MANUAL OF BACTERIOLOGY BIO LOST LIBRARY G LONDON : H. K. LEWIS, 136 GOWER STREET, W.C. SIR JOSEPH LISTER, BART., M.B., F.R.S., WHO HAS CREATED A NEW EPOCH IN MEDICINE AND SURGERY, BY APPLYING A KNOWLEDGE OF MICRO-ORGANISMS TO THE TREATMENT OF DISEASE AS A TOKEN OF ADMIRATION AND RESPECT BY THE AUTHOR. Vlll PREFACE TO THE THIRD EDITION. The author trusts that this manual will continue to be of use to students and practitioners in both the medical and veterinary professions. For references to recent literature, the author again takes the opportunity of calling attention to Professor Baumgarten's invaluable annual. EDGAR M. CROOKSHANK. 24, MANCHESTER SQUARE, LONDON, W. October 28?%, 1890. PREFACE TO THE SECOND EDITION. THE fact that a new edition of this manual was called for a few months after its publication, has induced the author to extend its scope in the hope of adding to its usefulness. The work has not only been revised throughout and brought up to date, but, in order to admit of a more concise arrangement of the species, the Systematic part has been recast. Additional chapters have been written upon the General Morphology and Physiology of Bacteria, upon Antiseptics and Disinfectants, and Immunity. Seventy-three illustrations have been added. Those not duly acknowledged as coming from other sources were drawn on the wood by the author from his own preparations. A list of references to works on Bacteriology, which was not ready for the first edition, has now been completed and extended. It has no pretension to be a complete bibliography, but being arranged X PREFACE TO THE SECOND EDITION. as much as possible in accordance with the chapters, and in chronological order, may be useful to those seeking further details. No doubt Professor Baum- garten's Jahresbencht, the first number of which has been issued this year, will be found a valuable guide to current literature in the future. The author desires again to express his ac- knowledgments to Professor Gerald Yeo and Mr. Herroun, of King's College, London. EDGAR M. CROOKSHANK. 24, MANCHESTER SQUARE, W. December, 1886. CONTENTS. INTRODUCTION. PAGE THE GERM THEORY i PART I . GENERAL METHODS. CHAPTER I. APPARATUS, MATERIAL, AND REAGENTS EMPLOYED IN A BACTERIOLOGICAL LABORATORY . . .23 a. Histological apparatus . . . . . 23 b. Reagents and materials employed in the processes of hardening, decalcifying, embedding, fixing, and cutting of tissues . . 25 c. Reagents for examining and staining microscopical preparations . 27 d. Reagents for mounting and preserving preparations . . 34 e. Drawing and photographic apparatus . > . . -34 f. Sterilisation apparatus . . . . . 36 g. Apparatus and material for preparing and storing nutrient gelatine and nutrient agar-agar . . . . . 38 h. Apparatus for employment of nutrient jelly in test-tube and plate- cultivations . . . . . . .41 i. Apparatus for preparation of potato-cultivations . * ' . 45 j. Apparatus for preparation of solidified sterile blood serum . 46 k. Apparatus for storing, and for cultivations in, liquid media . 48 /. Apparatus for incubation . . . . . . 49 m. Inoculating and dissecting instruments and apparatus in common use 58 M. General laboratory requisites ... . . '' 59 Xll CONTENTS. CHAPTER II. PAGE MICROSCOPICAL EXAMINATION OF BACTERIA IN LIQUIDS, IN CULTIVATIONS, ON SOLID MEDIA, AND IN TISSUES . . . '. . . .62 a. Examination in the fresh state ; ' : . ' . . -63 b. Cover-glass preparations : methods of Ehrlich, Babes, and His . 65 c. Cover-glass impressions . . . . . .69 CHAPTER III. PREPARATION AND STAINING OF TISSUE SECTION'S . 71 a. Methods of hardening and decalcifying preparations . 7 1 b. Methods of embedding, fixing, and cutting . . 72 c. General principles of staining bacteria in tissue sections : methods of Weigert, Gram, and Weigert- Ehrlich . . -75 CHAPTER IV. PREPARATION OF NUTRIENT MEDIA AND METHODS OF CULTIVATION . . . . . 80 SOLID MEDIA : a. Preparation of nutrient gelatine and nutrient agar-agar . . 82 b. Methods of employing nutrient jelly in test-tube and plate-culti- vations . . . . . . .87 c. Preparation and employment of sterilised potatoes, potato-paste, bread-paste, vegetables, fruit, and white of egg . . 99 d. Preparation and employment of sterile blood serum . . 104 LIQUID MEDIA : e. Preparation of sterilised bouillon, liquid blood serum, urine, milk, vegetable infusions, and artificial nourishing liquids . . 106 f. Methods of storing and employing liquid media ; Lister's flasks, Aitken's test-tubes, Sternberg's bulbs, Pasteur's apparatus, Miquel's bulbs; Drop-cultures; Warm stages . . . 109 CONTENTS. CHAPTER V. PAGE EXAMINATION OF AIR, SOIL, AND WATER i . . 125 Air . . . , . . '.'. . . . 125 Soil . . ..,.*. . . . . 130 Water . . . . . . . . 131 CHAPTER VI. EXPERIMENTS UPON THE LIVING ANIMAL . . 137 a. Inhalation of micro-organisms . . . . . 137 b. Administration with food . - . . . . . 137 c. Cutaneous and subcutaneous inoculation . '* . -v -138 d. Special operations . .-. . . i ^ . 139 CHAPTER VII. EXAMINATION OF ANIMALS EXPERIMENTED UPON, AND THE METHODS OF ISOLATING MICRO-ORGAN- ISMS FROM THE LIVING AND DEAD SUBJECT . 141 a. Method of dissection and examination .... 141 b. Isolation of micro-organisms from the living subject . -144 PART II. GENERAL BIOLOGY OF BACTERIA. CHAPTER VIII. GENERAL MORPHOLOGY AND PHYSIOLOGY . . 147 CHAPTER IX. ANTISEPTICS AND DISINFECTANTS . -. . .180 CHAPTER X. IMMUNITY - 192 XIV CONTENTS. PART III. SYSTEMATIC AND DESCRIPTIVE, WITH SPECIAL MICROSCOPICAL METHODS. CHAPTER XI. PAGE CLASSIFICATION OF BACTERIA . . . .205 CHAPTER XII. SYSTEMATIC AND DESCRIPTIVE . . . .224 GROUP I. COCCACEyE . . . . . . .224 Genus I. Streptococcus ...... 225 ,, II. Merismopedia . . . . . .241 ,, III. Sarcina .... . 243 ,, IV. Micrococcus ...... 245 ,, V. Ascococcus . . . . . . 257 GROUP II. BACTERIACE/E ...... 260 Genus I. Bacterium . . . . . .261 II. Spirillum . ..'. ... . . . 282 ,, III. Leuconostoc ., . . . . . 296 ,, IV. Bacillus ....... 299 ,, V. Vibrio . .' . . . . . 349 ,, VI. Clostridium . . . . . . 350 GROUP III. LEPTOTRICHE^: ...... 353 Genus I. Crenothrix ...... 354 II. Beggiatoa ... 35 ,, III. Phragmidiothrix ...... 360 ,, IV. Leptothrix . . . . . . 361 GROUP IV. CLADOTRICHE^E . " '.., . . , . . 362 Genus I. Cladothrix ...... 362 APPENDICES. A. Yeasts and moulds ....... 369 B. Actinomyces . . . * . . . . . 379 C. Flagellated protozoa in the blood ..... 397 D. Hsematozoa of malaria (Laveran) ..... 408 E. Chronological bibliography . . . . . -414 F. Table showing the magnifying power of Zeiss' objectives . . 452 V LIST OF ILLUSTRATIONS. WOOD ENGRAVINGS. FIG. PAGE 1. Koch's Steam-Steriliser . . . . '37 2. Hot-air Steriliser .- . . . . . -38 3. Section of Hot-air Steriliser . ' . ~ . . . 38 4. Hot-water Filtering Apparatus, with Ring Burner . '. -39 5. Wire Cage for Test-tubes . ' ' . . . . . . 42 6. Platinum Needles ... . ' . . .42 7. Damp Chamber for Plate-cultivations . . . . -43 8. Apparatus employed for Plate-cultivations . . . .43 9. Box for Glass Plates . . . . '.."..' -44 loandii, Glass Benches for Glass Plates or Slides . . -44 12. Israel's Case . . . . . . . . 45 13. Damp Chamber for Potato-cultivations . . . -45 14. Serum Steriliser . . . . .46 15. Serum Inspissator . . . * . . . -47 1 6. D'ArsonvaPs Incubator . . . . -5 17. Schlosing's Membrane Regulator . . . 51 1 8. Gas-burner protected with Mica-cylinder . . . 52 19. Moitessier's Gas-pressure Regulator . . / . -53 20. Koch's Safety Burner . . . . . ' . 53 21. Babes' Incubator . . . . . . . -54 22. Reichert's Thermo-regulator . . . . 56 23. Meyer's Thermo-regulator . . . . . -57 24. Siphon Bottle, with Flexible Tube, Glass Nozzle, and a Mohr's Pinchcock . . . . ' . . . -59 25. Dessicator . . . ; . . . .60 26. Method of making a Folded Filter . . . . -85 27. Method of Inoculating a Test-tube, containing Sterile Nutrient Jelly 88 28. Method of Inoculating Test-tubes in the preparation of Plate-culti- vations ....." . . . . -93 29. Method of dividing Potatoes . , . . . . . 101 30. Method of Forming a Simple Moist Chamber . . .114 31. Simple Warm Stage . . . . . ". . 115 32. Simple W T arm Stage shown in Operation . . . .116 33. Schafer's Warm Stage . . . . '. ' *".' H7 XVI LIST OF ILLUSTRATIONS. FIG. PAGE 34. Strieker's Warm Stage . ," . . - . .118 35. Section of Israel's Warming Apparatus and Drop-culture Slide . 118 36. Israel's Warming Apparatus . . . . . .119 37. Israel's Warming Apparatus in Operation . . . .120 38. Simple Gas Chamber . . . . . . .121 39. Gas Chamber in use with Apparatus for generating Carbonic Acid . 121 40. Strieker's Combined Gas Chamber and Warm Stage . \ . .122 41. Simple Moist Chamber adapted for transmission of Electricity . 122 42. Apparatus arranged for transmitting Electricity . . .123 43. Slide with Gold-leaf Electrodes . . . . .124 44. Hesse's Apparatus . . . . . .128 45. Apparatus employed for counting the Colonies on a Plate-cultivation 134 46. From a Preparation of Bacillus anthracis . . -149 47. Ascococcus Billrothii, x 65 (after Cohn) . >. . .152 48. Streptococcus and Sarcinacoccus from a Drop-cultivation, x 1200 . 153 49. Streptococcus in the Blood of a Rabbit, x 1200 . 153 50. Streptococcus of Progressive Tissue Necrosis in Mice (after Koch) . 153 51. Spirochseta from Sewage Water, x 1200 . . . 155 52. Bacteria showing Flagella . . .. . .. . I 57 53. Bacillus megatertiim . . . . . . 159 54. Clostridium butyricum, x 1020 . . . .160 55- A Thread of Bacillus anthracis with Spores in a Drop-cultivation, x 1400 . . 161 56. Leuconostoc Mesenteroides ; Cocci-chains with Arthrospores (after Van Tieghem and Cienkowski) . . . . .161 57. Spores of Bacillus anthracis, stained with Gentian-violet, after passing the Cover-glass twelve times through the Flame, x 1200 . 163 58. Spore-bearing Threads of Bacillus anthracis, Double-stained with Fuchsine and Methylene-blue, x 1200 . . . . -163 59. Tubercle Bacilli in Sputum, x 2500 (from photographs) ... .165 60. Leprosy Bacilli from a Section of Skin, x 1200 . . .165 61. Glanders Bacilli from a Section of a Glanders Nodule . x 1200 . 165 62. Bacterium of Chicken-cholera from Blood of Infected Hen, x 1200 . 166 63. Bacterium of Chicken-cholera from Muscle Juice of an Infected Hen, x 2500 (from a photograph) . . . . .166 64. Comma Bacilli in Sewage Water stained with Gentian-violet, x 1200 166 65. Vibrios in Water contaminated with Sewage, x 1200. . .167 66. Spirillum ^lndula, x 1200 . ... . . . . .167 67. Cladothrix dichotoma . . ._.-.. v . . . 214 68. Bacterium pneiimonice crouposce, x 1 500 (after Zopf) . ... .219 69. Emmerich's Bacterium, x 700 (after Emmerich) , . . 220 70. Colonies on Nutrient Gelatine, x 60 . . ., -. < . . 220 71. Colonies on Nutrient Agar-agar, x 60 . . , .221 72. Colony of Bacilhis anthracis, x 60 . . . , * .221 73. Bacterium of Rabbit Septicaemia . . . . .222 74. Streptococcus of Progressive Tissue Necrosis in Mice . -237 LIST OF ILLUSTRATIONS. XV11 FIG. PAGE 75. Micrococcus of Pyaemia in Rabbits ; Vessel from the Cortex of the Kidney, x 700 ....... 252 76. Ascococcus Billrothii (after Cohn) . . . \ V . 257 77. Bacterium Pneumonise Crouposae, from Pleurai Cavity of a Mouse, x 1500 (after Zopf) . . r'U: . -* ' .261 78. Bacterium Neapolitanum, x 700 (after Emmerich) . .- . 264 79. Bacteria of Rhinoscleroma, x 1400 (after Cornil) ; * " -. ' . 264 80. Bacterium of Chicken Cholera ; Blood of Inoculated Hen, x 1200 268 81. Bacterium of Chicken Cholera, from Muscle Juice of Inoculated Hen, x 2500 . - .- .- .- v : * . 268 82. Bacterium of Rabbit Septicaemia ; Blood of Sparrow, x 700 (after Koch) . . . .- ;/ ,. >x -''"'. - - < ' . 270 83. Bacterium Indicum ; Colonies on Nutrient Agar-agar, x 60 -275 84. Bacterium Zopfii ; Successive Changes in the same Thread, x 740 . 279 85. Cover-glass Preparation of the Edge of a Drop of Meat Infusion, x 600 (after Koch) : * . 1 * v. . 284 86. Colonies of Comma Bacilli on Nutrient Gelatine, natural size (after Koch) . . . " . . .< ' ..- . 285, 87. Colonies of Koch's Comma Bacilli, x 60 . .- ... . . 285 88. Cover -glass Preparation from the Contents of a Cholera Intestine, x 600 (after Koch) .. ' - 286- 89 Cover-glass Preparation of Cholera Dejecta on Damp Linen (two days old), x 600 (after Koch) . TJ ' :. v . ... . 286- 90. Section of the Mucous Membrane of a Cholera Intestine, x 600 (after Koch) . -., ,r '-..-. .' . . . 287 91. Pure Cultivation of Finkler's Bacillus, twenty-four hours old . 288 92. ,, ,, ,, ,, two days old . . 288 93. ,, ,, Koch's Cholera Bacillus, twenty-four hours old . 288- 94. ,, ,, ,, ,, two days old . . 288- 95. Comma-shaped Organisms with other Bacteria in Sewage-con- taminated Water, x 1200 . . . . . 289- 96. Pure Cultivation of Spirillum Finkleri in twenty-four hours . 292 97. ,, ,, thirty-six hours . . 292 98. Spirillum sputigenum, x 1200 . . . . 293. 99. Spirillum tyrogenum, x 1200 ..... 293 100. Spirillum plicatile (Marsh Spirochaete), x 1200 . . . 294 101. Spirillum undula, x 1500 . .... 295 1 02. Leuconostoc mesenteroides . . . . . -297 103. Leprosy Bacilli from a Section of Skin, x 1200 . . . 300 104. Bacillus typhosus from a Potato-cultivation, x 1500 . . 303 105. Bacillus tuberculosis from Tubercular Sputum, stained by Ehrlich's method, x 2500 ....... 306 106. Cultivation of the Tubercle Bacillus on Glycerine Agar-agar . 308- 107. Bacillus anthracis, x 1200 . . . 3 J 5 108. Pure Cultivation of the Bacillus anthracis in Nutrient Gelatine . 318 XV111 LIST OF ILLUSTRATIONS. FIG. PACK 109. Colonies in a Plate-cultivation, x 70. . . . 319 HO. Cover-glass Impression-preparation, x 70 . :>, . ,* . 320 111. Spores of Bacillus anthracis unstained, x 1500 . * . 324 112. Spores of Bacillus anthracis, x 1200 . . .. . 325 113. From a Double-stained Preparation of Bacillus anthracis > x 1200 . 325 114. Bacillus mallei, x 1200 ...... 326 115 and 116. Pure Cultivations of the Bacillus of Septicaemia of Mice . 331 117. From a preparation of Bronchial Mucus of a Pig (after Klein) . 333 118. Blood of Fresh Spleen of a Mouse, after inoculation with Swine Fever (after Klein) . . . ., ..*:. . 333 1 19. Bacilli from an Artificial Culture with Spores (after Klein) . . 333 1 20. Bacillus cyanogenus, x 650 (after Neelsen) . .'. . . 338 121. Bacillus megaterium (after De Bary). ;.''- ,.. *=-.'. * . 343 122. Pure Cultivation of Bacillus figurans in Nutrient Agar-agar . 344 123. Bacillus saprogenes, No. I (after Rosenbach) . . . 347 124. Vibrio rugula, x 1020 (after Prazmowski) . , -. . , 349 125. Clostridium butyricum (after Prazmowski) . ; , . 352 126. Crenothrix Kuhniana (after Zopf) . . .. , -355 127. Beggiatoa alba (after Zopf) . . . .:..,.,.> -357 128. Several Phase-forms of Beggiatoa Roseopersicina (after Warming) . 358 129. Cladothrix dichotoma (after Zopf) . .' . 4! , : , ; . 363 130. Parasites in the Blood of Rats (after Lewis) . ;-v- 397 131. Hcematomonas cobitis (after Mitrophanow) . . - . . 399 132. Organisms in the Blood of the Carp (after Mitrophanow) . . 400 133. " Surra " Parasites occurring singly and fused, x 1200 . . . 402 134. A Monad in Rat's Blood, x 3000 . . H. *;. . . 404 135. Monads in Rat's Blood, x 1200 . t ; . . 405 136. (stained) . . 406 137. Hsematozoa of Malaria; Non-pigmented Forms (after Marchiafava and Celli) . . . . . . . . 409 138. Pigmented Amoeboid Forms (after Golgi) . . . . 409 139. Semilunar Bodies of Laveran (after Golgi) . ;-*.- . 410 140. Rosette Forms with Segmentation (after Golgi) . . .411 141. Flagellated Forms (after Vandyke Carter) . <- . . .411 DESCRIPTION OF PLATES. PLATE I. (Facing page 18.) Bacteria, Schizomycetes, or Fission fungi. FIG. 1. Cocci singly and varying in size. 2. Cocci in chains or rosaries (streptococcus). 3. Cocci in a mass or swarm (zoogloea). 4. Cocci in pairs (diplococcus). 5. Cocci encapsuled (Bacterium pneiimonicz croupos Oc. 4). 29. From a cover-glass impression -preparation of a potato-cultivation of Bacillus anthracis (Zeiss' yg o.i. Oc. 4). 30. From a preparation of Bacillus anthracis^ cultivated in nutrient gelatine (torula-form). 31. Involution-form of Crenothrix (after Zopf). XX DESCRIPTION OF PLATES. FIG. 32. Involution-forms of Vibrio serpens (after Warming). 33. Involution-forms of Vibrio rugula (after Warming). 34. Involution-forms of Clostridium Polymyxa (after Prazmowski). 35. Involution-forms of the Spirillum cholera Asiatics, from an artificial cultivation. 36. Involution-forms of Bacterium aceti (after Zopf and Hansen). 37. Spirulina-form of Beggiatoa alba (after Zopf). 38. Various thread-forms of Bacterium merismopedioides (after Zopf). 39. False-branching of Cladothrix (after Zopf). PLATE II. (Facing page 76.) i. Micrococcus tetragonus. From a section of a kidney of a mouse which had died in eight days, after inoculation subcutaneously with a pure cultivation. Encapsuled tetrads, isolated and in masses, were found in the kidneys, lungs, and other organs. Stained with Gram's method (gentian-violet) without a contrast stain, x 1500. 2,. Micrococcus pyogenes aureus (Staphylococcus pyogenes aureus). From a section of the liver of a rabbit. A small vessel is shown plugged with cocci. From small abscesses in the liver, cultivations were obtained of the characteristic yellow coccus of pus. Stained with Gram's method (gentian- violet) without a contrast stain, x 1500. PLATE III. (Facing page 78.) 1. Bacillus tuberculosis. From a cover-glass preparation of tubercular pus. Stained with Ehrlich's method (fuchsine and methylene-blue). x 1500. 2. Bacillus leprae. From a section of a kidney from a case of leprosy. Stained with Ehrlich's method (fuchsine and methylene-blue). In the centre of the field is a glomerulus with a collection of the leprosy bacilli, x 400. PLATE IV. (Facing page 88.) 1. Spirillum cholerae Asiaticae (Comma-baccillus of Koch}. Tube inoculated from a plate-cultivation. The growth in this case was very striking. The funnel-shaped area of liquefaction, enclosing an air- bubble, and the white thread along the needle-track, are in marked contrast to the appearances, under similar conditions, of the comma- bacillus in Cholera nostras (p. 288). 2. Bacterium cholerae gallinarum (Microccus cholera galhnarum ; Microbe du cholera des poules). Tube inoculated from the blood of a hen which had died of so-called chicken-cholera. After several days the growth forms a very delicate, finely beaded thread. 3. Micrococcus cereus albus (Staphylococcus cereus albus). Tube inoculated from the pus of a subcutaneous abscess in a rabbit. The growth assumed a nodular appearance along the needle-track. DESCRIPTION OF PLATES. XXI PLATE V. (Following Plate 1. Micrococcus tetragonus. Tube inoculated from a plate-cultivation of bacteria in sputum. The cultivation consisted of a milk-white growth heaped up on the surface of the gelatine, and growing freely along the upper part of the needle-track. 2. Bacterium pneumoniae crouposae (Micrococcus pneumonia crouposa ; Friedldnder 1 s pneumo-coccus) . Tube inoculated from pneu- monic exudation. The growth, in nutrient gelatine, in the form of a round-headed nail, is not by itself distinctive. 3. Saccharomyces niger (Black toruld). Tube inoculated from an old contaminated nutrient-gelatine cultivation. The growth, isolated and reinoculated, formed a black crust on the surface of the gelatine. In some of the tubes little separate centres of growth occurred in the upper part of the track of the needle. PLATE VI. (Following Plate K) 1. Bacillus pyocyaneus (Bacterium aruginosum ; Bacillus fluorescens), Tube inoculated from pus. The gelatine was liquefied, and appeared green by transmitted and orange by reflected light. 2. Sarcina lutea. Tube inoculated from a colony which occurred on potato exposed to the air. The gelatine was partially liquefied, and a canary-yellow growth had subsided to the bottom of the liquefied layer. 3. Bacillus anthracis. Tube inoculated from the blood of a mouse which had died of anthrax. The typical growth which occurs in a few days is shown on p. 318. In this figure the appearance after three weeks is represented. The gelatine was completely liquefied, and a flocculent mass had subsided to the bottom of the tube. PLATE VII. (Following Plate VI.} 1. Sarcina lutea. In this tube and the two adjacent ones, the inocula- tions were made by thrusting the needle into the nutrient agar-agar. In all three cases the growth on the surface, freely exposed to air, developed a characteristic pigment, while the growth in the track of the needle was scanty and colourless. 2. Bacterium indicum (Micrococcus indicus, Koch ; Bacillus inaicus, Fliigge). 3. Saccharomyces rosaceus (Pink toruld). PLATE VIII. (Following Plate VII.) i. Bacterium indicum (Micrococcus indicus ; Koch ; Bacillus indicus > Fliigge). Tube inoculated from a nutrient agar-agar plate-cultivation. By plate-cultivation, or by succussive cultivation on potatoes, a pure cultivation can be obtained. The growth has then the colour of red XX11 DESCRIPTION OF PLATES. FIG. sealing-wax, and a peculiar crinkled appearance. After some days the growth loses its bright colour, and becomes purplish, like an old cultivation of Bacterium prodigiosum. 2. Bacillus cyanogenus (Bacterium syncyamim ; Bacillus of blue milk}. Tube inoculated from a potato-cultivation. The bacillus forms a whitish layer, and colours the nutrient agar-agar a smoky brown. 3. Bacterium prodigiosum (Monas prodigiosa ; Micrococcus prodi- giosus, Cohn ; Bacillus prodigiosus, Fliigge ; Blood rain). Tube inoculated from a potato-cultivation. The bacterium grows very rapidly, forming a blood-red growth, which gradually acquires a purplish colour. PLATE IX. (Following Plate VIII ^ 1. Sarcina lutea. Tube of nutrient agar-agar inoculated from a plate- cultivation. The canary-yellow colour forms a strong contrast to the colour of the growth in the adjacent tube. 2. Micrococcus pyogenes aureus (Staphylococcus pyogenes aureus). Tube inoculated from an abscess in a rabbit. 3. Bacillus pyocyaneus (Bacillus fiuorescens ; Bacteriiim ceruginosum) . Tube inoculated from a colony on a plate-cultivation. The growth formed a whitish, transparent layer composed of slender bacilli. The pigment diffused itself throughout the nutrient jelly. The growth appears green by transmitted light, owing to the colour of the medium behind it. The bacillus is now regarded as identical with the bacillus of green-blue pus. PLATE X. (Following Plate IX.) 1. Bacterium lineola. Tube inoculated from putrid blood-serum which swarmed with Bacterium lineola. The same bacteria were found in the cultivation. 