emulsions of organs containing the spirochetes into the peri- toneal cavity of a guinea-pig leads to an infection which is usually fatal in about seven to twelve days; the same holds with regard to the effect of pure cultures. The symptoms are conjunctival congestion, anemia, jaundice, hemorrhagic dia- thesis, and albuminuria. There is pyrexia, which towards the end is succeeded by subnormal temperature ; the jaundice occurs somewhat late, usually about twenty-four hours before death. Post mortem, there are hemorrhages in the lungs, intestinal walls, and retro-peritoneal tissue ; acute parenchymatous nephritis is present, and the spleen is large and congested. The hemor- rhages in the lungs occur as small and large spots, described as being “like the wing of a mottled butterfly.” The spirochztes are present in the blood and organs, and in the latter are chiefly interstitial in position, few being actually within cells. In this respect there is a difference from what obtains in the human disease. They are most abundant in the liver, where they may be arranged like a garland round the liver cells. The adrenals and the kidneys contain considerable numbers, but they are scanty in the spleen, bone-marrow, and lymphatic glands. The Japanese workers believe that, in the human disease, infection occurs chiefly through the alimentary tract, and they were able to produce the disease in the guinea-pig by feeding with material containing the organism or by introducing some of it into the RAT-BITE FEVER 529 rectum. They also showed that infection could take place through the apparently intact skin, and found that this occurred with comparative rapidity, as the application of an antiseptic five minutes after the infective material did not prevent infection. A highly important point with regard to the epidemiology of the disease is the common presence of the spirochzte in both house and field rats without any apparent disturbance of health. This has been established now with regard to rats in Japan, in the trenches at the front, and also in America; and it has been found that the proportion of infected rats is a high one, some- times over 30 per cent. In these animals the organisms are practically confined to the kidneys, and we have here a resem- blance to what is found in the human infection, at a later stage when immune-substances are present in the blood. The spiro- cheetes are passed in large number in the urine of the infected animals, and in this way contamination of the soil and various articles is brought about. The spirochetes obtained from rats are found to vary considerably in virulence. Rat-BiTE: FEVER. More than one form of infection may be produced by the bite of rats. In the form which is commonest in Japan, Futaki and his associates in 1915 found a spirochete in the skin lesion and in the lymph-glands. Their results have been confirmed by other Japanese workers, and the organism, now called the spirochete morsus muriwm, has been established as the cause of the infection. The clinical symptoms are inflammation of the bitten parts, paroxysms of fever of the relapsing type, swelling of the lymph glands, and eruption of the skin, all oc- curring after an incubation period usually of ten to twenty-two days, or longer. The spirochete, which also occurs in the blood as well as locally, is somewhat short, measuring 2-5 u, and has a few steep and fairly regular curves of 1 » each; it is, however, distinctly thicker than the sp. pallida. It has a distinct and fairly long flagellum at each end, and it is actively motile, the movements being very rapid, like those of a vibrio, and dis- tinguishing it from other pathogenic spirochetes. It has been cultivated outside the body and has been proved to be virulent to mice, rats, and other animals. It has been found in rats in the natural condition, in 3 per cent. of house rats, and the disease has been produced in the - guinea-pig by allowing a rat infected with the spirochete to bite it. The ' infection has been shown to be very amenable to treatment by salvarsan, and the blood of a convalescent patient has been found to possess protective properties. It is of interest to note, however, that Schottmiiller obtained a strepto- thrix by culture from the blood in a case of rat-bite fever, and Blake has found the same organism both in the blood and in the cardiac vegetations in another case. Further, Douglas, Colebrook, and Fleming have recently published a case in which the infection was due to a streptococcus. It is thus clear that definition in the nomenclature is required. 