2. Micrococcus rosaceous. Tube inoculated from an old cultivation on nutrient agar-agar which had become contaminated. The culti- vation, macroscopically resembling pink yeast, consisted of a pure cultivation of micrococci. 3. Micrococcus pyogenes citreus. Tube inoculated with cocci isolated from a subcutaneous abscess in a mouse. PLATE XI. (Following Plate X.) I. Bacillus anthracis (Bacteridie d^^ charbon ; Bacillus of splenic fever , wool-sorter's disease, or malignant pustule). Tube of nutrient agar-agar inoculated with the blood of a sheep which had died of anthrax. White flocculent patches developed, which were entirely composed of threads and spores of the bacilli. DESCRIPTION OF PLATES. XXlli FIG. 2. Bacillus subtilis. Tube inoculated with bacilli, isolated by plate- cultivation, from dust. The bacilli appeared to be identical with the hay-bacillus, but in this case formed a peculiar crinkled layer along the track of the needle. 3, Micrococcus cere us albus (Staphylococcus cereus albus). Tube inoculated from the discharge of a subcutaneous abscess in a rabbit. PLATE XII. (Facing page 96.) Spirillum of Finkler and Prior (Comma-bacillus of Finkler and Prior), This figure represents the appearance of a plate-cultivation of the comma-bacillus from Cholera nostras, when examined over a slab of blackened plate-glass. The colonies differ very markedly from the colonies of Koch's comma-bacilli (see p. 285). The drawing was made from a typical result of thinning out or attenuating * the colonies by the process of plate-cultivation. At this stage they were com- pletely isolated one from the other ; but later they became confluent, and produced complete liquefaction of the gelatine. PLATE XIII. (Following Plate XII.'} Spirillum of Finkler and Prior (Comma-bacillus of Finkler and Prior}. This figure represents the result obtained by a still further thinning out of the organisms than in the preceding case. The attenuation had been so far carried out that several of the colonies remained completely isolated for days. PLATE XIV. (Facing page 102.) 1. Bacterium prodigiosum (Monas prodigiosa ; Micrococcus prodigiosus, Cohn ; Bacillus prodigiosus ', Fliigge ; Blood rain). Growth on potato after three days. 2. Penicillium glaucum. A potato which had been freely exposed to the air was covered in three weeks by a growth of Penicillium glaucum. The surface of the growth is studded with dew-like drops of moisture. PLATE XV. (Following Plate XIV.} 1. Sarcina lutea. Growth on sterilised potato five days after inoculation from a tube-cultivation. Potatoes, especially old ones, have sometimes a tendency to become discoloured, and the brown appearance in this figure has nothing to do with the growth of the organisms. 2. Saccharomyces rosaceus (Pink torula). Growth on sterilised potato which had been inoculated from a colony contaminating a plate- cultivation. This yeast develops a coral-pink colour, but does not grow so luxuriantly as the chromogenic bacteria. * The term "attenuation" is applied also to a virus, in the sense of weakening or modifying its effect. To avoid confusion the term mitigation might be employed exclusively to express this, and attenuation used only in the sense indicated above. XXIV DESCRIPTION OF PLATES. PLATE XVI. (Folhioing Plate XV.} 1. Bacillus anthracis (Bacteridie du charbon ; Bacillus of splenic fever, wool-sorter's disease, or malignant pustule], The bacillus of anthrax grows very rapidly on sterilised potato, especially when placed in the incubator at the temperature of the blood. The growth forms a creamy-yellow layer, with copious spore-formation. 2. Bacterium indicum (Micrococcus indicus, Koch; Bacillus indicus, Fliigge). Sterilised potato inoculated with a pure growth obtained by successive cultivations. Unless the growth is quite free from the presence of other bacteria, the brilliant red colour is not obtained. PLATE XVII. (Following Plate i and 2. Bacillus cyanogenus (Bacterium syncyanum ; Bacillus of blue milk}. Potato inoculated from a cultivation in nutrient gelatine. In three days a peculiar bluish-green growth develops on the surface of the potato, and in nine days it has a heaped-up margin of a bluish- green colour, while the central portion has turned almost black. PLATE XVIII. (Facing page 104.) 1. Bacillus tuberculosis. Pure cultivation of the tubercle-bacillus on blood-serum solidified obliquely. 2. Pure cultivation on solid blood-serum in a glass capsule. 3. The same as Fig. 2, examined under a low power of the microscope, x 80. 4. Impression-preparation showing the peculiar serpentine growth of the colonies on blood-serum, x 700. (After Koch, MittheiL a. d. Kaiserl Gesundh. Amt.} PLATE XIX. {Facing page 228.) 1. Streptococcus pyogenes. From a microscopical specimen prepared from pus from a pysemic abscess. Stained with gentian-violet by the method of Gram, and contrast-stained with eosin. x 1200. Powell and Lealand's apochromatic T V Horn. imm. E. P. 10. 2. From a microscopical specimen of an artificial cultivation of the strepto- coccus in broth. Complex chains with elements dividing both longitudinally and transversely, and varying considerably in size in different lengths of the same chain. x 1200. Powell and Lealand's apochromatic -^ Horn. imm. E. P. 10. PLATE XX. (Facing page 300. ) i. Bacillus leprae. From a section of the skin of a leper. The section is, almost in its entirety, stained red, and, with moderate amplification, has a finely granular appearance. Stained with Ehrlich's method (fuchsine and methylene-blue). x 200. DESCRIPTION OF PLATES. XXV FIG. 2. Part of the same preparation with high amplification, showing that the appearances described above are due entirely to an invasion of the tissue by the bacilli of leprosy, x 1500. PLATE XXI. (Facing page 308.) 1. Bacillus tuberculosis. From a section of a lymphatic gland of a fcetal calf. The preparation was stained by the Ehrlich-Koch method (methyl-violet and bismarck-brown), and eosin. The giant cell takes the eosin stain, the nuclei are stained brown, and the bacilli blue. In the interior of the giant cell are numerous coloured grains, the significance of which is not known, and a number of tubercle-bacilli, x 1500. For the material from which this preparation was made the author is indebted to Professor Johne, by whom an account of this case was published, " Ein Zweifelloser Fall von Congenitaler Tuberkulose," Fortsch. d. Meet., 1885, No. 7, p. 198. 2. From a section of a lung of a rabbit after inoculation with tubercular sputum. Caseous areas are seen, and masses of bacilli showing distinct beading. Stained by the Ehrlich-Koch method (methyl-violet) without a contrast stain, x 1500. PLATE XXII. (Following Plate XXI.) 1. Bacillus tuberculosis. From a section of the liver of a tubercular hen. With a moderate power the areas of caseation and the topo- graphical distribution of the bacilli can be studied. Stained with the Ehrlich-Koch method (methyl- violet and bismarck-brown). x 400. 2. From the same preparation with high amplification, showing that the parts stained blue consist entirely of bacilli, x 1500. PLATE XXIII. (Facing page 316.) 1. Bacillus anthracis (Bacteridie Ju charbon ; Bacillus of splenic fever, wool-sorters disease, or malignant pustule}. From a section of the mucous membrane of the stomach of a mouse. The glandular capil- laries are mapped out by the bacilli. Stained by the method of Gram (gentian- violet), and eosin. x 500. 2. From a section of a kidney of a mouse. Under a low power the pre- paration has exactly the appearance of an injected specimen. Under higher amplification the bacilli are seen to have threaded their way along the capillaries between the tubules, and to have collected in masses in the glomeruli. Stained with Gram's method (gentian- violet), and eosin. x 500. C XXVI DESCRIPTION OF PLATES. PLATE XXIV. (Following Plate XXIII.') FIG. 1. Bacillus anthracis (Bacteridie du charbon ; Bacillus of splenic fever, wool-sorter's disease, or malignant pustule}. From a section of the liver of a mouse which had died after inoculation with a pure cultiva- tion of the bacillus. The bacilli are seen to have threaded their way between the liver-cells. The preparation is triple-stained by combining the methods of Weigert and Orth. x 500. 2. Bacillus anthracis and Micrococcus tetragonus. From a section of a lung of a mouse which had been inoculated with anthrax three days after inoculation with Micrococcus tetragonus. A double or mixed infection resulted. Anthrax-bacilli occurred in vast numbers, completely filling the small vessels and capillaries, and in addition there were great numbers of the characteristic tetrads. Stained with Gram's method (gentian-violet), and eosin. x 500. PLATE XXV. (Facing page 332.) 1. Bacillus of septicaemia of mice. From a section of a kidney of a mouse which had died after inoculation with a pure cultivation of the bacillus. With moderate amplification, the white blood-corpuscles have a granular appearance, and irregular granular masses scattered between the kidney tubules are seen. Stained with Gram's method and eosin. x 200. 2. Part of the same preparation with high amplification. The granular appearances are found to be due to the presence of great numbers of extremely minute bacilli, x 1500. PLATE XXVI. (Facing page 334.) 1. Bacillus of swine-erysipelas (Bacillus of German swine-fever}. Pure cultivation in nutrient gelatine. The growth is stated to be identical with that of the bacillus of septicaemia in mice. 2. Colonies on a plate-cultivation. 3. Cover-glass preparation of blood from an inoculated pigeon. (After Schiitz, Arb. a. d. Kais. Gesundh. Ami, Bd. I. PLATE XXVII. (Facing page 344.) 1. Bacillus figurans (Bacterium Z<$/?z). From an impression-prepara tion of a growth on the surface of nutrient gelatine, x 50. 2. Part of the same preparation with high amplification, showing that the coils and filaments of the growth are due to a peculiar and regular arrangement of the individual bacilli, x 1500. PLATE XXVIII. (Facing page 368.) i. Yeast fungi, or saccharomycetes ; and mould fungi, or hyphomycetes. Actinomyces teased out in the fresh state and stained with eosin. DESCRIPTION OF PLATES. XXvii no. 2. Torula cerevisia (after Rees). 3. Saccharomyces mycoderma, or oiditim albicans> from an artificial cultiva- tion (after Grawitz). 4. Saprolegnia (after Sachs). 5. Oiditim lactis (after Fliigg ). 6. Fungi of favus, or oiditim lactis (after Neumann). 7. Fenicillium glaucum (after Fliigge). 8. Aspergillus niger, from a preparation mounted in glycerine. 9. Aspergillus niger, from the same preparation (Zeiss y^- o.i.). 10. Aspergillus glaucus (after De Bary). ! 11. Botrytis Bassiana (after De Bary PLATE XXIX. (Facing page 384.) 1. Actinomyces (bovis). From a section of a maxillary tumour in a cow. Stained by Weigert's method (orseille and gentian-violet), x 900. 2. From a section of the lung of a cow. The rosettes are much smaller, possibly owing to their being more confined by their surroundings than when growing in the soft pulpy tissue of the maxillary tumour. They are here shown with high amplification, but under a power of about 50 diam. (Zeiss A.A. Oc. 2) the section of a lung resembles miliary tuber- culosis, and in the centre of a neoplasm the rosette appears about the size of a pin's head. Stained with Weigert's method (orseille and gentian-violet.) x 500. PLATE XXX. {Following Plate XXIX.} 1. Actinomyces (bovis). From a section of a maxillary tumour in a cow. Stained by Plaut's method (magenta and picric acid), x 90. Note. Neelsen's solution may be used instead of magenta solution. 2. Part of the same preparation, with higher amplification. The fungoid masses are very deeply stained by this method. The component club- shaped elements and their radiate arrangement are clearly shown. x 500. PLATE XXXI. (Facing page 390.) i. Actinomyces (hominis). From a preparation of the grains from an actinomycotic abscess in a boy. Examined in glycerine. The drawing has been made of a complete rosette, examined by focussing successively the central and peripheral portions. Towards the centre the extremities of the clubs are alone visible ; they vary in size ; and if pressed upon by the cover-glass, give the appearance of an irregular mosaic. Towards the periphery the clubs are seen in profile, and their characteristic form recognised. At one part there are several elongated elements, composed of separate links, x 1200. XXV111 DESCRIPTION OF PLATES. FIG. 2. Different forms of clubs from preparations in which the rosettes have been flattened out by gentle pressure on the cover-glass, x 2500. (a) Single club. (b) Bifid club. (c) Club giving rise to four secondary clubs. (d) Four clubs connected together, recalling the form of a branch of bananas. (e) Mature club with a lateral bud. (f) Apparently a further development of the condition represented at (e}. (g) Club with a lateral bud and transverse segmentation. (h) Single club with double transverse segmentation. (t) Club with oblique segmentation. (/) Collection of four clubs, one with lateral gemmation, another with oblique segmentation. (k) Club with lateral buds on both sides, and cut off square at the extremity. (/) Club with a daughter club which bears at its extremity two still smaller clubs. (m) Club divided by transverse segmentation into four distinct elements. () Elongated club composed of several distinct elements. (o) and (p) Clubs with terminal gemmation. (q) Palmate group of clubs. (r) Trilobed club. (s) Club with apparently a central channel. (/) Filament bearing terminally a highly refractive oval body. PLATE XXXII. (Facing page 392.) 1. Actinomyces (hominis). From cover-glass preparations of the fungus teased out of the new growths produced by inoculation of a calf with pus from a boy suffering from pulmonary actinomycosis. Stained by Gram's method and orange-rubin. The threads are stained blue and the clubs crimson. In the younger clubs the thread can be traced into the interior of the club. In some of the older clubs the central portion takes a yellowish stain, and in others the protoplasm is not continued as a thread, but is collected into a spherical, or ovoid, or pear-shaped mass. In others, again, irregular grains stained blue are scattered throughout the central portion. 2. From a section of a portion of the growth removed from a boy during life, The tissue was hardened in alcohol, and cut in celloidin. The section was stained with Gram's method and orange-rubin. There are several rosettes, surrounded by granulation tissue. 3. A mass of extremely fine filaments occupies the central part of the rosette. Many of the filaments have a terminal enlargement. The marginal part shows a palisade of clubs stained by the orange-rubin. x 500. BACTERIOLOGY, INTRODUCTION. THE GERM THEORY.. THE researches of Pasteur into the role played by micro-organisms in the processes of fermentation and putrefaction, and in diseases such as anthrax, the silkworm malady, pyaemia, septicaemia, and chicken cholera, have invested the science of Bacteriology with universal interest and vast im- portance. The further researches of the practical mind of Lister, with the resulting evolution of antiseptic surgery, have demonstrated the necessity for a more intimate acquaintance with the life- history of these micro-organisms ; while the more recent investigations which have established the intimate connection between bacteria and infective diseases, and more especially the discovery by Koch of the tubercle and cholera bacilli, have claimed the attention of the whole thinking world. Those bacteria which are connected with disease, and more especially those which have been proved 2 BACTERIOLOGY. to be the causa, if not the actual materies morbi, are of predominant interest and importance. The first attempt to demonstrate the germ theory of disease dates back almost to the discovery of the microscope. Athanasius Kircher, nearly two and a half centuries ago, expressed his belief that there were definite micro-organisms to which dis- eases were attributable. The microscope had revealed that all decomposing substances swarmed with countless micro-organisms which were invisible to the naked eye, and Kircher sought for similar organisms in diseases which he considered might be due to their agency. The microscope which he described * obviously could not admit of the possibility of studying, or even detecting, the micro- organisms which are now known to be associated with certain diseases ; and it is not surprising that his teachings did not at the time gain much attention. They were destined, however, to re- ceive a great impetus from the discoveries which emanated from " the father of microscopy." Antony van Leeuwenhoek had learned as a youth to grind and polish lenses, and later in life employed his spare time in constructing micro- scopes, and in conducting those researches which have made for him a name which is familiar to all microscopists. His researches were published in a series of letters to the Royal Society. In 1675 ne described extremely minute organisms in * Ars Magna Lucis et Umbrcs, 1646. THE GERM THEORY. 3 rain-water, well-water, infusions of pepper, hay, and other vegetable and animal substances, in saliva, and in scrapings from the teeth ; and, further, he was able to differentiate these minute living things by their size, their form, and the character of their movements. In 1683 these discoveries were illus- trated by means of woodcuts, and there can be little doubt, from the drawings of these micro- organisms, that they are intended to represent leptothrix filaments, vibrios, and spirilla. Indeed, we can almost recognise these micro-organisms as bacteria from Leeuwenhoek's graphic descriptions, apart from his figures. They were described as moving in the most characteristic manner, pro- gressing with great rapidity, or spinning round like a top, and as so excessively minute that they were only perceived with great difficulty. The smallest forms could hardly be examined indivi- dually; but, viewed en masse, they closely resembled a swarm of gnats or flies. In another communica- tion, published in 1692, he gives some idea of the size of these animalcules by stating that they were a thousand times smaller than a grain of sand. Others which, comparatively speaking, were of considerable length, were characterised by their peculiar mode of progression, bending and rolling on themselves, movements which, he adds, created both delight and astonishment in the mind of the observer. Leeuwenhoek himself was not disposed to believe in the possibility of such organisms 4 BACTERIOLOGY. being found in the blood in disease ; but as soon as he had proved the actual existence of such minute creatures, theoretical physicians were not wanting who at once attributed various maladies to their agency. Among these, Nicholas Andry is made conspicuous by his work published in 1701.* Andry classed the minute organisms dis- covered by Leeuwenhoek as worms. In 1718 Lancisi believed that the deleterious effect of the air of malarial districts depended upon animalcules, and others considered that the plague in Toulon and Marseilles in 1721 arose from a similar cause. In fact, by some, all diseases were attributed to vermicules, and this led to the theory being ridiculed and dis- credited. In spite of adverse criticism, the theory of con- tagium vivum survived, and Linnaeus acknowledged it by placing the micro-organisms discovered by Leeuwenhoek, the contagia of specific fevers, and the causes of putrefaction and fermentation, into one order " chaos." The theory was further sup- ported by the writings of Plenciz, who, in 1762,! very ably discussed the nature of contagium, as well as the relation of animalcules to putrefaction and disease. However, no proofs in support of these theories were forthcoming, and gradually the * De la Generation des Vers dans le Corps de VHomme. t Opera Medico- Physica, Tractatio de Contagio, de Lue Bovina, de Variolis ; de Scarlatina. THE GERM THEORY. 5 idea of contagium vivum fell into obscurity, and indeed came to be regarded by some as an absurd hypothesis. Though a causal relation of animalcules to dis- eases was for a time discredited, the natural history of these micro-organisms was studied with increas- ing interest. In 1778 Baron Gleichen* described and figured a great number of micro-organisms which he had discovered in various vegetable infusions. Joblot, Lesser, Reaumur, Hill, and many others worked at the same subject. Hillf remarked that there was hardly the least portion of matter or the least drop of fluid of any kind naturally found in the earth which was not in- habited by multitudes of animalcules. But these observers inclined rather to searching for new forms than to studying more thoroughly those which had been already discovered ; and, as a result, but little scientific progress was made until the time of Miiller, of Copenhagen. Miiller, in 1786, criticised the work of these previous writers, and pointed out that they had been too much occupied with merely finding new micro-organisms. Miiller took into account the form of the micro- organism, its mode of progression, and other biological characters, and on such data based a classification. Thus the scientific knowledge of * Dissertation sur la Generation, les Animalcules Sfiermatiques et ceux d? Infusions . f Essays in Natural History and Philosophy. O BACTERIOLOGY. these minute beings was considerably advanced by his writings and his illustrations. The subject which now eclipsed all others in interest was the origin of these micro-organisms. Two rival theories were widely discussed spon- taneous generation and development from pre-exist- ing germs ; and the researches that were made in the course of this discussion, and the discoveries which resulted, indirectly materially advanced the germ theory of disease, and explain many of the phenomena in the life-history of the pathogenic microbes which have been brought to light in recent years. Spontaneous development of micro-organisms in putrescible infusions was believed in by many, but was supported by no one with greater per- sistency than Needham. Needham found, if meat infusion were boiled and transferred to a well- stoppered flask, that animalcules readily developed, and he could only explain this by supposing that they originated spontaneously from the material of the infusion. In 1768 Bonnet* strenuously op- posed these conclusions on purely theoretical grounds, and maintained that it was far more probable that the ova of the animalcules were present in the infusions or were suspended in the air enclosed in the flask. Spallanzani was the first to demonstrate by ex- periment the correctness of Bonnet's arguments * Considerations sur les Corps Organises. THE GERM THEORY. 7 It occurred to him to boil the infusion in flasks, and to seal the vessels during the process of boiling. As a result the flasks remained free from putre- faction, and animalcules only developed when the infusion was exposed to the air by making a hole in the flask. That Spallanzani's experiments were reliable, and his conclusions correct, was evidenced by the fact that his simple precaution led to great practical results, as Francois Appert introduced on this principle the method of preserving meats, vegetables, and other provisions. The disciples of Needham nevertheless brought forward counter objections. Treviranus urged that a certain quantity and quality of air was necessary for the spontaneous development, of these infusoria, and that by sealing the flasks too small a quantity of air was in contact with the infusion, and, further, that this air had become changed in quality by the process of boiling. Spallanzani argued against these objections, but did not support his opinions by further experi- ments, so that the question remained for a time undecided. In 1836 Francis Schulze devised an experiment which brought still further evidence against Need- ham's theory. Schulze filled a glass vessel half full with distilled water and different animal and vegetable substances. This was plugged with a doubly bored cork, and through each perforation a glass tube was introduced, bent at a right angle. 8 BACTERIOLOGY. On boiling the flask, steam issued freely from each tube, and all parts were thoroughly sterilised. Each tube was then connected with a bulbed tube, one bulb containing concentrated sulphuric acid, and the other a solution of potash. By aspiration fresh air was drawn into the flask, and this was deprived of any germs which might be present by its passage through the sulphuric acid. The result was that the infusion remained without any development of micro-organisms. If, on the other hand, air were admitted without first being drawn through the sulphuric acid, the infusion in a short time teemed with animalcules. In other words, Schulze demonstrated that in spite of free access to air, which had not been heated, the infusions remained free from germs. Schwann, in 1837, arrived at similar results. He found that putrescible substances remained sterile if exposed to an abundant supply of air which was heated by being passed through a melted mixture of metals. This convinced him that the cause of the decomposition which would otherwise have oc- curred must exist in the air. The objection remained that in the experiments of Schulze and Schwann, the air which was ad- mitted to the flasks had undergone either a chemical or a thermal change, and therefore the theory of Needham was not yet entirely disposed of. In 1854 the final blow was dealt by Schroeder and Van Dusch. These investigators demonstrated THE GERM THEORY. 9 that decomposition could be obviated without resort- ing either to thermal or chemical treatment of the air, as simple filtration of the air through cotton- wool was shown to be efficacious in excluding germs. Finally, Hoffman, in 1860, and, indepen- dently, Chevreuil and Pasteur, in 1861, showed that even cotton-wool could be dispensed with, as a sterile solution might be kept sterile if the neck of the vessel were bent with an S-shaped curve. Micro-organisms in the air entering the flask, were deposited by gravitation in the bend of the tube. The advocates of spontaneous generation were ready with fresh objections. They now urged that the medium lost its power of undergoing decom- position by being boiled. This objection was at once set aside by the fact that if unfiltered air were admitted to the infusion decomposition set in. Additional evidence was brought against spon- taneous generation by the experiments of Pasteur, Burdon Sanderson, Lister, and others, in which it was shown that blood, urine, and milk would remain without decomposition, if all precautions were adopted to avoid contamination in filling the sterilised flasks. Even at this stage of this great scientific con- troversy fresh difficulties arose, for it was found that in certain solutions which had been boiled and hermetically sealed in flasks micro-organisms made their appearance. In 1872 Charlton Bastian 1O BACTERIOLOGY. published a research * which was to prove that spontaneous generation actually took place. Decoctions of turnip and cheese which had been filtered, neutralised, and boiled for ten minutes, and hermetically sealed during the boiling, were found after a time to contain micro-organisms. These re- sults, however, were before long explained by the fact that in milk, infusions of hay, and certain other decoctions, the spores of bacilli are present, which are much more resistent than the bacilli themselves. In such cases mere scalding or boiling for a few minutes will not sterilise the solution. The bacilli are destroyed, but not their spores ; and if the latter remain unhurt, they will germinate, increase, and multiply. But if, as Tyndall found, the boiling be repeated a second and a third time, all the spores will be destroyed ; for in the intervals between the boilings the spores sprout into bacilli, and the bacilli at the next boiling perish ; so that after three or four repeated boilings the infusion is rendered perfectly free from germs. While this discussion was occupying the attention of the whole scientific world, some investigators had been again following up the theory of a connection between micro-organisms and disease. In 1837 Cagniard Latour and Schwann inde- pendently made the discovery that the yeast plant was a living organism, and the true cause of the fermentation. The close analogy between the * Proceedings of the Royal Society. THE GERM THEORY. II processes of fermentation and of certain diseases had long been held ; and, therefore, when it was proved that fermentation was due to a micro-organism, fresh advocates appeared in support of the theory that diseases were produced by similar agencies. Boehm, in 1838, described certain organisms in cholera which was at that time raging in Europe ; but the publication of Bassi, who a year previously had discovered the cause of a disease of silkworms, attracted much greater attention. Bassi discovered .that in this disease extremely minute spores existed on the bodies of the worms, which were conveyed from the sick to the healthy. They destroyed the healthy worms by germinating in their skins and growing into their bodies. These discoveries may be said to have brought the theory of contagium vivum to life again ; and Henle, in reviewing the facts of the case in 1840, came to the conclusion that the cause of all contagious diseases must be of a living nature, and this he maintained, although he had searched in vaccine and small-pox lymph, in the desquamation of scarlet fever, and in other diseases without success. Bassi's discovery and Henle's doctrine encouraged a number of investigators, and remarkable results followed. In favus, in herpes tonsurans, in pity- riasis versicolor, fungus threads and spores were found, and were regarded as being of etiological importance, inasmuch as the morbid lesions corre- sponded with the growth of the particular fungus. 1 2 BACTERIOLOGY. Cholera became especially a subject for research. Swaine, Brittan, and Budd found micro-organisms in choleraic dejecta. Davaine described certain monads in the intestinal contents, but no causal connection was established between these organisms and the disease ; and when the cholera disappeared the interest in contagium vivum waned, and was eclipsed by the question of fermentation. The discoveries which followed in this subject had, as we shall see, a very important bearing on the micro-parasitic origin of communicable diseases. Pasteur followed up the researches of Cagniard Latour and Schwann, and in 1857 demonstrated that the lactic, acetic, and butyric fermentations were also produced by micro-organisms. Previous to this, in 1850, Davaine and Rayer had noted the existence of little rod-like or filamentous bodies about the size of a blood corpuscle in the blood of a sheep that had died of splenic fever. Pollender had seen similar bodies in the blood of cows. Davaine did not pay much heed to his discovery ; but in 1863 he thoroughly reinvestigated the sub- ject, and conducted a series of experiments which led him to the conclusion that the actual cause of splenic fever was an organised being whose presence and multiplication in the blood produced changes in that fluid of the nature of fermentation, resulting in the death of the animal. THE GERM THEORY. 13 These conclusions were not accepted by all ; and, indeed, the evidence was so far incomplete that the sceptic was justified in considering that these experiments afforded only a working hypothesis. But Davaine's comparison between this disease and fermentation attracted the attention of Pasteur whose mind had been fully trained for entering upon this investigation by the researches which he had been carrying on in the interval between Davaine's publications of 1857 and 1863. Pasteur, as already mentioned, had been working at fermentation, and his attention was next directed to studying the so-called diseases of wines, and subsequently to a contagious disease which com- mitted ravages among silkworms. By laborious researches Pasteur was able to confirm the belief that the disease was due to the presence of micro- organisms only discernible with the aid of the microscope. These oval shining bodies in the moth, worm, and eggs had been previously observed by Cornalia, and described by Naegeli as Nosema bombycis, and by Lebert as Panhistophyton. But it was reserved for Pasteur to introduce a means of combating the disease. Pasteur showed that if a silkworm, whose body contained these micro- organisms, were pounded up with water in a mortar, and the mixture painted with a brush on the leaves on which healthy worms were fed, they would all without fail succumb to the disease. As the contagious particles were transmitted to 14 BACTERIOLOGY. the eggs, a method for preventing the spread of the disease suggested itself. Each female moth was kept separate from the others, and allowed to deposit her eggs on a small linen cloth. The moth was then pinned to the corner of the cloth, and left for future examination. When the time for this arrived, the moth was crushed up with water in a mortar, and a drop examined under the microscope. If any trace of corpuscular matter were found to be present, the cloth with its collection of eggs was burnt ; and if not, the eggs were set aside for use. Complete as this appears to be as a demonstra- tion of a causal connection between the micro- organisms and the disease, it could obviously be objected that there was no distinct proof that the corpuscular bodies constituted the actual contagium. There was no isolation of the organisms, no artifi- cial cultivation of them apart from the diseased moth or worm, and subsequent production of the disease by means of the isolated organisms. The same ob- jection was applicable to Davaine's investigations. Davaine found the rods in association with the disease, and maintained that they were causally related ; but others stated that it was possible to inoculate animals with anthrax blood containing rods, and to produce the disease without being able to detect the rods again in the blood of the animal experimented upon. It was also urged that it was possible to infect with anthrax blood after the rods THE GERM THEORY. 15 had disappeared, and to find a reappearance of the bacilli in the blood of the inoculated animal. What appeared also to lend great support to these objections was the well-known fact that anthrax or splenic fever was especially prevalent in certain seasons and certain localities. The disease, in fact, was regarded by some as originating from peculiar conditions of climate and soil. The fallacies in these objections were, however, rapidly dispelled. Bollinger, in 1872, pointed out that the blood, from which the rods had disappeared, was still virulent owing to the presence of the spores of the bacillus, and that it was owing to the soil being impreg- nated with these spores that the disease broke out in certain localities. But there still remained many who refused to regard these particles as living bodies, some looking upon them simply as crystals, and the question of their importance remained un- decided for several years. In 1877 Robert Koch published a memoir in which he fully described the life-history of the anthrax or splenic fever bacillus, and gave a com- plete demonstration of the life-history of the micro- organism, and the definite proofs of its pathogenic properties. He pointed out how quickly the rods grew in the blood and tissues by lengthening and by cross division. Further, that in the blood or in serum or in aqueous humour they grew into long leptothrix filaments, and formed numerous shining spores. He traced, by continuous observation on 1 6 BACTERIOLOGY. the warm stage, the whole life cycle from the fission of the rods to the formation of spores and the sprouting of the spores into fresh rods. Further, he carried on the disease by inoculating from mouse to mouse for several generations, and observed that in the blood of the animal and in the swollen spleen the glass-like rods were always to be found. Pasteur also studied the microbe of splenic fever, and amply confirmed and extended the observations of Koch by his researches on the attenuation of the anthrax virus. Pasteur also met with adverse criticism. Paul Bert argued that the bacilli were of no importance, because he could destroy them by exposing material containing them to great pressure, and yet the ma- terial produced the disease on inoculation. But such measures did not destroy the spores ; and finally Paul Bert was convinced of his error when Pasteur demon- strated cultures of the anthrax bacillus in urine, from which successive generations were started, and that with such cultivations the disease could always be produced. It was, however, principally the researches of Koch which placed the doctrine of contagium vivum on a scientific basis. Koch's improvements in the methods of cultivation, his recommendation of the necessary microscopical apparatus, his histological methods for examining these minute organisms, and his famous postulates for proving beyond controversy the existence of THE GERM THEORY. I/ specific pathogenic micro-organisms, elevated the theory of contagium vivum to a demonstrated and established fact. The chain of evidence regarded by Koch as essential for proving the existence of a pathogenic organism was composed as follows : 1. The micro-organism must be found in the blood, lymph, or diseased tissue of man or animal suffering from or dead of the disease. 2. The micro-organisms must be isolated from the blood, lymph, or tissues, and cultivated in suitable media, i.e., outside the animal body. These pure cultivations must be carried on through successive generations of the organism. 3. A pure cultivation thus obtained must, when introduced into the body of a healthy animal, pro- duce the disease in question. 4. Lastly, in the inoculated animal the same micro- organism must again be found. It is, however, impossible by localising one's knowledge to pathogenic species to thoroughly understand the life-history of these particular forms, or to be able to grasp and appreciate the various arguments and questions that arise in comparing their life-history with the progress of disease. Nor is it sufficient to know only how to recognise and artificially cultivate a bacterium associated with disease ; we must endeavour to establish the exact relationship of the bacterium to the disease in question. 1 8 BACTERIOLOGY. The determination of the true pathogenic microbe is beset with fallacies. In many diseases bacteria have been regarded as the actual contagia, until a searching inquiry by other investigators has shown that the evidence was most unsatisfactory or entirely mis- leading. For example, in diseases with lesions of the external or internal linings of the body, extra- neous micro-organisms may get into the circulation and be swept into the internal organs, where they either perish in the battle with the healthy tissues which are opposed to their existence, or they may gain the upper hand and set up destructive processes. Such organisms, when found in association with these diseases, may be discovered in the blood and internal organs ; and though accidental epi- phytes, often associated with septic complication, they may only too readily be accepted by the enthusiast as the actual contagium of the disease in question. It is only when such fallacies are exposed that we are brought once more face to face with the fact that the nature of the contagium in many diseases, such as hydrophobia, variola, vaccinia, scarlet fever, and measles, is at the present day undetermined. Such cases invite further research ; but these fallacies must be borne in mind, or the application of bacteriology to the elucidation of the germ theory of disease will not only be misleading, but tend to retard rather than assist the progress of science. Plate 1 fcucing p.18. P>/VCT~RIA, SCHIZOMYCETES OR FISSION- FUNGI. K I , ' "r-cokshavnJc. del. Vwuxnblfroo'ks, Doty & Son. THE GERM THEORY. 19 Koch's postulates naturally suggest a sequence in the various processes which must be adopted in a practical study of micro-organisms associated with disease. Inasmuch, however, as these processes embrace those which are employed in the isolation, cultivation, etc., of non-pathogenic species, we shall, in studying the bacteria as a whole, adopt the order suggested. After an introduction to the apparatus commonly employed in a bacteriological laboratory, we shall describe the methods of examining liquids, tissues, etc., and the means of recognising micro- organisms. Then will follow the methods of isolat- ing these micro-organisms from such liquids, tissues, etc., and of carrying on pure cultivations in nutrient media. Lastly, we shall refer briefly to experimental researches on the living animal, and the means of isolating micro-organisms from the liquids and tissues of the body after death. In Part II. will be found chapters upon the General Biology of bacteria, and in Part III. a chapter upon their classification, followed by a description of each species, more particularly of those of pathological interest, with a detailed account of the special methods of examination and of staining employed for demonstrating the different species. In the Appendix a descriptive list of important yeasts and moulds will be given, with any special technique required in their case. Yeasts and moulds are constantly encountered in the investigation of 2O BACTERIOLOGY. the bacteria in air, soil, and water ; and several are of interest in being, like many bacteria, micro- organisms associated with disease. An account is given of Actinomycosis, of Flagellated Protozoa which have been found in the blood of animals, and of the micro-organisms associated with malaria. PART I. GENERAL METHODS. CHAPTER I. APPARATUS, MATERIAL, AND REAGENTS EMPLOYED IN A BACTERIOLOGICAL LABORATORY. (A) HISTOLOGICAL APPARATUS. Microscope. For the investigation of micro- organisms a good microscope with oil-immersion system and a condenser, such as Abbe's, is essential. Such instruments are supplied by Leitz, Zeiss, and Hartnack in Germany, and Powell & Lealand, and Swift & Son, in England. Zeiss' microscope, with y 1 ^ and ^ 8 oil-immersion lenses, or Powell & Lealand's, with T T ^ and -f^, is recommended for investigators ; while Swift's or Leitz', with y 1 ^, is a serviceable and economical one for students.* In addition to the usual microscopic fittings, Zeiss supplies a micrometer eyepiece, with directions for use. Some such arrangement is essential for the measurement of bacteria. Other accessories to the microscope are A large bell-glass for covering the microscope when not in use. About a foot square of blackened plate-glass. * Leitz', with ^, costs about ^15 ; Zeiss', with the same, ^30, and with T V, 20 more. Refer to foot-note on p. 61. 24 BACTERIOLOGY. A white porcelain slab of the same size. Glass bottles with ground-glass stoppers, for alcoholic solutions of aniline dyes, etc. Glass bottles with funnels, for aqueous solutions of the dyes, and others provided with pipettes. A small rod-stoppered bottle of cedar oil. This is recommended by Zeiss in preference to other oils for his immersion lenses. Set of small glass dishes or capsules, and watch-glasses for section staining, etc. Stock of best glass slides, in packets of fifty. Several boxes of round and square thin cover-glasses, in various sizes, of the best quality. Needle-holders, with a couple of platinum needles, and a packet of ordinary sewing-needles. Glass rods drawn out to a fine point ; useful for manipu- lating sections when acids are employed. Copper lifters, preferably plated. One pair of small brass or spring-steel platinum-pointed forceps, for holding cover-glasses. One pair of brass tongs. Collapsible tubes for containing Canada balsam ; very serviceable for transport and general use. Turn-table, for sealing cover-glass preparations with rings of cement. Boxes for preparations, book-form. Tickets and labels, various sizes. Soft rags or old pocket-handkerchiefs, for removing cedar oil after use of immersion lens, cleaning cover-glasses, etc. Chamois leather for wiping lenses. Microtome. Schanze's is much in favour in Germany; but Jung's, of Heidelberg,* though a * Price lists may be obtained from any of the above-mentioned firms, from which an idea of the instruments can be formed, and a comparison of the prices made. APPARATUS, MATERIAL, AND REAGENTS. 25 somewhat cumbrous instrument, is preferred by others. Smaller accessories, which should be within reach, are A small can of sewing-machine oil. A soft rag and chamois leather, for wiping the knives immediately after use. Stone and leather for setting and sharpening the same. Two or three camel's-hair brushes. A Freezing Microtome is very useful. The method of embedding in celloidin may be used with advantage in combination with the ordinary process of freezing. (B) REAGENTS AND MATERIAL EMPLOYED IN THE PROCESSES OF HARDENING, DECALCIFYING, EM- BEDDING, FIXING, AND CUTTING OF TISSUES. Alcohol, absolute. Bergamot oil. Celloidin. Dissolved in equal parts of ether and alcohol. Cork, or stock of ready-cut corks. Ebner's solution. A mixture in the following proportions : Hydrochloric acid . . ...y B ,$jc '5 Alcohol . . f . [f ' . . 