34 CHAPTER XXI. PATHOGENIC FUNGI. In pathological bacteriology, besides the bacteria themselves, higher organisms belonging to the group of fungi not in: frequently claim attention. On the one hand, cultures may be contaminated with the spores of the omnipresent terrestrial forms growing in all decaying material, and on the other hand, fungi of the same type are known to be the causal agents in certain diseases. Before considering the latter, with which we are more intimately concerned, we shall first give a short account of the group of fungi as a whole and of some of the common saprophytic forms. For this we are indebted to the kindness of Professor Percy Groom. The overwhelming majority of fungi consist of tubular branched fila- ments, termed hyphee, each of which has a thin continuous wall within which are the protoplasmic and other contents. The whole body of the fungus thus composed of hyphe is termed the mycelium. This may be loose and web-like in texture, as in the case of common moulds, or may assume the form of a compact skin or mass which is produced by the copious branching and close interweaving of the hyphe, as in ordinary toadstools. P In the Phycomycetes, a lowly organised group of fungi, the hyphe are typically continuous tubes devoid of any cross septa, excepting where re- productive organs or cells occur ; whereas in the more highly organised fungi, Mycomycetes, the hyphe are segmented by transverse walls. Inasmuch as fungi have descended from alge, which are mainly aquatic, those fungi that are most alga-like betray in their life-history signs of the aquatic mode of existence. Thus in a number of Phycomycetes the ends of certain hyphe become shut off by a transverse wall. The terminal chamber becomes swollen and its abundant protoplasm divides into a number of cells, which, by rupture of the outer wall, escape as naked ciliated swarm-spores. Each of these swims about in water (raindrops and so forth), eventually clothes itself with a thin cell-wall, and, emitting a hypha which grows and branches, develops into « new plant. The terminal organ within which these aseaual spores arise is termed a sporangium. In other types of Phycomycetes, for instance Mucor mucedo (Fig. 162), the spores arising in the same manner inside a sporangium acquire a cell-wall betgee rupture of the sporangium wall: 5 “ PATHOGENIC FUNGI 531 in this case the walled spores are not swarm-spores, but are adapted for dispersal through the air. Some of the Phycomycetes can produce spores asexually in an entirely different manner, namely, externally by abstriction from the end of a hypha. Such asexual spores externally cut off are termed conidia, and the special hypha bearing the conidia, if different in form from the vegetative hyphe, is termed a cunidiophore. Each conidium can emit one or more hyphe and thus give rise to a new plant. Other forms of asexual spores occurring in these simple fungi include oidia, in which a hypha undergoes cross septation into a number of short segments, each of which acts as an asexual spore. A hypha in this oidial condition has aresemblance toa greatly magnified row of bacteria ; indeed according to one theory bacteria represent merely oidial conditions of very degenerate fungi. Finally, as opposed to the thin-walled asexual spores so far mentioned, thick-walled asexual spores (often termed chlamydospores) occur in some of these simple fungi, and are endowed with greater powers of resistance to hostile external conditions and act as resting-spores. Phycomycetes also reproduce sexually. In the simplest case, as repre- sented by Mucor mucedo, the ends of two hyphe come into contact and the terminal parts of the hyphe are segmented off by a transverse wall. The wall at the region of contact of the two hyphe is dissolved, and the protoplasmic contents of the two terminal compartments fuse and produce around the resultant mass a thick wall. This thick-walled structure is capable of growing out to produce a new plant. As it is pro- duced by the fusion of two similar sexual cells it is termed a zygospore. Those Phycomycetes that have no marked structural distinction between male and female cells or organs, and whose sexually produced cells are therefore zygospores, are grouped together to form the class Zygomycetes. In other Phycomycetes there is a very clear distinction between, on the one hand, the large female organ, which encloses one or more female cells, the ova or oospheres, and, on the other hand, the usually smaller but differently shaped male organ, which contains the equivalent of a number of male cells. The union of some of the protoplasm of the male organ with an oosphere results in the production of a fertilised egg-cell or oospore. Those Phycomycetes having this mode of sexual reproduction are grouped together to form the class Oomycetes. Sexually produced cells, zygospore and oospore, germinate vegeta- tively to produce a new mycelium or in a fructificative manner to pro- duce a sporangium. Now the number of spores inside a sporangium of a Phycomycete is not only great but is at least often variable in the same species. Thus if a plant of Mucor mucedois starved, the number of spores produced in each sporangium is greatly reduced. Similarly in the Phycomycetes the number of conidia produced on a conidiophore is considerable and variable. Sporangia and conidiophores, then, are in- definite in type in these simple fungi. The more highly organised fungi, the Mycomycetes, differ from the Phycomycetes in that (1) their sporangia or conidiophores are definite ; (2) the hyphe are septate, with numerous cross partitions ; (3) the sexual process, organs, and cells are so modified as to be more or less difficult of recognition, or even perhaps unrecognisable as such. In any case, the Mycomycetes never have a sexually produced zygospore or oospore capable of developing into an independent vegetating fungus. ; Two main series are recognisable in the Myconiycetes. In one serics 532 PATHOGENIC FUNGI Fic. 162,—A, Mucor mucedo ; I, 2, 3, stages in formation of a zygospore. 4, a sporangium containing spores. B, Oidium lactis. C, Aspergillus glaucus (De Bary). 1, mycelium. 2 and 5, gonidiophore-bearing spores. 3, 4, a perithecium (4 contains rudimentary asci). 6, piece of gonidiophore ; a, sterigma; /, spore. D, branched gonidiophore of Penicillium glaucum bearing spores. E, F, Saccharomyces cerevisix, cells are budding. G, ditto, formation of endospores (after Hansen). ZYGOMYCETES 533 the sporangium has become definite in type, as it produces inside it a number of spores that is definite and constant to the species. The number of spores is usually eight, but a few species produce other multiples of two. This definite sporangium is termed an ascus, the spores are asco- spores, and the group of fungi having asci is the Ascomycetes. In some of the Ascomycetes the asci are grouped together and forma kind of fructi- fication (ascocarp), which, to give an example, is a closed spherical body in Aspergillus and Penicillium (vide infra). In the other series of Mycomycetes it is the conidiophore that has become definite in type, being constant and defined in form and numbers of conidia produced. The conidiophore usually bears four conidia or, in a few species, two or a multiple of two. Such a conidiophore is termed a basidiwm, and characterises the class Basidiomycetes, of which the common toadstools are examples. There are some groups of fungi whose characters are sufficiently well known and defined as to be capable of diagnosis, and yet do not accord in characters with any of the classes already mentioned. One of these groups —that of the true rust-fungi, Ustilaginacee—belongs to the Mycomycetes : among the salient features belonging to the members is their capacity to produce thick-walled asexual resting-spores, which in germination give rise to a minute plant that buds off indefinite numbers of conidia. The other group, the Chytridiales, on the contrary is a collection of minute fungal parasites so exceedingly low in organisation as to have feebly denoted or no filamentous hyphe. The life-histories of some fungi placed in the groups already enumerated are incompletely known, yet certain characteristic stages are known, so that it is possible to refer these types to their correct systematic position and class. But there still remain many kinds of fungi that are known only in their conidial stage, and the conidiophores are indefinite in type (not basidia). These imperfectly known fungi cannot be placed in their natural classes and have to be empirically grouped according to the arrangement and form of their conidiophores, structure and colour of their conidia, and so forth. They form the large unnatural group Fungi Imperfecti. Finally, there remain a few parasitic fungi known only in a sterile mycelial condition. We now give examples of common non-pathogenic types. Zygomycetes: Mucor mucedo (and other species of Mucor).—This form occurs on damp bread, horse dung, and other organic substrata. To the naked eye it appears as a white or smoky mould composed of fine filamentous usually non-septate hyphwe spreading over the substratum. Here and there arise erect hyphe which in a saturated atmosphere may attain a length of several inches, but which are very much shorter in ordinary air. Each erect hypha ends in a spherical sporangium whose proto- plasm is separated off from that of the supporting hypha by a transverse wall, which bulges greatly into the cavity of the sporangium and forms the so-called columella. The'protoplasm of the sporanginm divides into many masses, each of which acquires a cell-wall and’is then a spore. The spores escape by the rupture of the wall of the sporangium. (The needle-like bodies often seen outside the wall of the sporangium are crystals of calcium oxalate.) The less frequent sexual method of repro- duction and the formation of the zygospore has already been described. The infrequency of the sexual mode of reproduction is due partly to the fact that the individual plants are sexually differentiated and might be termed male and female. Zygospore and asexual spore alike 534 PATHOGENIC FUNGI germinate to produce a new mycelium. In rich culture media or old cultures the mycelium may become septate. Cultivated under water some species (including Chlamydomucor racemosus) enter into an oidial -condition. Ascomycetes: (1) Aspergillus herbariorum (=A. niger).