100 Distilled water . . ' .' " ' . " 20 Chloride of sodium . . . .5* 26 BACTERIOLOGY. Gelatine. Melted in a small porcelain capsule, and set aside ready to be re-melted when required for use. Glycerine gelatine (Klebs). Best well-washed gelatine . . 10 Add distilled water, allow gelatine to swell up, pour off excess of water, melt gelatine with gentle heat, add Glycerine . . . . , , \ v - 10 Lastly, a few drops of phenol for preservation. Gum. Kleinenberg's solution. Saturated watery solution of picric acid . . ^ < 01 " 3:/ y 100 Strong sulphuric acid ., . ^ 2 Filter and add Distilled water . . . v 300 Miiller's fluid. Bichromate of potash . a! ( >V{q? 2 Sulphate of sodium . r . . i Distilled water . ' . . " '. ' 100 Osmic acid. Distilled water .. , .. .' '- . 100 Osmic acid . ., -. . . ->/i ! -5 Paper trays (small or glass-capsules). Paraffine. APPARATUS, MATERIAL, AND REAGENTS. 27 Spermaceti. Xylol. Hardening and decalcifying solutions should be kept in stock in quantities according to require- ment. A jacket of brown paper should be pasted round a well-stoppered bottle to contain osmic acid to efficiently protect it from light, and it should be kept in a cool place. The celloidin solution may be kept in stock in a wide-mouthed glass bottle, from which small wide-mouthed bottles may be filled according to the number required. To put several pieces of different tissues in the same bottle leads to confusion. (c) REAGENTS FOR EXAMINING AND STAINING MICROSCOPICAL PREPARATIONS. 1. Acetic acid, strong. 2. Alcohol, absolute. 3. Alcohol, 60 per cent. 4. Alcohol, acidulated. Alcohol . . . . . :. > .100 Hydrochloric acid . . . . I 5. Alum carmine (Grenacher). Carmine . . * . ^ fc r.& :[ i '.. Five per cent, solution of alum . 100 Boil twenty minutes, filter when cold. 28 BACTERIOLOGY. 6. Ammonia, strong. 7. Aniline. 8. Aniline water. Distilled water ..,,.; 100 Aniline . . . , .... . .,- , , :. .- 5 Shake well and filter emulsion. 9. Bismarck brown. (a) Concentrated solution in equal parts of gly- cerine and water. (6) Aqueous solution. Bismarck brown ' . . ; '. <: i 2 Alcohol . . . ' . . . 15 Distilled water . . ; . :/ . 85 10. Borax carmine (Grenadier). Borax . . " . . ". " . 2 Carmine . . . ' . / . 5 Distilled water .ar -:'^ b'?<. . 100 To the dark purple solution add a 5 per cent, solution of acetic acid until a red colour is produced ; set aside twenty-four hours, filter, and add a drop of carbolic acid. 11. Cedar oil. 12. Eosin. (a) Saturated alcoholic solution. (b) Aqueous solution. Distilled water > C-v L : . - : v : ' * K 1 - 100 Eosin . . &&* '%r7;f^-- ir^ APPARATUS, MATERIAL, AND REAGENTS. 29 13. Ether. 14. Fuchsine. (a) Saturated alcoholic solution. (d) Aqueous solution. Fuchsine ..... 2 Alcohol . . . '..-' : .- . 15 Water . . . . . ^ . 85 15. Gentian violet. a) Saturated alcoholic solution. Aqueous solution. Gentian violet . . . . 2*25 Distilled water . . . .100 1 6. Gibbes' solution, for double staining. Take of Rosaniline hydrochlorate ... 2 Methylene blue i Triturate in a glass mortar, Dissolve aniline oil . . . . 3 In rectified spirit . . . .15 and add slowly to the above. Lastly, slowly add distilled water . ... 15 Keep in stoppered bottle. 17. Glycerine, pure. 30 BACTERIOLOGY. 18. Hsematoxylin solution. Hsematoxylin ..... 2 Alcohol .... ?' . 100 Distilled water .;.*. . . 100 Glycerine . . . . .100 Alum ...... 2 19. Iodine solution. Iodine, pure . . . . . i Iodide of potassium ... 2 Distilled water . . . . 50 20. Iodine solution (Gram). Iodine ...... i Iodide of potassium . . . 2 Distilled water . . . . 300 21. Lithium-carmine solution (Orth). Saturated solution of carbonate of lithium . , . / , . 100 Carmine . . . . \ *.' t . 2*5 22. Magenta solution (Gibbes). Magenta . . , ; -v^ . . 2 Aniline oil . . . .r. . . 3 Alcohol (Sp. Gr. '830) '. > . 20 Distilled water f ; ; v . 20 23. Methylene blue. (a) Concentrated alcoholic solution. APPARATUS, MATERIAL, AND REAGENTS. 3! (6) Aqueous solution. Methylene blue .... 2 Alcohol . . . '1~ . 15 Water - . . Min . $& ^ 85 (c) Koch's solution. Concentrated alcoholic solution of methylene blue ,. ,. . .- i Ten per cent, potash solution . V' v 2 Distilled water . . :..,-, f , M r . 200 (a) Loffler's solution. Concentrated alcoholic solution of methylene blue . . . * 30 Solution of potash i 10,000 . C V r 100 24. Methyl violet. (a) Concentrated alcoholic solution. (b) Aqueous solution. Methyl violet . . ;> _ }J > : : -,;. 2*25 Distilled water -. . . . 100 (c) Koch's solution. Aniline water .... ' ; . i; 100 Alcoholic solution of methyl violet ! : iil n Absolute alcohol . . . .10 25. Neelsen's solution. Dissolve fuchsine . . . . i In alcohol ..... 10 Add a 5 per cent, watery solution of carbolic acid. 100 32 BACTERIOLOGY. 26. Nitric acid, pure. 27. Orseille (Wedl). Dissolve pure ammonia-free orseille in Absolute alcohol . . . .20 Acetic acid . . ... . 5 Distilled water .' V u /'."' '. 40 until a dark red liquid results : filter. 28. Picric acid. (a) Concentrated alcoholic solution. (b) Saturated aqueous solution. 29. Picro-carmine (Ranvier). Carmine . . *. ,-. i Distilled water .:, . . 10 Solution of ammonia . . 3 Triturate, add cold saturated solution of picric acid . .-' . : . 200 30. Picro-lithium-carmine (Orth). To above-mentioned lithium-carmine solution add Saturated solution of picric acid . 2 "3 31. Potash solution. (a) i to 3 per cent. (6) 10 (') 33 , APPARATUS, MATERIAL, AND REAGENTS. 33 32. Safranine. (a) Concentrated alcoholic solution. (b) Watery solution . /.. i per cent. 33. Sulphuric acid, pure. 34. Salt solution . . . 0*8 per cent. 35. Turpentine. 36. Vesuvin. (a) Concentrated alcoholic solution. (6) Watery solution. Water, distilled. Water, sterilised. } Distilled water can be kept for use in a wash bottle, or far better in a siphon apparatus. Steril- ised water is convenient in plugged sterile test-tubes, which may be kept close at hand in a beaker, or tumbler, with a pad of cotton wool at the bot- tom. The numbered reagents can be conveniently arranged on shelves within easy reach. Alcoholic solutions of the aniline dyes and other special preparations should be kept in bottles with ground- glass stoppers. Aqueous solutions of the dyes may be kept in bottles with funnel filters, and the solution filtered before use. To both aqueous and alcoholic solutions a few drops of phenol, or a 3 34 BACTERIOLOGY. crystal of thymol, should be added as a pre- servative. For the rapid staining of cover-glass preparations, it is convenient also to have the most frequently used stains (fuchsine, methyl violet) in bottles provided with pipette-stoppers. (D) REAGENTS FOR MOUNTING AND PRESERVING PREPARATIONS. Acetate of potash. Concentrated solution. Asphalte lac. Canada balsam. Dissolved in xylol. Glycerine gum (Farrant's solution). Glycerine. Water. Saturated solution of arsenious acid. Equal parts, mix and add of picked gum arabic half a part. Hollis' glue. Zinc-white. (E) DRAWING AND PHOTOGRAPHIC APPARATUS. Camera Lucida. The camera lucida of Zeiss is an excellent instrument, though many prefer APPARATUS, MATERIAL, AND REAGENTS. 35 the pattern made by Nachet of Paris. Combined with the use of a micro-millimeter objective, it affords also a simple method for the measurement of bacteria. For drawing macroscopical appearances, and for illustrating microscopical specimens with or without the use of a camera lucida, the following materials should be within reach : Pencils. Etching Pens. Prepared Indian Ink. Water-colour Paints and Brushes. Ordinary and tinted drawing paper and other usual accessories. Photo-micrographic Apparatus. Zeiss of Jena, Seibert & Kraft of Wetzlar, Nachet of Paris, and Swift & Son of London, may all be recom- mended for constructing an arrangement in which the photographic camera is combined with the microscope. For illumination either sunlight or artificial light may be employed. In the case of sunlight a helio- stat is necessary to procure the best results ; but as sunlight is not always available by day, and it is also more convenient for many to work at night, it is better to have recourse altogether to artificial light. Excellent results may be obtained with an ordinary paraffine lamp, or with magnesium, oxycal- cium, or electric light. Specimens are preferably stained yellow, brown, or black, and for mounting 36 BACTERIOLOGY. the preparations Koch * recommends a saturated solution of acetate of potash ; but there is little or no objection to the use of Canada balsam dissolved in xylol. Hauser,t who employed the electric light, obtained some excellent pictures of preparations mounted in balsam. Van Ermengem J first recom- mended the isochromatic dry plates, and produced most successful results with the lime-light from objects stained with fuchsine and methyl violet. The author also has investigated the applicability of photographic processes for illustrating micro- organisms. Numerous preparations have been satisfactorily depicted by means of the isochromatic plates without any reference to the staining reagents employed. For a full description of the apparatus and methods employed the reader is referred to the author's publication. (F) STERILISATION APPARATUS. Steam-steriliser. A cylindrical vessel of tin about half a metre or more in height, jacketed with thick felt, and provided with a conical cap or lid (Fig. i). The lid is also covered with felt, has handles on either side, and is perforated * Koch, Verfahren zur Untersuchung zum Conserviren und Photographiren der Bacterien . 1877. t Hauser, Ube r Faulniss Bacterien und deren Beziehungen zur Septicamie. 1885. J Van Ermengem, BulL de la Soc. Beige deMicroscojbie, No. X., pp. 170-2. 1884. Photography of Bacteria. 1887. APPARATUS, MATERIAL, AND REAGENTS. 37 at the apex to receive a thermometer. Inside the vessel is an iron grating or diaphragm about two-thirds the way down, which divides the in- terior into two chambers the upper or "steam chamber," and the lower or " water-chamber." A gauge outside marks the level of the water in the lower chamber ; this should be kept about two- thirds full. The apparatus stands upon three legs, and is heated from below with two or three Bunsen, or better, a Fletcher's burner. It is employed for sterilising nutrient media in tubes or flasks, for cook- ing potatoes, or hastening the filtration of agar-agar. When the thermometer indicates 100 C. the lid is removed, and test- tubes are lowered in a wire basket by means of a hook and string, and the lid quickly replaced. Potatoes or small flasks are lowered into the cylinder in a tin receiver with a perforated bottom, which rests upon the grating and admits of its contents being exposed to the steam. Hot-air Steriliser. A cubical chest of sheet iron with double walls, supported on four legs ; it may also be suspended on the wall of the laboratory, with a sheet of asbestos intervening (Figs. 2 and 3). It is heated with a rose gas-burner from below, FIG. i. KOCH'S STEAM-STERILISER. BACTERIOLOGY. and the temperature of the interior indicated by a thermometer inserted through a hole in the roof; in a second opening a gas regulator can be fixed. Test-tubes, flasks, funnels, cotton wool, etc., may be sterilised by exposure to a tem- perature of 150 C. for an hour " or more. FIG. 2. HOT-AIR STERILISER. FIG. 3. SECTION OF HOT-AIR STERILISER. (G) APPARATUS AND MATERIAL FOR PREPARING AND STORING NUTRIENT GELATINE AND NUTRIENT AGAR-AGAR. Water-bath. A water-bath on tripod stand is required for boiling the ingredients of nutrient jellies and for general purposes. The lid may be conveniently composed of a series of concentric rings, so that the mouth of the vessel may be graduated to any size required. APPARATUS, MATERIAL, AND REAGENTS. 39 Test-tube Water-bath. This consists of a circular rack for test-tubes within a water-bath. It is sometimes employed instead of the steam cylinder for sterilising nutrient jelly in tubes, by boiling for an hour for three successive days. Hot-water Filter. A copper funnel with double walls, the interspace between which is filled with hot water. A glass funnel fits inside the copper cone, the stem of the glass funnel passing through and being tightly gripped by a perforated caoutchouc plug, which fits in the opening at the apex of the cone. The water in the cone is heated by applying the flame of a burner to a tubular pro- longation of the water chamber. In a more re- cent model, as represented in Fig. 4, this prolonga- tion is dispensed with, and FIG the temperature is main- HOT-WATER FILTERING APPARATUS WITH RING BURNER. tained by means of a circular burner which acts at the same time as a funnel ring. Glass Vessels. A number of glass vessels should be kept in stock according to requirements. 40 BACTERIOLOGY. Bohemian hard glass flasks are employed in several sizes, for boiling nutrient media. The conical forms are especially used in the larger sizes for storing nutrient jelly. Glass funnels large and small are necessary, not only in the processes of preparing nutrient jelly, but for filtering solutions of aniline dyes and for general purposes. A liberal supply of test-tubes should always be kept in stock, as they are not only employed for the tube-cultivations, but can be conveniently used for storing bouillon, sterilised water, etc. Cylindrical glasses graduated in cubic centi- metres, 10 ccm., 100 can., 500 ccm., are required for measuring the liquid ingredients of nutrient jelly, and also in preparing the various staining solutions. A large wide-mouthed glass jar, with a glass cover, is extremely useful. It must be padded at the bottom with cotton wool for containing a stock of tubes of sterilised nutrient jelly, and should be placed within reach on the working table. Balance and Weights. A balance, with large pans and set of gramme weights, is constantly required. Cotton Wool. The best or " medicated " cotton wool should be procured. Gelatine. The gelatine for bacteriological purposes must be of the very best quality (gold .label). APPARATUS, MATERIAL, AND REAGENTS. 41 Agar-agar. This is also called Japanese Isin- glass ; it consists of the shrivelled filaments of certain Algse (Gracilaria lichenoides and Gigartina speciosa).^ Peptonum Siccum (German). Table Salt. Prepared table salt can be ob- tained in tins or packets. Litmus Papers. Blue or red litmus paper in cheque books, for testing the gelatine mixture, etc. Carbonate of Soda. A bottle, containing a saturated solution of carbonate of soda, and provided with a pipette-stopper, may be kept, especially for use in the preparation of nutrient jelly. Lactic Acid. Filter Paper. For filtering gelatine stout Swedish filter paper of the best quality is recom- mended. Flannel or Frieze. This is employed as a substitute for, or combined with, filter paper in the preparation of nutrient agar-agar. (H) APPARATUS FOR EMPLOYMENT OF NUTRIENT JELLY IN TEST-TUBE AND PLATE-CULTIVATIONS. Wire Cages. These cages or crates are used for containing test-tubes, especially when they are * Hueppe, Die Methoden der Bakterien Forschung. 1885. 42 BACTERIOLOGY. to be sterilised in the hot-air steriliser ; or for lowering tubes of nutrient jelly into the steam steriliser, etc. (Fig. 5). Test-tube Stands. The or- dinary wooden pattern, or the metallic folding stands, are called into use for holding cultivations. FIG. 5. WIRE CAGE Pegged racks are also recom- FOR TEST-TUBES. , 1 r , . . , mended for draining test-tubes after washing. Caoutchouc Caps. These are caps for fitting over the cotton-wool plugs, and may be used in different sizes for test-tubes and stock-flasks. Platinum Needles. A platinum needle for inoculating nutrient media, examining cultivations, n rzrrij:- FIG. 6. PLATINUM NEEDLES ; STRAIGHT, HOOKED, LOOPED. etc., consists of two or three inches of platinum wire, fixed to the end of a glass rod. Several of these needles should be made, with platinum wire of various thicknesses. A piece of glass rod, about seven inches long, is heated at the extreme point in the flame of a Bunsen burner, and a piece APPARATUS, MATERIAL, AND REAGENTS. 43 of platinum wire, held near one extremity with forceps, is then fused into the end of the rod. Some needles should be perfectly straight, and kept especially for inoculating test-tubes of FIG. 7. DAMP CHAMBER FOR PLATE -CULTIVATIONS. nutrient jelly. For other purposes the needles may be bent at the extremity into a small hook, and others provided with a loop (Fig. 6). Tripod Levelling-stand. A triangular wooden FIG. 8. APPARATUS EMPLOYED FOR PLATE-CULTIVATIONS. Tripod Stand ; Glass Dish, filled with cold or iced water ; Sheet of Plate-glass ; Spirit Level, and Glass Bell. frame supported upon three screw-feet which enable it to be raised or lowered to adjust the level. Large Glass Plate. A piece of plate-glass, or a pane of ordinary window glass, about a foot square. Spirit Level. Glass Bells and Dishes. Shallow glass bells 44 BACTERIOLOGY. and dishes, for making a dozen or more damp chambers (Fig. 7), and for completing the appa- ratus for pouring out liquefied nutrient jelly on glass plates or slides (Fig. 8). Iron Box. A box of sheet-iron (Fig. 9) for con- taining glass plates during their sterilisation in the hot-air steriliser, and for storing them until required for use. Glass Plates. Small panes of glass, about six inches by four. Not less than three dozen are required for a dozen damp chambers. Glass Benches. These are BOX ^OR GLASS necessary for arranging the glass plates or slides in tiers in the damp chambers (Fig. 10). Metal shelves may be substi- tuted for them, but the former are to be preferred. FIGS. 10, ii. GLASS BENCHES FOR GLASS PLATES OR SLIDES. They can be easily made, in any number required, by cementing a little piece of plate-glass at either end of a glass slip (Fig. n). Glass Rods. One dozen or more glass rods, twelve to eighteen inches in length. They are employed for smoothly spreading out the liquefied nutrient gelatine or agar-agar on the glass plates, etc. Thermometers. Two or three centigrade ther- mometers. APPARATUS, MATERIAL, AND REAGENTS. 45 FIG. 12. ISRAEL'S CASE. (l) APPARATUS FOR PREPARATION OF POTATO- CULTIVATIONS. Israel's Case. Sterilising instruments in the flame of a Bunsen burner is most destructive. It is better, there- fore, to have a sheet-iron case (Fig. 12) to contain potato- knives, scalpels, and other instru- ments, and to sterilise them by placing the case in the hot-air steriliser for an hour at 1 50 C. The box can be opened at the side, and each instrument withdrawn with a pair of sterilised forceps when required for use. Glass Dishes. Several shallow glass dishes are required for preparing damp chambers for potato - cultivations (Fig. 13). The upper, being the larger, fits over the lower, and having no handle, admits of these damp chambers being placed, if necessary, in the incubator in tiers. The large size may also be used in the same way for plate-cultivations. Potato Knives. A common broad smooth- FIG. I 3. DAMP CHAMBER FOR POTATO- CULTIVATION. 4 6 BACTERIOLOGY. bladed knife set in a wooden handle is sold for this purpose. Scalpels. Half a dozen scalpels, preferably with metal handles, may be kept especially for inoculating sterilised potatoes. Brush. A common stout nail-brush, or small scrubbing brush, is essential for cleansing potatoes. (j) APPARATUS FOR PREPARATION OF SOLIDIFIED STERILE BLOOD SERUM. Glass Jar. A tall cylindrical glass jar, on foot, with a broad ground stopper, for receiving blood. Pipette. An ordinary or graduated pipette for transferring the serum from the jars to sterile test-tubes or glass cap- sules. Serum Steriliser. A cylindrical case, with double walls forming an interspace to contain water, closed with a lid, also double walled and provided with a tubular prolongation of the en- closed water chamber (Fig. 14). The water in the cylinder is heated from below, and that in the lid by means of the prolongation. FIG. 14. SERUM STERILISER. APPARATUS, MATERIAL, AND REAGENTS. 47 In the centre of the cylinder is a column which communicates with the water chamber of the cylinder, and from it pass four partitions, which serve to support the test-tubes. In the lid are three openings, one of which com- municates with the water chamber in the lid by which the latter is filled, and into which a thermo- meter is then fixed. In the centre an opening admits a thermometer, which passes into the central pipe of the cylinder ; through a third open- ing a thermometer passes to the cavity of the cylinder. The cylinder and cover are jacketed with felt, and the apparatus is supported on iron legs. Serum Inspissator. A shallow tin case with glass cover, both case and cover jacketed with felt (Fig. 15). The case is double walled, and the water contained in the interspace is heated from below. It is supported on four legs, and the two front ones move FIG I5 ._ SERUM INSPISSATOR . in grooves in the case, so that the latter can be placed obliquely at the angle required, and secured in position by screw-clamps. It is employed for coagulating sterile liquid serum, and for solidifying nutrient agar-agar so as to give them a sloping surface. 48 BACTERIOLOGY. Glass Capsules. Small capsules or hollowed- out cubes of crystal glass are employed for cultiva- tions on solid blood serum, on nutrient gelatine, and on agar-agar. They may be procured of white and blackened glass, and are provided with glass slips as covers. (K) APPARATUS FOR STORING, AND FOR CULTI- VATIONS IN, LIQUID MEDIA. Lister's Flasks. Lister devised a globe-shaped flask with two necks ; a vertical and a lateral one. The lateral one is a bent spout, tapering towards its constricted extremity. When the vessel is restored to the erect position after pouring out some of its contents, a drop of liquid remains behind in the end of the nozzle, and prevents the regurgitation of air through the spout. A cap of cotton wool is tied over the orifice, and the residue in the flask kept for future use. The vertical neck of the flask is plugged with sterilised cotton wool in the ordinary way. Sternberg's Bulbs. Sternberg advocates the use of a glass bulb, provided with a slender neck drawn out to a fine point and hermetically sealed.* Aitken's Test-tube. This is an ingenious device for counteracting the danger of entrance of atmospheric germs on removal from the ordinary * Magnin and Sternberg, Bacteria. 1884. APPARATUS, MATERIAL, AND REAGENTS. 