—This, with other varieties of the same group, is of frequent occurrence, especially on dead vegetable matter. It grows readily on gelatin and, to the naked eye, consists of a mass of filaments which microscopically are seen to form a septate branching mycelium. Two forms of reproduction occur, the variety depending largely on the nutrition of the plant. The less common form is effected by means of structures known as ascocarps, which owe their formation to a sexual process. From a mycelial branch there arises a hypha which becomes specially coiled and transversely septate at its end. From the base of the lowest coil of the spiral two or three hyphe grow up towards its apex, where one of these fuses with the coiled hypha and represents the male organ. The others by branching copiously produce a mass of closely woven hyphe forming a closed wall to this structure, which is the ascocarp referred to. Within it numerous asci arise as the ultimate ramifications of branches given off by the central coiled hypha. Inside each ascus eight ascospores are produced. Ultimately all the structures lying within the ascocarps, save the spores, undergo disintegration, so that the mature ascocarp consists of a small hollow sphere within which lie the loose spores. These latter are ulti- mately freed by the decay of the wall of the ascocarp and develop into new individuals. The commonest method of reproduction is by the formation of spores in the form of conidia, which are clearly of non-sexual origin. A filament grows out, and at its termination a rounded swelling is formed on which a series of little finger-like processes called sterigmata are perched. At the free end of each of these, rows of oval conidia are suc- cessively abstricted. Hach conidium, on becoming free, can give rise to a new individual, just as can an ascospore. (2) Penicillium crustaceum (=Penicillium glaucum).—-This is perhaps a composite species and is the most common of all fungi met with in bacteriological work. It is the common green cheese mould, and its extraordinary versatility and powers of resistance make its spores practically omnipresent. The mycelium is like that of the Aspergillus. Ascocarp formation takes place, but the commonest mode of reproduction is by the conidia. A filament (the conidiophore) grows out, and at its end frays out into a pencil of finger-like branches. On the point of each of these a peg-like sterigma is developed. On the end of this a row of oval conidia is successively cut off ; these break off and can give rise to new iridividuals. (3) Saccharomyces or Yeasts (Torula, Mycoderma).—These organisms have been subjected to much investigation in consequence of their economic importance in brewing and baking. They occur in nature chiefly in connection with fruits, such as the grape, which contain fermentable sugars. They consist of round or oval cells, 3 to 5 mw in longest diameter, and under ordinary conditions reproduce themselves by budding, in which process a portion of the cell protrudes, increases in size, and finally becomes separated from the parent cell so as to form a new individual. In a number of other fungi belonging to the various groups, the conidium, when cultivated in a liquid, has the power of budding off conidia which behave in like manner; such fungi, therefore, have a yeast-like stage in their life-history. Under certain conditions of TINEA. FAVUS 535 moisture and oxygen supply, endogenous sporulation occurs. As the spores produced are definite in number—two in some species and four in others—the sporangium is an ascus and Saccharomyces is a degenerate ascomycete. While in yeasts generally the oval cell represents the vege- tative unit, in certain species elongated tube-like bodies may be formed which suggest an attempt at hyphal formation. In Saccharomyces myco- derma, the vegetative cells are so elongated and linked as to form a kind of simplified mycelium. Fungi imperfecti: Oospora lactis (Fres:) (=Oidium lactis),—This is a common fungus in sour milk and sour bread, and can easily be cultivated on gelatin, where the colonies consist of short and fine septate filaments radiating from a centre. Here and there the hyphe are divided, especially at the ends, into short oval or cylindrical segments, po oidia, which act as spores. No other method of reproduction is