49 test-tube of the cotton-wool plug. Each test-tube is provided with a lateral arm tapering to a fine point, which is hermetically sealed. Drop culture Slides. About a dozen or more thick glass slides with a circular excavation in the centre are required for drop-cultures. Vaseline. A small pot of vaseline with a camel's- hair brush should be reserved especially for use in the preparation of drop-cultures. Bulbed Tubes. Glass vessels such as test- tubes, flasks, and pipettes, which are used in dealing with liquid media, have already been mentioned under other headings ; but bulbed tubes, Pasteur's bulbs, and various other forms are also required for special experiments. (L) APPARATUS FOR INCUBATION. There are several forms of incubator, each of which has its advocates. They are mostly rect- angular chests, with glass walls, front and back, or in front only. A cylindrical model is preferred by some. Two only will be described here D'Arson- val's and Babes'. The former admits of very exact regulation of temperature, and the latter is a very practical form for general use. D'Arsonval's Incubator. The " Etuve UAr- sonval" (Fig. 16) is a very efficient apparatus, and is provided with a heat regulator, which enables the temperature to be maintained with a minimum varia- 4 BACTERIOLOGY. tion. It consists of a cylindrical copper vessel, with double walls, enclosing a wide interspace for con- taining a large volume of water. The roof of the water-chamber is oblique, so that the wall rises higher on one side than on the other. This admits of the inter- space being com- pletely filled with water. At the highest point is an opening fitted with a perforated caou- tchouc stopper, through which a glass tube passes. The mouth of the H cylinder itself is horizontal, and is closed by a lid, which is also double- walled to contain water. In the lid are four openings ; one serves for filling its water-chamber, and the others for thermometers and for regulating the air supply in the cavity of the cylinder. The cylinder is continued below by a cone, also double- walled, and there is a perforated grating at the line of junction of the cylinder and cone. The cone FIG. 1 6. D'ARSONVAL'S INCUBATOR. APPARATUS, MATERIAL, AND REAGENTS. 51 terminates in a projecting tube provided with an adjustable ventilator. The apparatus is fixed on three supports united to one another below. One of them is utilised for adjusting the height of the heat- ing apparatus. Situated above this leg is the heat- regulating apparatus (Fig. 17), attached to a circular, lipped aperture in the outer wall of the incubator. To the lip is fixed with six screws the correspond- ing lip of a brass box, with a tightly stretched diaphragm of india-rubber intervening. Thus the diaphragm separates the Cavity Of the box from the FIG. 17. SCHLOSING'S MEMBRANE REGULATOR. water in the interspace of the incubator. The cap of the box, which screws on, is bored in the centre for the screw-pipe, by which the gas is supplied. Another pipe entering the box from below is connected with the gas-burners. Around the end of the screw-pipe a collar loosely fits, and is pressed against the diaphragm by means of a spiral wire spring. Close to the mouth of the screw-pipe a small opening exists, so that the gas supply to the burners is not entirely cut off even when the diaphragm completely occludes the mouth of the screw-pipe. To work the apparatus the tube and plug must be removed, and the water-chamber filled completely with distilled or rain water at the temperature required. The BACTERIOLOGY. caoutchouc plug is replaced and the tube placed in position. Gas enters through d (Fig. 17), and passes through the opening at its extremity into the chamber of the box. Thence it passes through the vertical exit which is connected with the gas-burners. As the temperature rises the water rises in the tube, and at the same time exercises a pressure on every part of the walls of the incubator, and hence on the diaphragm. In consequence of this, the diaphragm bulging outwards approaches the end of the tube d, and gradually diminishes the gas supply. As a result the temperature falls, the water contracts and sinks in the tube, and the diaphragm receding from d, the gas supply is again increased. By adjusting the position of the tube d to the diaphragm, any required temperature within the limits of the working of the apparatus can be regulated to the tenth of a degree; provided, (i) that the gas supply is rendered independent of fluctua- tions of pressure, by means of a gas-pressure regulator, (2) that the height of the water in the tube is controlled daily by the with- drawal or addition of a few drops of distilled water, and (3) that the apparatus is kept in a place with as even a temperature as pos- sible, and sheltered from currents of air. The burners in Fig. 16 are pro- tected with mica cylinders similar to the burner represented in Fig. 1 8. The flames of these burners can be turned down to the smallest length without danger of extinction, and the temperature may be regulated very satisfactorily without using the heat regulator just described, if the gas first passes through a pressure regulator (Fig. 19). To provide against the danger resulting from accidental extinction of FIG. i 8. GAS-BURNER PROTECTED WITH MICA CYLINDER. APPARATUS, MATERIAL, AND REAGENTS. the gas, Professor Koch has devised a self-acting apparatus (Fig. 20), which, simultaneously with the extinction of the flame of the burner, shuts off the supply of gas. Babes' Incuba- tor. The pattern of Dr. Babes is very simple, and is recommended by the author in pre- ference to all others FlG - MOITESSIERS GAS-PRESSURE REGULATOR. (Fig. 21). It consists of a double-walled chest with sides and FIG. 20. KOCH'S SAFETY BURNER. roof jacketed with felt. Water fills the interspace 54 BACTERIOLOGY. between the walls, and on the roof are two apertures one for a gas regulator, and the other for a thermometer. In front, the chest is closed in by a sheet of felt, a glass door, and a sliding glass panel. The apparatus can be sus- pended on the wall or sup- ported on legs, and is heated from below by means of protected burners. The gas should pass first through a pressure regulator, and then through a thermo-regulator to the burners. Moitessier's Gas-pressure Regulator. This apparatus is best explained by reference to the diagram (Fig. 19). In the bottom of the cylinder A are the entrance (/) and exit (/) gas tubes. The tap (m) regulates the size of the flame. The cover (n n) roofs in the cylinder A. The bell (B) supports by means of e and/" the ball valve d, which lies in the cover c c. The gas, entering by k, passes through the valve d, and is thence conducted by the tube a to the tube /. The bell B and the weighted dish h are screwed on to the connecting rod g. To diminish as much as possible the friction of g in 2, g only touches i by three projecting ridges. FIG. 21. BABES' INCUBATOR. APPARATUS, MATERIAL, AND REAGENTS. 55 Section of i and g is shown at s. To put the ap- paratus in use it is first levelled, then h is screwed off and the cover nn removed. A mixture of two parts of pure acid -free glycerine to one of distilled water is poured into the cylinder until it flows out at q, which is then closed, and the cover n n replaced. The manometers are filled with coloured water, and k and / connected with the entrance and exit gas tubing respectively. The pressure of the incoming gas raises the bell B ; and with it the valve d is raised towards the opening at c c. The weight k, which is replaced on g, by its downward pressure counteracts this upward pressure of the gas and opens the valve cc. Thus the flame is best regu- lated in the morning, when the pressure is at a minimum ; then supposing an increase of pressure occurs, the weight of h is overbalanced, B is raised and with it d, and the gas supply proportionately diminished by the gradual closing of the valved opening. Reichert's Thermo-regulator. This regu- lator (Fig. 22) consists of three parts a hollow T piece, a stem, and a bulb. The T piece fits like a stopper in the upper widened portion of the stem. One arm of the T is open, and connected with the gas supply ; the vertical portion terminates in a small orifice, and is also provided with a minute lateral opening. The stem is provided with a lateral arm, and this arm, the stem, and the bulb contain mercury. The regulator is fixed in the 56 BACTERIOLOGY. roof of the incubator, so that the bulb projects either into the interior of the incubator or into the water chamber. When the incu- bator reaches the required temperature, the mercury is forced up by means of the screw in the lateral arm, until it closes the orifice, at the extremity of the vertical portion of the T. The gas which passes through the lateral orifice is sufficient to maintain the apparatus FIG. 22. at the required temperature. If the REICHERT'S . THERMO- temperature ot the incubator tails the REGULATOR. mercury contracts, and gas passing through the terminal orifice of the T, increases the flame of the burner, and the temperature is restored. Page's Thermo - regulator resembles the above, but instead of the T piece there are two pieces of glass tubing. The outer tubing envelops the upper part of the stem of the regulator, and admits of being raised or lowered. The upper end of this tubing is closed by a cork, which is perforated to admit the narrow glass tubing, which represents the vertical arm of the T passing within the stem of the regulator. This has a terminal and a lateral opening, and is the means of entrance for the gas. This regulator is adjusted by noting when the thermometer indicates the desired temperature, and then pushing down the outer tube until the terminal opening of the inner tube, which is carried down with it, is obstructed by the mercury. APPARATUS, MATERIAL, AND REAGENTS. 57 Meyer's Thermo-regulator is represented in Fig. 23. No. I. shows the construction of the regulator ; its inner tube terminates in an oblique FIG. 23. MEYER'S THERMO-REGULATOR. opening, and is also provided with a minute lateral aperture, which prevents the complete shutting off of the gas supply. No. II. illustrates the method of introducing the mercury by suction through a filling tube, which is substituted for the 58 BACTERIOLOGY. inner tube of the regulator. No. III. represents Franke!' s modification of the same instrument. (M) INOCULATING AND DISSECTING INSTRUMENTS AND APPARATUS IN COMMON USE. Mouse Cages. As mice are the animals most frequently employed for experimental purposes, mouse cages have been especially introduced, con- sisting simply of a cylindrical glass jar \vith a weighted wire cover. Dressing Case. A small surgical dressing case, with its usual accessories forceps, knives, small straight and curved scissors, needles, silk, and so forth will serve for most purposes. Pravaz' Syringe. Koch's modification of Pravaz' syringe admits of sterilisation by exposure to 150 C. for a couple of hours. Special Instruments and Material. Instru- ments required for special operations and the materials necessary for strict antiseptic precautions need not be detailed here.* Dissecting Boards. Slabs of wood in various sizes, or gutta-percha trays, provided with large- headed pins, are employed for ordinary purposes. Dissecting Case. A dissecting case fitted with scalpels, scissors, hooks, etc., should be reserved entirely for post-mortem examinations. * Vide Cheyne, Antiseptic Surgery. 1882. APPARATUS, MATERIAL, AND REAGENTS. 59 (N) GENERAL LABORATORY REQUISITES. Siphon Apparatus. Two half-gallon or gallon glass bottles, with siphons connected with long flexible tubes provided with glass nozzles and pinchcocks (Fig. 24), should be employed for the FIG. 24. SIPHON BOTTLE, WITH FLEXIBLE TUBE, GLASS NOZZLE, AND A MOHR'S PINCHCOCK. following purposes : One is used to contain distilled water, with the nozzle hanging down conveniently within reach of the working table ; the other is to contain a solution of corrosive sublimate (i in 1000), and may be placed so that the nozzle hangs close to the lavatory sink or basin. The former replaces the use of the ordinary wash bottle, in washing off 60 BACTERIOLOGY. surplus stain from cover-glasses, etc., and thel atter is conveniently placed for disinfection of vessels and hands after cleansing with water. They should be placed on the top of a cupboard, or on a high shelf. Desiccator. The desiccator (Fig. 25) consists of a porcelain pan containing concentrated sulphuric acid, and covered over with a bell-glass receiver. FIG. 25. DESICCATOR. The sheet of plate-glass upon which the pan rests is ground upon its upper surface, and the rim of the glass bell is also ground and well greased. In the centre of the pan is a column supporting a .circular frame, which is covered with wire gauze. Slices of potatoes, upon which micro-organisms have been cultivated, are rapidly dried by the action of sulphuric acid in confined air. A cultivation of Micrococcus prodigiosus, for example, APPARATUS, MATERIAL, AND REAGENTS. 6 1 may be dried in this way, and preserved for subsequent experiments. Other items commonly in use in a research laboratory cannot be detailed here, and a descrip- tion of air-pumps, refrigerators, etc., access to which is nevertheless necessary for some special investigations, must be sought for elsewhere.* * All bacteriological apparatus, as employed by Professor Koch, may be obtained from Dr. Muencke, 58, Louisen Strasse, Berlin. Nearly all the figures of apparatus here given are from blocks, kindly lent to me by Dr. Muencke. Griffin & Son, 22, Garrick Street, Covent Garden, W.C., will make to order any bacteriological ap- paratus required, and from them all glass vessels and chemical apparatus of home manufacture can be obtained. All histological instruments and material, such as microscopes, microtomes, aniline dyes, celloidin, gelatine, agar-agar, etc., are supplied by G. Konig, Berlin, N.W., 35, Dorotheen Strasse. Chemicals, staining reagents, and ready-prepared nutrient gelatine can also be obtained from Dr. Georg Griibler, Leipsig, 17, Dufour Strasse. Solutions of lithium carmine, picro-lithium carmine (Orth), picro-carmine (Weigert), alum and borax carmine (Grenacher), etc., ready for use, are pre- pared by Becker & Co. , 34, Maiden Lane, Covent Garden, London, W.C. The latter firm also keep in stock bacteriological apparatus and glass ware of the German pattern. Mr. Baker, of High Holborn, W.C., is recommended for the supply of microscopes and the ordinary objectives by continental makers, and the new apochromatic objectives recently introduced by Zeiss. Objectives made of the new glass are also constructed by Powell and Leland, but though invaluable to the specialist their expense places them beyond the reach of the general student. Messrs. Swift & Son have recently introduced an excellent T V oil. imm. for five guineas, and are prepared to supply a microscope completely equipped for bacteriological work at a very low price. CHAPTER II. MICROSCOPICAL EXAMINATION OF BACTERIA IN LIQUIDS, IN CULTIVATIONS ON SOLID MEDIA, AND IN TISSUES. Preliminary Remarks. In conducting bacterio- logical researches, the importance of absolute clean- liness cannot be too strongly insisted upon. All instruments, glass vessels, slides, and cover-glasses should be thoroughly cleansed before use. A wide- mouthed glass jar should always be close at hand, containing refuse alcohol for the reception of rejected slide preparations or dirty cover-glasses. When required again for use, slides can be easily wiped clean with a soft rag. Cover-glasses require further treatment, for, unless they are perfectly clean, it is difficult to avoid the presence of air bubbles when mounting specimens. They should be left in strong acid (hydrochloric, sulphuric, or nitric) for some hours ; they are then washed, first with water and then with alcohol, and carefully wiped with a soft rag. The same principle applies in the preparation and employments of culture media ; any laxity in the processes of sterilisation, or insufficient attention to minute technical details, MICROSCOPICAL EXAMINATION OF BACTERIA. 63 will surely be followed with disappointing results in the contamination of the cultures, resulting in the loss of much time. When using platinum needles, either for inoculating fresh tubes in carry- ing on a series of pure cultures, or in transferring a small portion of a cultivation to a cover-glass for examination under the microscope, the careful sterilisation of the needle by heating the platinum wire till it is white hot in every part, and heating also as much of the glass rod as is made to enter the test-tube, must be carried out with scrupulous care. Indeed it is a good plan to let it become a force of habit to sterilise the needle before and after use on every occasion, whatever may be the purposes for which it is employed. (A) EXAMINATION IN THE FRESH STATE. Liquids containing micro-organisms, such as pus, blood, juices, culture-fluids, can be investigated by transferring a drop with a looped platinum needle or a capillary pipette to a slide, covering it with a clean cover-glass, and examining without further treatment. If it is desirable to keep the specimen under prolonged observation, a drop of sterilised water or salt solution must be run in at the margin of the cover-glass to counteract the tendency to dry. Cultures on solid media can be examined by transferring a small portion with a sterilised needle to a drop of sterilised water on a slide, thinning 64 BACTERIOLOGY. it out, and covering with a cover-glass as already described. A more satisfactory method, by which one can keep micro-organisms under observation and study their movements, spore-formation, etc., will be described under " Drop-cultures." Tissues in the fresh state may be teased out with needles in sterilised salt solution, and pressed out into a sufficiently thin layer between the slide and cover- glass. Glycerine may in many cases be substituted for salt solution, especially for the examination of micro-organisms such as Actinomyces, Aspergilli, etc. There is, as a rule, no difficulty in recognising the larger micro-organisms such as those just mentioned ; but where we have to deal with very small bacilli, bacteria, and micrococci, they may possibly be mistaken for granular detritus or fat- crystals, or vice versa. They are distinguished by the fact that fatty and albuminous granules are altered or dispersed by acetic acid, and changed by solution of potash ; alcohol, chloroform, and ether dissolve out fat-crystals or fatty particles ; on the other hand, micro-organisms remain unaffected by these reagents. This micro-chemical reaction is made the basis of Baumgarten's method. MICROSCOPICAL EXAMINATION OF BACTERIA. 65 (B) COVER-GLASS PREPARATIONS. The method next to be described is the most commonly employed ; in addition to its value as a means of examining liquids, etc., it affords the additional advantage of enabling one to make, if necessary, a large number of preparations which when dried can be preserved, stained or unstained, in ordinary cover-glass boxes ; they are then in a convenient form for transport, and can be mounted permanently at leisure. The method is as follows : A cover-glass is smeared with the cut surface of an organ, or pathological growth, or with sputum ; or a drop of blood, pus, or culture-fluid is conveyed to it with a looped platinum needle. By means of another cover-glass, the juice, or fluid, is squeezed out between them into a thin layer, and on sliding them apart each cover-glass bears on one side a thin film of the material to be examined. They are then placed with the prepared side upwards and allowed to dry. After a few minutes, they are held with a pair of flat-bladed or spring forceps, with the prepared side uppermost, and passed rapidly three times through the flame of a spirit lamp or Bunsen burner. To stain them, put two or three drops of an aqueous solution of fuchsine or methyl violet over the film, and after a minute or two wash off the surplus stain with distilled water by means of the siphon apparatus or a 5 66 BACTERIOLOGY. wash-bottle. Turn the cover-glass on to a slide, remove excess of water with filter-paper, and wipe the exposed surface ; examine with a power of about 250 diams., and if a higher power be required, which is usually the case, place a droplet of cedar oil on the cover-glass, and examine with an im- mersion lens. If the specimen is to be made permanent, fix the cover-glass at one corner with the thumb, and with a soft rag carefully wipe off the cedar oil ; then float off the cover-glass by running in distilled water at its margin, and having made a little ledge with a strip of filter-paper, place the cover-glass up against it upon one of its edges and leave it to dry. When perfectly dry mount in Canada balsam, or put it away in a cover-glass box provided with a label of contents. A minute portion of a culture may be stained and examined in the same way after spreading it out with a hooked platinum needle into a thin film, with or without the addition of a droplet of sterilised water. In many cases it is necessary or preferable to apply the stain for a much longer period. This is effected by pouring some of the staining solution into a watch-glass, and allowing the cover-glasses to swim on the surface, with their prepared side, of course, downwards. Throughout all these manipu- lations it is necessary to bear in mind which is the prepared surface of the cover-glass. MICROSCOPICAL EXAMINATION OF BACTERIA. 67 Double Coloration of Cover-glass Prepara- tions can also be obtained as in Ehrlich's method for staining tubercular sputum, or by staining with eosin after treatment by the method of Gram. Ehrlich's Method is as follows : Five parts of aniline oil are shaken up with one hundred parts of distilled water, and the emulsion filtered through moistened filter-paper. A saturated alcoholic solu- tion of fuchsine, methyl violet, or gentian violet, is added to the filtrate in a watch-glass drop by drop until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen minutes to half an hour, then washed for a few seconds in diluted nitric acid (one part nitric acid to two of water), and then rinsed in distilled water. The stain is removed from everything except the bacilli ; but the ground substance can be after- stained, brown if the bacilli are violet, or blue if they have been stained red (Plate III., Fig. i). Double staining with eosin after the method of Gram is described under tissue staining. The cover-glass preparations are treated by the same processes as employed with sections ; superfluous oil of cloves can be removed by gently pressing the cover-glass between double layers of filter-paper. Babes' Method affords a very rapid means of examining cultivations, etc. A little of the growth, removed by means of a sterilised platinum hook or small loop, is spread out on a cover-glass into as thin a film as possible : when almost dry, a drop or 68 BACTERIOLOGY. two of a weak aqueous solution of methyl violet is allowed to fall from a pipette upon the film. The cover-glass with the drop of stain is, after a minute, carefully turned over on to a slide, and the excess of stain gently and gradually removed by pressure with a strip of filter-paper. It affords a rapid means of demonstration for example, of such a cultivation as Koch's comma bacilli in nutrient gelatine enabling the microbes to be seen in some parts of the preparation both stained and in active movement. His' Method. The staining of fresh prepara- tions, especially those with no coagulable albumen to fix them, may be also carried out by His' method. A slide is prepared as already described in the examination of micro-organisms in the fresh state. The reagents are then applied by placing them with a pipette drop by drop at one margin of the cover-glass, and causing them to flow through the preparation by means of a strip of filter-paper placed at the opposite margin. To stain Spores, the method described on p. 65 is somewhat modified. The cover-glass prepara- tions may be either passed as many as twelve times through the flame, or heated to a temperature of 210 for half an hour, or exposed to the action of strong sulphuric acid for a few seconds, and then stained with a watery solution of the dye. To double-stain Spore-bearing Bacilli. The cover-glass preparations may be floated for twenty MICROSCOPICAL EXAMINATION OF BACTERIA. 69 minutes on a fuchsine aniline-water solution, as used in Ehrlich's method, which has been heated to boiling-point. The fuchsine is removed from the bacilli either by simply rinsing in water, in alcohol, or in weak acid, according to the species, and then the preparations are floated for a few minutes on solution of methylene blue, rinsed in water, dried, and mounted. To stain Flagella. Koch recommends floating the cover-glasses on a concentrated watery solution of haematoxylin. From this they are transferred to a 5 per cent, solution of chromic acid or to Muller's fluid, by which the flagella obtain a brownish-black coloration. The author has succeeded in demonstrating and photographing flagella, by staining with a drop of a saturated solution of gentian violet in absolute alcohol. Before the alcohol has time to evaporate, the cover-glass is rinsed in water, and then allowed to dry, and finally mounted in balsam. A very in- tense staining of the whole preparation results. (c) COVER-GLASS IMPRESSIONS. One of the most instructive methods for examin- ing micro-organisms is to make an impression- preparation. This enables us, in many cases, to study the relative position of individual micro- organisms one to another in their growth on solid cultivating media, and in some cases produces the 70 BACTERIOLOGY. most exquisite preparations for the microscope. A perfectly clean, usually small- sized, cover-glass is carefully deposited on a plate- or potato-culture, and gently and evenly pressed down. One edge is then levered up carefully, with a needle, and the cover-glass lifted off by means of forceps. It is then allowed to dry, passed through the flame three times, and stained as already described. In the case of plate-cultures, especially where no lique- faction has taken place, the growth is bodily transferred to the cover-glass, and a vacant area left on the gelatine or agar-agar, corresponding exactly with the form and size of the cover-glass employed (Plate XXVII., Figs, i and 2). CHAPTER III. PREPARATION AND STAINING OF TISSUE SECTIONS. (A) METHODS OF HARDENING AND DECALCIFYING PREPARATIONS. To harden small organs, such as the viscera of a mouse, they must be placed on a piece of filter- paper at the bottom of a small, wide-mouthed glass jar, and covered with about twenty times their volume of absolute alcohol. Larger organs, patho- logical growths, etc., are treated in the same way, but must first be cut into small pieces, or cubes, varying from a quarter of an inch to an inch in size. Miiller's fluid may also be employed, and methylated spirit may be substituted for alcohol, from motives of economy. Tissues hardened in absolute alcohol are ready for cutting in two or three days, and those hardened in Miiller's fluid in as many weeks. Teeth, or osseous structures, must first be placed in a decalcifying solution, such as Kleinenberg's. When sufficiently softened, they are allowed to soak 72 BACTERIOLOGY. in water, to wash out the picric acid, and then transferred through weak spirit to absolute alcohol. Ebner's solution also gives excellent results, espe- cially when the structures to be decalcified are placed in fresh solution from time to time. (B) METHODS OF EMBEDDING, FIXING, AND CUTTING. Material to be cut with the freezing microtome, if hardened in spirit, must be well soaked in water before being frozen ; if hardened in M tiller's fluid, it can be frozen at once. If Williams' microtome is employed, the hard- ened tissues must first be well soaked in gum mucilage, then frozen, and cut. For cutting with Jung's microtome, the tissues are embedded in paraffine, or celloidin, and mounted on cork ; or, if firm enough, they may be fixed upon cork without any embedding material at all. Paraffine, dissolved in chloroform, will be found very serviceable as an embedding material, but celloidin is more commonly employed now. The pieces of tissue to be embedded are placed, after the process of hardening is completed, in a mix- ture of ether and alcohol for an hour or more. They are then transferred to a solution of celloidin in equal parts of ether and alcohol, and left there, usually for several hours. Meanwhile, corks ready cut for the clamp of the microtome are smeared PREPARATION AND STAINING OF TISSUE SECTIONS. 73 over with the solution of celloidin ; this is applied with a glass rod to the surface which is to receive the piece of tissue. The corks are then set aside for the film of celloidin to harden. The pieces of tissue are allowed to remain in the celloidin solution for from one to twenty -four hours, the time varying according to the structure of the specimen. Better results are obtained in the case of lung, or de- generated broken-down tissue, if left for a much longer time than is found to be sufficient for firmer structures. The specimen, when ready, is removed from the celloidin solution with forceps and placed upon a prepared cork. A little of the solution, which is of syrupy consistence, is allowed to fall on the piece of tissue to cover it completely, and the mounted specimen is finally placed in 60 to 80 per cent, alcohol to harden the celloidin. The specimen will be ready for cutting next day. The specimen may be more neatly embedded by fixing it with a pin in a small paper tray, pouring the celloidin solution over it, and then placing the tray in alcohol to harden the celloidin. The em- bedded specimen is then fixed on a cork, which has been cut for the clamp of the microtome. The celloidin in the section disappears in the process of clearing with clove-oil. Material infiltrated with paraffine must be cut per- fectly dry, and the sections prevented from rolling up by gentle manipulation with a camel's-hair brush. They must then be picked off the blade of 74 BACTERIOLOGY. the knife with a clean needle, and dropped into a watch-glass containing xylol. This dissolves out the paraffine ; the sections are then transferred to alcohol to get rid of the xylol, and then to the staining solution. In the case of specimens embedded in celloidin, or mounted directly on a cork, the tissue, as well as the blade of the knife, should be kept constantly bathed with alcohol, and the sections transferred from the blade with a camel's-hair brush, and floated in alcohol. For fixing small organs and pieces of firm tissue directly on cork, such as the kidneys of a mouse, or liver, we may employ gelatine, or glycerine gela- tine, liquefied over a Bunsen burner in a porce- lain capsule. The cork, with specimen affixed, is placed in alcohol, and is ready for cutting sections next day. The advantage of glycerine gelatine consists in that it may be used for fixing irregular pieces of tissue, as it does not become of a consistency that would injure the edge of the knife. Embedding in Celloidin and Freezing. This method gives excellent results. Celloidin is used as already described. The piece of tissue is then placed in a glass capsule, and some of the celloidin solution poured over it. After hardening in spirit, the tissue is cut out and put in water until it sinks. It is then transferred to gum, and frozen and cut in the ordinary way. PREPARATION AND STAINING OF TISSUE SECTIONS. 75 (c) GENERAL PRINCIPLES OF STAINING BACTERIA IN TISSUE SECTIONS I METHODS OF WEIGERT, GRAM, AND WEIGERT-EHRLICH. Sections of fresh tissues made with the freezing microtome are to be floated and well spread out in *8 per cent, salt solution, and then carefully transferred, well spread out on the copper lifter, to a watch-glass containing absolute alcohol. Simi- larly, sections selected from those cut with Jung's microtome may be transferred from the spirit to absolute alcohol. The sections may be then stained by any of the methods to be described. It is often advisable to employ some method which will enable one to study the structure of the tissue itself. In the same way with sections, however prepared, one should always examine with a low power (one inch) first ; this enables one to recognise the tissue under examination in most cases, and even to examine in many cases the topographical distribution of masses of bacteria. With a power of about 250 diams. (one sixth), very many bacteria can be distinguished ; and with the oil immersion lenses the minutest bacilli and micrococci can be recognised, and the exact form of individual bacteria accurately determined. As most good modern instruments, like Zeiss' micro- scopes, are provided with a triple nosepiece, there 7 6 BACTERIOLOGY. is no loss of time in examining a preparation suc- cessively with these different powers. Weigert's Method. A very useful method for staining both the tissue and the bacteria is as fol- lows : Place the sections for from six to eighteen hours in a i per cent, watery solution of any of the basic-aniline dyes (methyl violet, gentian violet, fuchsine, Bismarck brown). To hasten the process, place the capsule containing the solution in the incubator, or heat it to 45 C. A stronger solution may also be employed, in which case the sections are far more rapidly stained, and are easily over- stained. In the latter case they must be treated with a half-saturated solution of carbonate of potash. In either case the sections are next washed with distilled water, and passed through 60 per cent, alcohol into absolute alcohol. When almost decolorised, spread out the section carefully on a copper lifter and transfer it to clove-oil, or stain with picro-carmine solution (Weigert's) for half an hour, wash in water, alcohol, and then treat with clove-oil. After the final treatment with clove-oil, transfer with the copper lifter to a clean glass slide. Dry the preparation by pressure with a piece of filter-paper folded four times, and preserve in Canada balsam dissolved in xylol. Gram's Method. In the method of Gram the sections are stained for three minutes in aniline- gentian-violet solution. This is prepared by shak- ing up one ccm. of pure aniline with twenty-four PLATE 2. faarufp.76. I < * : V-V. :!*' % :.*' Kgl. Fig Crtcksha.ni tic.^Lpi, !.on,!,.!i,t'nt>li.ak Pin.-e: Loiidon.Publur!ied bv H . K Lewi.-,,!3G,Gower Street . PLATE 5. fott plate. 4. Kg' 2. Pig 3. Vincent BmoTcj.Day &Scn.,Uih . London.Pabli3hedbv H.K.LewiG^SG.Gower Street . PLATE 6. Hg2. H.Cmokahank Pinx; Vincent BmoTcs.Day IfSon. London.PuhKsliedW H.K.Lewis, 136. Gcr^r Street . PLATE 7. fM plate 6. Pig 1. Tig 2 Londo.n,r\itdi3hedlv H ,R.Lewio,136,'-Jver Street . PLATES ic U, Uiie 'I. Vincent Bwlcs.Day * Sen,l0i . ..K.Lewis, 136, Gowr Street . London, Pub.! is hed V H . K . ] ,.-vio , 1 3 G , Gower S trect . Vinrrrit Brr,c.Tc?.Day *< PLATE 10 Vincent Brfoh^Day o7i,LLth. . l.ondon.Rihlished by H .K.Lewis, 13 6. Gower Street . PLATE II. H.Cmokahank Pin*' Lorvaon.ftibhshedbvH.K.Lewio.l36.Gay Son. Zvtk. SOLID BLOOD SERUM. 105 ferred to plugged sterile test-tubes. These should be filled with a sterilised pipette for about a third of their length, and are then placed in Koch's slow steriliser with the temperature maintained for an hour at 58 C. The same process is repeated for six successive days, the temperature on the last day being gradually raised to 60. This completes the sterilisation, but to solidify the serum it is necessary to arrange the tubes in the inspissator at the angle required. The temperature of this apparatus is kept between 65 and 68 C. Directly solidification takes place the tubes must be removed, and they should then present the character of being hard, solid, of a pale straw colour, and transparent. A little liquid collects at the lowest point, and the serum is sometimes milky in appearance at its thickest part. The serum may not only be employed in test-tubes, but also in small flasks, glass capsules, or other vessels, all of which must be cleansed and sterilised in the usual way. Hydrocele fluid and other serous effusions may be prepared in the same manner, or gelatine may be added to the serum in the proportion of 5 per cent. Inoculation of the Tubes. A small portion of the material to be inoculated is taken up with a sterilised platinum needle, and drawn in lines over the sloping surface of the serum ; or a minute piece of tissue, tubercle, etc., may be introduced into the tube and deposited on the surface of the nutrient medium. The precautions that are to be observed 106 BACTERIOLOGY. in isolating the material to be inoculated will be referred to later (p. 141). LIQUID MEDIA. (E) PREPARATION OF STERILISED BOUILLON, LIQUID BLOOD SERUM, URINE, MILK, VEGETABLE INFU- SIONS, AND ARTIFICIAL NOURISHING LIQUIDS. Nutrient liquids are still largely employed, and by some observers even in preference to the solid media advocated by Koch. It must not be supposed, however, that the methods of cultivation in liquids are discarded entirely by the modern school, for there is no more instructive method than the employment of so-called drop-cultures. For inocu lation experiments where the presence of gelatine is undesirable, for studying the physiology and chemistry of bacteria and where for any object a rapid growth of micro-organisms is necessary, the employment of liquid media is not only advis- able, but is absolutely necessary. Liquid media comprise two distinct groups natural and artificial. The natural group includes meat broths, blood, urine, milk, and vegetable infusions ; the artificial are solutions built up from a chemical formula representing the essential food constituents. NATURAL MEDIA. Bouillon. A broth of bouillon of beef, pork, or chicken may be made in the same manner as LIQUID MEDIA. 1 07 described for the preparation of nutrient gelatine, with simply omission of the gelatine. After the neutralisation with carbonate of soda solution drop by drop, the flask of broth is placed in the steam steriliser for half an hour at 100 C. A clear liquid results on filtration, which is transferred to plugged sterilised flasks or test-tubes, and sterilisa- tion effected by exposing them in the steam steriliser for half an hour at 100 C. for two or three succes- sive days. Liquid Blood Serum. The preparation of sterile blood serum has already been described. It may be used for cultivation, especially in the form of drop-cultivations, before the final treatment by which it is solidified, Hydrocele fluid, peritonitic and pleuritic effusions, can also be employed after sterilisation in the steam steriliser. The fluid should be withdrawn with a sterilised trocar and canula, and received into plugged sterilised flasks. Urine. In order to obtain urine free from micro- organisms the following precautions must be ob- served : The orifice of the urethra must be thoroughly cleansed with sublimate solution. The first jet of urine should be rejected, and the rest received into sterilised vessels, which must be quickly closed with sterile plugs. If these precautions be not attended to the urine must be rendered sterile by the means described for the sterilisation of bouillon. Milk. If milk has been drawn into sterile flasks after thoroughly cleansing and disinfecting the teats IO8 BACTERIOLOGY. and hands, it may be kept without change. If pro- cured without these precautions, it must be steamed in the steriliser for half an hour for five successive days. Vegetable Infusions. Infusions of hay, cucumber, and turnip are used for special purposes, and more rarely decoctions of plums, raisins, malt, and horse-dung. They are mostly prepared by boiling with distilled water, after maceration for several hours. The filtrate is received into sterile flasks and sterilised in the usual way in the steam steriliser. ARTIFICIAL FLUIDS. Pasteur's Fluid. This solution is prepared by mixing the ingredients in the following propor- tions : Distilled water . . . .100 Pure cane sugar . . . .10 Ammonium tartrate i Ash of yeast -075 Cohn-Mayer Fluid. Mayer's modification of the nourishing fluid employed by Cohn is as follows : Distilled water .... 20 Phosphate of potassium . . . i Sulphate of magnesium . . . *i Tribasic calcium phosphate . . 'Oi Ammonium tartrate . *2 LIQUID MEDIA. 1 09 (F) METHODS OF STORING AND EMPLOYING LIQUID MEDIA; LISTER'S FLASKS, AITKEN'S TEST-TUBES, STERNBERG'S BULBS, PASTEUR'S APPARATUS, MIQUEL'S BULBS ; DROP-CULTURES. Cultivations in liquid media can be carried on in test-tubes, but it is more satisfactory to employ special forms of flasks, bulbs, U tubes, etc. As test-tubes and flasks containing liquid media cannot be inverted, inoculation with a sterilised needle must be carried out as rapidly as possible, with the addi- tional precaution of closed windows and doors. Lister's Flasks. These flasks (p. 48) were especially introduced by Lister as a means of storing liquid nutrient media. They are so constructed that after removal of a portion of the contents, on re- storing the vessel to the vertical position, a drop of liquid always remains in the extremity of the nozzle, which prevents the regurgitation of unfiltered air. Sternberg's Bulbs. The method of introducing liquid into the bulbs employed by Sternberg, and of sterilising and inoculating it, is as follows : The bulb is heated slightly over the flame, and the extremity of the neck, after breaking off the sealed point, is plunged beneath the surface of the liquid. As the air cools the liquid is drawn into the bulb, usually filling it to about one-third of its capacity. The neck of the flask is again sealed up, and the 110 BACTERIOLOGY. liquid which has been introduced is sterilised by repeatedly boiling the flasks in the water-bath. They should then be placed in the incubator for two or three days ; and if the contents remain trans- parent and free from film, they may be set aside as stock-bulbs, to be used when required. To inoculate the liquid in the bulb the end of the neck is heated to sterilise the exterior, the bulb is gently warmed, and the extremity of the neck nipped off with a pair of sterilised forceps. The open extremity is plunged into the liquid containing the micro-organism, a minute quantity enters the tube and mingles with the fluid in the bulb, without fear of contamination by atmospheric germs. The extremity of the neck is once more sealed up in the flame of a Bunsen burner. Aitken's Tubes. These tubes are plugged and sterilised, and the nutrient medium introduced as into ordinary test-tubes. Instead of withdrawing the cotton-wool plug, they are inoculated by means of the lateral arm. The sealed extremity of the arm is nipped off with sterilised forceps, and the inoculating needle is carefully introduced through the opening thus made. It is directed along the arm until it touches the opposite side of the test- tube, where it deposits the material with which it was charged. The needle is withdrawn, and the end of the lateral arm again sealed up in the flame ; the test-tube is then tilted until the liquid touches the deposited material ; on restoring the tube to LIQUID MEDIA. Ill the vertical the material is washed down into the body of the nutrient liquid. Pasteur's Apparatus. Special forms of tubes, bulbs, and pipettes are employed by the school of Pasteur. The tubes are provided with lateral or with curved arms drawn out to a fine point, and with slender necks plugged with cotton-wool. A double form shaped like a tuning-fork, each limb with a bent arm, is a convenient form for storing sterilised bouillon. The sealed end of an arm is nipped off with sterilised forceps, the sterile bouillon aspirated into each limb, and the arm again sealed in the flame ; a series of such tubes can be arranged upon a rack on the working table.* Bulbs with a vertical neck drawn out to a fine point, others with a neck bent at an obtuse angle, plugged with cotton-wool, and a lateral curved arm drawn out to a fine point, are also employed. For a description of these various vessels and their special advantages, the works of Pasteur and Duclaux must be consulted. Miquel's Bulbs. The tube a boule of Miquelf is also a very useful form. It consists of a bulb of 50 cc. capacity blown in the middle of a glass tube. The part of the tube above the bulb is con- tracted about half-way between the bulb and its extremity, and can either be left quite straight or * Duclaux, Ferments et Maladies. 1882. t Miquel, Les Organismes Vivants de V Atmosphere. 1883. I I 2 BACTERIOLOGY. can be made to curve slightly. On either side of the contraction the tube is plugged with asbestos. The portion of the tube below the bulb is S shaped, and drawn out at its extremity into a fine point. The bulbs are charged with nutrient liquid and inoculated by aspiration, and the point of the S tube sealed in the flame of a Bunsen burner. Drop-cultures. This method of cultivation has already been referred to as a particularly in- structive one. It enables us to study many of the changes which take place during the life history of micro-organisms. This is illustrated, for example, by the anthrax bacillus, where we can watch the gradual growth of a single bacillus into a long filament, and the subsequent development of bright oval spores. It is necessary carefully to observe the minutest details to maintain the cultivation pure. An excavated slide is thoroughly cleaned, and then sterilised by being held with the cupped side downwards in the flame of the Bunsen burner. A ring of vaseline is painted round the excavation, and the slide is then placed under a glass bell. Meanwhile a carefully cleansed cover-glass is also sterilised by passing it through the flame, and should be deposited on the plate of blackened glass. With a sterilised looped needle, a drop of sterile bouillon is transferred to the cover-glass, and this is inoculated by touching it with another sterilised needle charged with the material, without disturbing the form of the drop. It is quite sufficient just to LIQUID MEDIA. 113 touch the drop instead of transferring a visible quantity of blood, juice, or growth, as the case may be. The slide is then inverted and placed over the cover-glass, so that the drop will come exactly in the centre of the excavation, and is gently pressed down. On turning the slide over again the cover- glass adheres, and an additional layer of vaseline is painted round the edges of the cover-glass itself. The slide must be labelled, and, if necessary, placed in the incubator, and the results watched from time to time. Instead of bouillon, liquid blood serum may also be employed in this form of cultivation. If it be required to preserve the drop-cultivation as a microscopic preparation, the cover-glass is gently lifted off and allowed to dry. Any vaseline ad- hering to the cover-glass should be wiped off, and the cover-glass can then be passed through the flame and stained in the usual manner. Moist Chambers. Unless drop-cultures are very carefully prepared, they are liable to dry up, if kept for examination for several days. Many therefore prefer employing a moist chamber. There are several different forms in use. The drop-culture slide may be converted into a moist chamber by having a deep groove cut round the circumference of the concavity. This groove is filled with sterilised water by means of a pipette. A ring of vaseline is painted with the earner s-hair brush outside the groove, and the cover-glass, with the drop-cultivation, is inverted and placed over the 8 114 BACTERIOLOGY. concavity. This form is very useful, as the slide can be easily cleansed and effectually sterilised by holding it in the flame of the Bunsen burner. A very simple form of moist chamber, which may be used in some cases, but possesses the disadvan- tage of not admitting of sterilisation by heat, may be constructed as follows*: A small piece of putty or modelling wax is rolled into a cord about two inches long and ^ inch thick. FIG. 30. METHOD OF FORMING A SIMPLE MOIST CHAMBER. By uniting the ends a ring is formed, which is placed on the middle of a clean glass slide (Fig. 30). A drop of water is placed in the centre of the ring, and the cell roofed in by applying the cover-glass. A somewhat similar cell in form, which has the advantage of permitting of thorough cleansing, may be constructed by cementing a glass ring with flat surfaces to an ordinary slide. Vaseline is applied with a camel's-hair brush to the upper surface of * Schafer's Course of Practical Histology. 1877. LIQUID MEDIA. 115 the ring, and one or two drops of water placed with a pipette at the bottom of the cell. The cover- glass, with the preparation, is then inverted over the cell and gently pressed down upon the glass ring. The vaseline renders the cell air-tight, and, to a certain extent, fixes the cover-glass to the ring. Warm Stages. To apply warmth while a pre- paration is under continuous observation, we must either place the microscope bodily within an in- cubator, with the eyepiece protruding through an FIG. 31. SIMPLE WARM STAGE. opening, so that we may observe what is going on without moving the preparation, or we must em- ploy some means of applying heat directly to the preparation. A simple warm stage may be made of an oblong copper plate, two inches long by one inch wide, from one side of which a rod of the same material pro- jects. The plate has a round aperture in the middle, half an inch in diameter, and is fastened to an or- dinary slide with sealing wax. The drop to be examined is placed on a large-sized cover-glass and covered with a smaller one. Olive oil or vaseline n6 BACTERIOLOGY. is painted round the edge of the smaller cover-glass to prevent evaporation, and the preparation is placed over the hole in the plate (Fig. 31). The slide bearing the copper plate is clamped to the stage of the microscope (Fig. 32). The flame FIG. 32. SIMPLE WARM STAGE SHOWN IN OPERATION. of a spirit-lamp is applied to the extremity of the rod, and the heat is conducted to the plate and thence transmitted to the specimen. That the temperature of the copper plate may be approxi- mately that of the body, the lamp is so adjusted that a fragment of cacao butter and wax placed close to the preparation is melted. LIQUID MEDIA. 117 For more accurate observations, the apparatus shown in Fig. 33 may be employed. The vessel /, filled with water which has been boiled to expel the air, is heated by means of a gas-flame at g. The warmed water ascends the india-rubber tube c to the brass box a. The box is pierced by a tubular aperture to admit light to the object, and has an FIG. 33. SCHAFER'S WARM STAGE. exit tube c', by which the cooled water from the stage returns to be reheated by the flame g. At d is a gas-regulator, so that a constant temperature at any desired point can be maintained. Another form, in which warm water or steam can be used for heating, and by the employment of iced water also used for observing the effects of cold, is shown in Fig. 34. It consists of a hollow rectangular box, with a central opening (C) permitting n8 BACTERIOLOGY. the passage of light. The water makes its exit and entrance at the side tubes a, a, and the temperature is indicated by a thermometer in front. CL on a FIG. 34. STRICKER'S WARM STAGE. Israel's Warming Apparatus. It is obvious that in employing very high powers a difficulty will be presented by the warm stages just described, FIG. 35. SECTION OF ISRAEL'S WARMING APPARATUS AND DROP-CULTURE SLIDE. owing to their interference with the illumination. To overcome this an apparatus has been constructed by which the slide is warmed from above.* * Israel, Zeitsch.f. Wiss. Mikrosc, ii., pp. 459-63. 1885. LIQUID MEDIA. 119 The drop-culture slides are provided with a shallow groove *i mm. deep and i mm. broad, cut round the concavity. Into this the cover-glass fits, so that its upper surface is flush with that of the slide. The heating apparatus consists of a flat disk-shaped box with a central conical aperture (Fig. 35). The entrance and exit pipes are fixed on at a FIG. 36. ISRAEL'S WARMING APPARATUS. right angle to the side (Fig. 36). The former, z, is of metal, and the latter, a, of glass fitted with a thermometer, the bulb of which, k, is contained within the box. A partition, s, keeps up a current between the openings of the pipes, which are supported on a stand and connected by tubing with the hot- water supply (Fig. 37). A mixture of paraffine and vaseline is recom- mended for indicating the temperature of the chamber, and experience has shown that if a T2O BACTERIOLOGY. temperature of 37 C. be required the temperature of the water in the box must range between 42 and 47 C. Gas Chambers. To investigate the action of FIG. 37. ISRAEL'S WARMING APPARATUS IN OPERATION. gases or vapours upon micro-organisms, a modi- fication of the simple moist chamber (Fig. 30) may be employed (Fig. 38). A piece of glass tubing is first fixed to the slide by means of sealing wax, and the ring of putty is LIQUID MEDIA. 121 so placed as to include the end of this, leaving a small interval at the side, or a little notch is | FIG. 38. SIMPLE GAS CHAMBER. made in the putty opposite, so as to afford an exit for the gas or vapour (Fig. 39). FIG. 39. GAS CHAMBER IN USE WITH APPARATUS FOR GENERATING CARBONIC ACID. A more complicated apparatus, combining both a warm stage and a gas chamber, is shown in Fig. 40. This consists of a rectangular piece of ebonite (EE) 122 BACTERIOLOGY. fixed to a brass plate which rests on the stage of the microscope. On the upper surface of the ebonite is n< FIG. 40. STRICKER'S COMBINED GAS CHAMBER AND WARM STAGE. another brass plate (/), with an aperture (c] leading into a brass tube closed below by a piece of glass. To heat the apparatus the copper wire B is placed FIG. 41. SIMPLE MOIST CHAMBER ADAPTED FOR TRANSMISSION OF ELECTRICITY. on the tube a, and its extremity heated by the flame of the lamp. The nearer the lamp to the stage the higher the temperature, which is indicated by the thermometer t. To employ it as a gas chamber LIQUID MEDIA. 123 the wire B is laid aside, and the gas is conducted into the chamber by the tube a ', and escapes by the tube a. Application of Electricity. To study the effect of electricity we may prepare a drop-culture in the moist chamber (Fig. 41). The cover-glass to be used is provided with two strips of tinfoil, FIG. '42. APPARATUS ARRANGED FOR TRANSMITTING ELECTRICITY. which are isolated from the brass of the microscope, and so arranged that a current of electricity may be passed through them (Fig. 42). A much simpler plan, which may also be employed, is to take an ordinary glass slide and coat the sur- face with gold-size. The slide is then pressed firmly down on gold-leaf or tinfoil and allowed to dry. When dry, the metal is scraped away, leaving 124 BACTERIOLOGY. two triangles with a small interval between them, as in Fig. 43. FIG. 43. SLIDE WITH GOLD-LEAF ELECTRODES. IS The liquid containing the micro-organisms placed between the electrodes, covered with a cover- glass, and then subjected to the electric current. CHAPTER V. EXAMINATION OF AIR, SOIL, AND WATER. Am. THE air, as is well known, contains in suspension mineral, animal, and vegetable substances. The mineral world is represented by such substances as silica, silicate of aluminium, carbonate and phosphate of calcium, which may be raised from the soil by the wind, and particles of carbon, etc., which gain access from accidental sources. Belonging to the animal kingdom we find the ddbris of perished creatures, as well as sometimes living animals. The vegetable world supplies micrococci, bacilli, and other forms of the great family of bacteria, spores of other fungi, pollen seeds, parts of flowers, and so forth. The air of hospitals and sick rooms has been found to be especially rich in vegetable forms, e.g., fungi and spores have been observed as present in particularly large numbers in cholera wards, spores of tricophyton have been discovered in the air of hospitals for diseases of the skin, and achorioni n wards with cases of favus. The tubercle-bacillus is said to have been detected in the breath of patients suffering from phthisis. 126 BACTERIOLOGY. These points indicate that, in addition to the interest for the microbiologist, considerable import- ance, from a hygienic point of view, must be attached to the systematic examination of the air. Especially a knowledge of the microbes which are found in the air of marshy and other unhealthy dis- tricts, and in the air of towns, dwellings, hospitals, workshops, factories, and mines, will be of practical value. Miquel,* who has particularly studied the bac- teria in the air, has found that their number varies considerably. The average number per cubic metre of air for the autumn quarter at Montsouris is given at 142, winter quarter 49, spring quarter 85, and summer quarter 105. In air collected 2,000 to 4,000 metres above the sea-level, not a single bac- terium or fungus spore was furnished, while in 10 cubic metres of air from the Rue de Rivoli (Paris) the number was computed at 55,000. The simplest method for examining the organisms in air consists in exposing plates of glass or micro- scopic slides coated with glycerine, or a mixture of glycerine and glucose, which is stable, colourless, and transparent. Nutrient gelatine spread out on glass plates (p. 91), may be exposed to the air for a certain time, and then put aside in damp chambers for the colonies to develop. Sterilised potatoes, prepared in the usual way (p. 99), may be similarly exposed. In both the last-mentioned methods * Miquel, Organismes Vivantes de r Atmosphere. EXAMINATION OF AIR. 127 separate colonies develop, which may be isolated as already described, and pure cultivations carried on in various other nutrient media (p. 97). Nutrient gelatine has also been employed in the special methods of Koch and Hesse. Koch's Apparatus. This consists of a glass jar, about six inches high, the neck of which is plugged with cotton- wool. In the interior is a shallow glass capsule, which can be removed by means of a brass lifter. The whole is sterilised by exposure to 150 C. for an hour in the hot-air steriliser. The nutrient gelatine in a stock-tube is liquefied, and the contents emptied into the glass capsule. The jar is exposed to the air to be examined for a definite time, the cotton-wool plug replaced, and the apparatus set aside for the colonies to develop. Hesse's Apparatus (Fig. 44), The advantage of this apparatus consists in that a known volume of air can be examined. A glass cylinder, 70 cm. long and 3-5 cm. in diameter, is closed at one end by an india-rubber cap, perforated in the centre. Over this fits another cap, which is not perforated. The opposite end of the cylinder is closed with a caoutchouc stopper, perforated to admit a glass tube plugged with cotton-wool. The tube can be con- nected by means of india-rubber tubing with an aspirating apparatus. This apparatus consists of a couple of litre-flasks, suspended by hooks from the tripod-stand which supports the whole apparatus. 128 BACTERIOLOGY. The cylinder, caps, and plug are washed with solu- tion of corrosive sublimate, and then with alcohol. After being thus cleansed, 50 ccm. of nutrient gela- tine are introduced, and the whole sterilised by steaming for half an hour for three successive days. FIG. 44. HESSE'S APPARATUS. After the final sterilisation, the cylinder is rotated on its long axis, so that the nutrient medium solidifies in the form of a coating over the whole of the interior. When required for use, the cotton- wool plug is removed from the small glass tube, and the latter connected with the upper flask by means of the india-rubber tubing. EXAMINATION OF AIR. 1 29 The apparatus is placed in the air which is to be examined, the outer india-rubber cap removed from the glass cylinder, and the upper flask tilted until the water begins to flow into the lower one. The emptying continues by syphon action, and air is drawn in along the cylinder to replace the water. When the upper flask is empty, the position of the two is reversed, and the flow again started. When a sufficient volume has been drawn through the cylinder, the outer cap and the cotton-wool plug are replaced, and it is set aside for the colonies to develop. As an example, twenty-five litres of air from an open square in Berlin gave rise to three colonies of bacteria and sixteen moulds ; on the other hand, two litres from a schoolroom just vacated by the scholars gave thirty-seven colonies of bacteria and thirty-three moulds. Various forms of " aeroscopes " and " aeroni scopes " have from time to time been employed. Pouchet's aeroscope consists of a small funnel, drawn out to a point below which is a glass slip coated with glycerine. The end of the funnel and the glass slip are enclosed in an air-tight chamber, from which a small glass tube passes out, connected by india-rubber tubing with an aspirator. The air passing down the funnel strikes upon the glycerine, which arrests any solid particles. For a description of the more exact apparatus em- ployed by Maddox, Cunningham, and Miquel, reference should be made to the writings of these 9 I3O BACTERIOLOGY. authors, and particularly to the treatise published by the last-named. EXAMINATION OF SOIL. Surface soil, or mould, is exceedingly rich in bacteria. Miquel, e.g., has computed that there exists in a gramme of soil an average of 750,000 germs at Montsouris, 1,300,000 in the Rue de Rennes, and 2,100,000 in the Rue de Monge. As agents of putrefaction and fermentation they play a very important role in the economy of nature; but there exist in addition bacteria in the soil which are pathogenic in character. Pasteur has succeeded in isolating the bacillus of anthrax from the earth, and sheep, sojourning upon a plot of ground where animals with anthrax had been buried, succumbed to the disease. Pasteur considered that the spores were conveyed by worms from buried beasts to the surface soil. The bacillus of malignant cedema is also present in soil, and Nicolaier has cultivated a bacillus from earth which produces tetanus in mice, rabbits, guinea-pigs, and other animals. To obtain a cultivation of the microbes in soil a sample of the latter must be first dried and then triturated. It may then be shaken up with distilled water, and from this a drop transferred to. sterilised bouillon. The employment of solid media is, how- ever, much more satisfactory : a sample of earth is collected, dried, and triturated, and a small quantity EXAMINATION OF WATER. 13! sprinkled over the surface of nutrient gelatine pre- pared for a plate-cultivation. In another method the gelatine is liquefied in a test-tube, the powder added, and, in the usual way, distributed throughout the medium, which is then poured out upon a glass plate. Just in the same way the dust which settles from the air in houses and hospitals, or food sub- stances in powder, may be distributed over nutrient gelatine, and the micro-organisms which develop studied, both as to their morphological and biological characteristics. EXAMINATION OF WATOR. As in the case of air, so, too, in that of water a knowledge of the micro-organisms which may be present is not only of interest to the microbiologist, but of the greatest importance in practical hygiene. Common putrefactive bacteria and vibrios may not be hurtful in themselves, but they indicate the probability of the presence of organic matter in some of which there may be danger.* The Microzyme Test, which was introduced for their detection, consisted in adding three or four drops of the sample of water to i or 2 ccm. of Pasteur's fluid, the nourishing fluid having been previously boiled in a sterilised test-tube. If the microzymes or their germs existed in the water, the liquid in a few days became milky from the * Parkes, Manual of Practical Hygiene. 1883. 132 BACTERIOLOGY. presence of countless bacteria. This test is of no real value, for it does little more than indicate that bacteria are present, which we know to be the case in all ordinary water, and even in ice. On the other hand, the bacteriological test of Koch is a most valuable addition to the usual methods of water-analysis. It enables us not only to detect the presence of bacteria, but to ascertain approximately their number, and to study very minutely their morphological and biological characteristics. The importance of a thorough acquaintance with the life-history of the individual micro-organisms cannot be too strongly insisted upon. For example, by such means the spirillum of Asiatic cholera can be distinguished from other comma-shaped organisms, and inasmuch as its presence may be an indication of contamination with choleraic discharges, such water should be condemned for drinking purposes, even though we may not yet be in a position to affirm that the microbe is the cause of the disease. The test, in short, consists in making plate-cultiva- tions of a known volume of water, counting the colonies which develop, isolating the micro- organisms, and studying the characters of each individual form. Collection and Transport of Water Samples. Erlenmeyer's conical flasks of about 100 ccm. capacity may be employed with advantage for collecting the samples of water. They are cleansed, plugged, and sterilised in the hot-air EXAMINATION OF WATER. 133 steriliser. When required for use, the plug is removed and held between the fingers, which must not touch the part which enters the neck of the flask. About 30 ccm. of the water to be examined are introduced into the flask, and the plug must be quickly replaced and covered with a caoutchouc cap. If collected from a tap, the water should first be allowed to run for a few minutes, and the sample should be received into the flask without the neck coming into contact with the tap. From a reservoir or stream the flasks may be filled by employing a sterilised pipette. During transport contact between the water and cotton-wool plug must be avoided, and if likely to occur the sample must be collected and forwarded in a Sternberg's bulb. Examination by Plate - cultivation. The. apparatus for plate-cultivation should be arranged as already described. Crushed ice may be added to the water in the glass dish to expedite the setting of the gelatine, so that the plate may be transferred as quickly as possible to the damp chamber. The caoutchouc cap is removed from the flask, and the cotton-wool plug singed in the flame to prevent contamination from adventitious germs on the out- side of the plug. The flask is then held slantingly in the hand, and the plug twisted out and retained between the fingers. With a graduated pipette a drop of the sample is transferred to a tube of liquefied nutrient gelatine, and the plugs of the flask 134 BACTERIOLOGY. and of the tube quickly replaced. I f the water be very impure, it may be necessary to first dilute the sample with sterilised water. The inoculated tube must be gently inclined backwards and forwards and rolled as already explained, to distribute the germs through- out the gelatine, and the gelatine finally poured on a plate. When the gelatine has set, the plate is trans- ferred to a damp chamber, which should be carefully labelled and set aside in a place of moderate tem- perature. In about two or three days the cultiva- FIG. 45. APPARATUS FOR ESTIMATING THE NUMBER OF COLONIES IN A PLATE-CULTIVATION. tion may be examined. In some cases the colonies may be counted at once ; more frequently they are so numerous that the plate must be placed on a dark background, and a special process resorted to. A glass plate, ruled by horizontal and vertical lines into centimetre squares, some of which are again subdivided into ninths, is so arranged on a wooden frame that it can cover the nutrient-gelatine plate without touching it (Fig. 45). A lens is added to assist in discovering minute colonies. If then the colonies are very numerous, the number in some EXAMINATION OF WATER. 135 small divisions is counted, if less in some large ones, and an average is obtained from which the number of colonies on the entire surface is calculated. A separate calculation of the liquefied colonies should be also made, and their number, as well as the total number of colonies present in i ccm. of the sample, recorded. Any peculiar macroscopical appearances, colour, etc., should be noted, and then the micro- scopical appearances of the colonies studied. Lastly, examination of the individual organisms should be made by cover-glass preparations, and by inoculation of nutrient gelatine, potatoes, and other media. Examination by Test-tube-cultivation. A drop of the sample of water may also be added to liquefied nutrient gelatine in a tube, the organisms distributed as already explained (p. 93), and the gelatine allowed to solidify in the tube. A rough comparison of water samples may be made in this way. Microscopic Examination. A drop of the water may be mounted and examined in the way described under drop-cultivations (p. 1 1 2), or a drop is allowed to evaporate on a cover-glass placed under a bell-glass. This is then passed three times through the flame, and stained in the usual manner. The examination of rain water, drinking water, tap water, sea water, various liquids and infusions, etc., by these methods, opens up a wide field for research. Pettenkofer has shown that impregnation with 130 BACTERIOLOGY. carbonic acid of water containing many bacteria diminishes the number of the latter. The examina- tion of waters before and after filtration, or after addition of chemical substances, are matters which require further investigation. CHAPTER VI. EXPERIMENTS UPON THE LIVING ANIMAL. To carry out the last of Koch's postulates, and so complete the chain of evidence in favour of the causal relation of micro-organisms to disease, and to study the mode of action of a pathogenic bac- terium, it is necessary to introduce into a living animal a pure cultivation of the micro-organism in question. For this purpose various animals are employed such as mice, guinea-pigs, rabbits, pigeons, and fowls. Inhalation of Micro-organisms. The animals may be made to inhale an atmosphere impregnated with micro-organisms by means of a spray. In this way Friedlander succeeded in administering the bacteria of pneumonia to mice, and the production of tuberculosis by experimental inhalation has thrown light upon the clinical records of cases reported as instances of the infectiousness of phthisis. Administration with Food. A sheep fed upon potatoes which have been the medium for the cultivation of the anthrax bacillus dies in a 138 BACTERIOLOGY. few days. Similarly, animals fed upon the nodules of bovine tuberculosis become tubercular, and even the flesh and milk of tuberculous animals will occasionally set up tuberculosis. Cutaneous and Subcutaneous Inocula- tion. Cutaneous inoculation may be carried out by making a superficial wound, and inoculating it with a sterilised platinum needle, charged with the micro-organisms to be inoculated. An- other simple method is to take a sterilised knife, infect the point with the material to be inoculated, and then make a minute wound or incision. In either case a situation should be selected, such as the root of the ear, which cannot be licked by the animal after the operation. Subcutaneous inoculation is very simple and effectual, and consequently the method most fre- quently employed. The animal selected for example, a guinea-pig is held by an assistant, who covers it with a towel, leaving only the hinder extremities exposed. By so doing, and gently laying it upon its back, with its head low, a guinea-pig passes apparently into a state of hypnotism, and the trivial operation can be per- formed with little or no movement on the part of the animal. From a spot on the inner side of the thigh the hair is cut close with a small pair of scissors curved on the flat, and the skin must be thoroughly purified with sublimate solu- tion. A small fold of skin is then pinched up EXPERIMENTS UPON THE LIVING ANIMAL. 139 with a pair of sterilised forceps, and with a pair of sharp sterilised scissors, or with a tenotomy knife, a minute incision is made. A sterilised platinum loop is charged with the material to be inoculated, and the loop is gently inserted under the skin, forming a small pocket in the subcutaneous tissue. The needle is then with- drawn, and the sides of the wound gently pressed into apposition. In a mouse the same process is adopted, with the exception that the root of the tail is the usual site of the operation. In a method suggested by Koch an assistant can be dispensed with : a glass bell reversed is placed as a cover to a wide-mouthed glass jar, in which a mouse is held by the tail with a pair of forceps, while the cover is so placed over the mouth of the jar as to leave a small interval near the rim uncovered. The mouse rests with its head down- wards and with its feet against the inner wall of the jar, and in the interval between the cover and the rim the root of the tail is exposed, and must be cleansed and treated as already described. Special Operations. In many cases it is absolutely necessary to perform an operation of greater severity. After the administration of an anaesthetic, infective material may be introduced into the peritoneal cavity by the performance of abdominal section, or injected into the duodenum in the manner employed in the case of Koch's 140 BACTERIOLOGY. comma bacilli by Nicati and Rietsch. In such cases antiseptic precautions must be rigidly followed, and use made of iodoform and other antiseptic dressings. The disinfection of the skin of the animal, of the instruments employed, and of the hands of the operator, are details essential to secure success. To inoculate tubercular matter, sputum may be rubbed up with distilled water, filtered, and the filtrate injected into a tracheal fistula, or the first steps of the operation of iridectomy may be performed, and tubercular material inserted in the anterior chamber of the eye. The advantage of the latter method consists in that it enables the results and changes to be observed from day to day. A cultivation of micro-organisms may also be mixed with sterilised water, and then injected with a syringe directly into the circulation. In rabbits this may be performed without difficulty by injecting the large vein at the base of the ear with a Pravaz' syringe. Before every inoculation the instruments must be sterilised, as already ex- plained, by employing an Israel's case, and after each operation all instruments should be placed in strong sublimate solution (i in 100), wiped dry, and sterilised in the hot-air steriliser, before they are put away. If these precautions be not observed, instances of accidental infection are sure to occur. CHAPTER VII. EXAMINA TION OF ANIMALS EXPERIMENTED UPON AND THE METHODS OF ISOLATING MICRO- ORGANISMS FROM THE LIVING AND DEAD SUBJECT. METHOD OF DISSECTION AND EXAMINATION. ALL animals that die after an experimental inocu- lation should be examined immediately after death. Every precaution must be taken, in conducting the dissection, to exclude extraneous micro-organ- isms, and all instruments employed must have been sterilised in the hot-air steriliser, or heated in the Bunsen burner. If a mouse, for example, has died after an inoculation, it should be at once pinned out by its feet on a slab of wood or in a gutta- percha tray, and bathed with sublimate solution. In the same way, before examining a dead rabbit, a stream of sublimate should be directed over it to lay the fur, which otherwise interferes with the dissection. The hair should be cut away with sterilised scissors from the seat of inoculation, which is the first part to be examined, and any suppura- tion, haemorrhage, oedema, or other pathological 142 BACTERIOLOGY. change should carefully be noted. From any pus or exudation that may be present, material for inocu- lations should at once be taken, and cover-glass- preparations made for microscopical examination. To examine the internal organs and to make inoculations from the blood of the heart or spleen, the skin is cut through from below upwards in the median line of the abdominal and thoracic regions. The abdominal cavity is then opened, and the walls pinned back on either side of the animal. Any abnormal appearances should be noted, and espe- cially the state of the spleen should be examined, by turning the intestines aside. After noting its appearances it should be removed with sterilised forceps and scissors, and deposited upon a sterilised glass slide. After washing it with sublimate solution by means of a camel's hair brush or strip of filter paper, it should be incised with sterilised scissors ; the pulp may be squeezed out from the cut surface, and test-tubes of nutrient gelatine and agar-agar can be inoculated from it, and, if necessary, potato- and drop-cultivations also established. Precisely the same care must be taken in examining lym- phatic glands, tubercles, or pathological nodules; any chance putrefactive micro-organisms on the surface are destroyed by the sublimate solution, and a section is then made, and a minute fragment snipped out of the centre of the nodule, to be examined or transferred to the nutrient medium. The examination of the thorax is made by cutting EXAMINATION OF ANIMALS EXPERIMENTED UPON. 143 through the ribs on either side of the sternum with sterilised scissors, and turning the sternum up where it will be out of the way. The pericardium is then opened, and the right auricle or ventricle pierced with the point of a sterilised scalpel, and inoculations and cover-glass-preparations are made from the blood which escapes. The lungs also require to be especially studied. They should be incised with a sterilised scalpel, and inoculations and cover-glass-preparations made from the cut surface. It may be necessary to embed a piece of lung or fragment of spleen, so that it shall be free from air. This may be done by isolating a fragment with the precautions just de- scribed and depositing it upon the surface of a test- tube of nutrient agar-agar. The contents of another tube, which have been liquefied, and allowed to cool almost to the point of gelatinisation, must then be poured over it. From a potato a little cube must be cut, the tissue deposited in the trough thus formed, and the cube replaced. Blood may also be taken directly from a vein by laying it bare by dissection, making a small section with sterilised scissors, and inserting a looped platinum needle, the needle of a Pravaz' syringe, a capillary tube, or the extremity of the capillary neck of a Sternberg's bulb. If the cultivation be contaminated by the presence of putrefactive or other micro-organisms, they must be isolated subsequently by carrying out a series of plate-cultivations. 144 BACTERIOLOGY. Having completed the dissection, the organs of such a small animal as a mouse may be removed m masse and transferred to absolute alcohol for subsequent examination. In other cases it may be only necessary to reserve portions of each organ. In any case it should be remembered that with a virulent micro-organism, e.g., anthrax, any remain- ing part of the animal should be cremated, and the hands and all instruments should be thoroughly disinfected. Isolation of Micro-organisms from the Living Subject. Micro-organisms in the living subject may be isolated from pus of abscesses, or other discharges, and from the blood and tissues. Abscesses should be opened, and other operations performed, when practicable, with Listerian precau- tions, and a drop of the discharge taken up with a looped needle or capillary pipette, as already explained. To make a cultivation from the blood of a living person, the tip of a finger must be well washed with soap and water and bathed with strong sublimate, or i in 20 carbolic, solution. Venous conges- tion is produced by applying an elastic band or ligature to the finger, which is pricked with a sterilised sewing needle. From the drop of blood which exudes the necessary inoculations and ex- aminations can be made. PART II. GENERAL BIOLOGY OF BACTERIA. CHAPTER VIII. GENERAL MORPHOLOGY AND PHYSIOLOGY. BACTERIA may be considered as minute vegetable cells destitute of nuclei. They are distinguished from animal cells by being able to derive their nitrogen from ammonia compounds, and they differ from the higher vegetable cells in being unable to split up carbonic acid into its elements, owing to the absence of chlorophyll. Von Engelmann and Van Tieghem include among the bacteria certain organisms, named by them Bacterium chlorinum, Bacterium viride, and Bacillus virens, which are coloured green by this substance ; but further researches are required before any conclusions are definitely arrived at as to the place of these parti- cular organisms in the vegetable kingdom. It is quite possible that they may be Algae, and they will, therefore, find no place in the classification which will be here adopted. Chemical composition. For our knowledge of the composition of bacteria we are chiefly in- debted to Nencki. Their constituents are found on 148 BACTERIOLOGY. analysis to vary slightly, according to whether the bacteria are in zooglcea or in the active state. In the latter condition they are said to consist of 83*42 per cent, of water. In one hundred parts of the dried constituents there are the following: A nitrogenous body . . . 84*20 Fat . . ... . 6 04 Ash . . . . . . 472 Undetermined substances . ,,. , 5-04 This nitrogenous body is called Mycoprotein^ and consists of Carbon . . ^ . . . 52' 32 Hydrogen . 7-55 Nitrogen . , . . H'75 but no sulphur or phosphorus. The nitrogenous body appears to vary in different species, for in Bacillus anthracis a substance has been obtained which does not give the reactions of mycoprotein, and, therefore, is distinguished as anthraxprotein. Considering bacteria as cells, we may speak of the cell-wall and the cell-contents. Cell-wall. The cell-wall consists of cellulose, or according to Nencki in the putrefactive bacteria of mycoprotein. It may be demonstrated by the action of iodine, which contracts the protoplasmic contents, and renders the cell-wall visible. The author has taken advantage of the action of iodine to differentiate by staining the sheath of \hzBacillus GENERAL MORPHOLOGY AND PHYSIOLOGY. 149 anthracis from its contents. If we stain cover-glass preparations of this bacillus by the method of Gram, we get the following results. By the first solution the rods are uniformly stained blue; by subjecting them to the iodine solution, the proto- plasmic contents are contracted, while the next solution, alcohol, decolorises the sheath, which may be then stained in contrast with eosin. The cell-wall may be either pliable or rigid. Pliability is ob- FlG< 46 - served in the long filaments, which are endowed with a slow vermi- THRACIS, DOUBLE - ... . . STAINED WITH GEN- cular movement, while rigidity TIAN VIOLET AND accounts for the maintenance of EOSIN \ I ! heS 1 hea * was stained pink, and the characteristic form of several the ceil - contents , . .,, blue. X 1200. species, such as spirilla. Cell - contents. The cell - protoplasm yields mycoprotein. In some it is homogeneous, and in others granular. The action of the aniline dyes indicates a close relation to nuclear protoplasm, though all nuclear stains are not suitable for bacteria. In some cases also, the bacteria remain stained under the influence of a reagent, which removes the colour from nuclei. The power of fixing the stain is not always present, and indicates a difference in the protoplasm of different species. Thus in staining phthisical sputum, the nitric acid removes the stain from all bacteria and bacilli I5O BACTERIOLOGY. present, with the exception of the tubercle bacillus. This difference in the protoplasm of different species is also illustrated by the necessit}^ in many cases of using- special processes, owing to the ordinary methods being unsatisfactory or not pro- ducing any result. The protoplasm of some bacteria contains starch granules ; thus Clostridium butyricum gives the starch reaction with iodine. Sulphur granules are present in some species of Beggiatoa which thrive in sulphur springs. The colouring matter of the pigment bacteria is probably external to the cell as a rule ; for example, in micrococcus prodigiosus the pigment granules are distinctly between the cells ; on the other hand, in Beggiatoa roseo- persicina, or the peach-coloured bacterium, the special pigment bacterio-purpunn appears to be dissolved in the cell protoplasm. In Bacillus pyocyaneus the pigment is certainly not localised entirely in the cell ; for it becomes rapidly diffused in the surrounding medium, considerably beyond the confines of the growth itself. Gelatinous envelope. In several species, either as a result of a secretion from the cell, or of the absorption of moisture and swelling up of the outer layer of the cell-wall, a mucinous or gelatinous envelope develops around them. This envelope may form a capsule, such as we meet with in certain bacteria found in the rusty sputum of pneumonia, and in Micrococcus tetragonus ; or it may GENERAL MORPHOLOGY AND PHYSIOLOGY. occur as a continuous sheath around a chain of bacteria, which by its disappearance sets the individual links free. The capsule is soluble in water, and under some circumstances is difficult tc demonstrate. In the pneumo-coccus of Friedlander the capsule disappears on cultivation, but reappears in preparations made from an inoculated animal. In the pleuritic fluid of a mouse these cocci are often found with a strikingly well-marked capsule, and in other capsuled cocci the extent of the envelope has been observed to vary considerably in the same species of bacterium. When this gelatinous material forms a matrix, in which numbers of bacteria are congregated in an irregular mass, we have what is termed a zooglcea. Thezooglcean stage is a resting stage, often preceded or followed by a motile stage. Thus bacteria may be present in a solution in an active state, and after a time a scum or pellicle forms on the surface of the liquid, which consists of zooglcea. At the edges of the zooglcea individuals may be seen again to become motile, detaching themselves from the edges of the mass, and swimming off in the surrounding fluid. The same may be observed sometimes in culti- vations started in nutrient gelatine. The inoculated bacteria grow and multiply, and liquefy the gelatine, and after a time a zooglcean film appears on the surface of the liquefied layer. On potatoes the appearances are very varied. In a bacillus which '52 BACTERIOLOGY. readily develops on unsterilised potatoes, the zoo glcea may spread over the cut surface, forming a pellicle which can be raised en masse like a delicate veil. Another bacillus forms a zooglcea, consist- ing of a tenacious layer which can be drawn out in long stringy threads. In Ascococcus Billrothii the gelatinous envelope develops to such an enormous extent that it forms the characteristic feature of the species (Fig. 47). FIG. 47. Ascococcus BILLROTHII, x 65. [After Cohn.] Form. The individual cells vary in form, and may either remain isolated or attached to each other. Round cells and egg-shaped cells are called cocci. The spherical form is the most common, but cocci are occasionally exclusively ovoid, as in Streptococcus bombycis. The giant cocci of some species are spoken of as megacocci, to distinguish them from the ordinary cocci, such as micrococci. The fission by which the cocci increase may take place in one direction only, and if the two resulting GENERAL MORPHOLOGY AND PHYSIOLOGY. 153 cells remain attached to each other, they form a diplococcus. If these two cells again divide, and the resulting cells remain linked together, we get a chain or rosary, or streptococcus (Figs. 48, 49, 50). FIG. 48. STREPTOCOCCUS AND SARCINACOCCUS FROM A DROP-CULTIVATION, X 1200. FIG. 49. STREPTOCOCCUS IN THE BLOOD OF A RABBIT, x 1 200. These chains may consist of a few individuals linked together, or of several hundreds, in which case the chains are generally curved or twisted. If the division occur in two directions, so that FIG. 50. STREPTOCOCCUS OF PROGRESSIVE TISSUE NECROSIS IN MICE, [After Koch.] four cocci result, a tetrad or merismopedia is formed. If the division occur in three direc- tions, one coccus divides into eight, and we get a packet form or sarcinacoccus. Immediately after division, the daughter cells are not perfectly 154 BACTERIOLOGY. circular, but are flattened or facetted where they are opposite to each other. They gradually become rounded off, and each daughter cell is then ready to divide in its turn. In other cases the cocci after division only form irregular heaps or collections like bunches of grapes. This form is sometimes distinguished as ^taphylococcus^ but it cannot be considered an important feature. Where we find irregular masses or balls embedded in a copious gelatinous matrix, the extent of the latter affords a characteristic condition described as ascococcus. Another type is the rod, characteristic of bac- terium and bacillus. The rods may vary con- siderably in length. The very short rods with rounded ends are very difficult to distinguish from the oval cocci, but differ in that a rod, however short it may be, must have at least two sides parallel. The vibrio or bent rod may be considered as the connecting link between the rods and the corkscrew forms or spirilla. Lastly we have the filamentous forms, which may be straight, leptothrix, or wavy, spirochata (Fig. 51), or the wavy thread may be looped and entwined on itself, spirulina (Plate I., Fig. 37). By involution forms we signify certain irregular shapes which result especially in exhausted culti- vations. They are peculiar, oval, pear-shaped, or irregular enlargements (Plate I, Figs. 31 to 36). Movement. Many bacteria are devoid of move- GENERAL MORPHOLOGY AND PHYSIOLOGY. 155 ment throughout the whole of their life history. Others, during certain stages of their life cycle, and possibly some forms always, are endowed with locomotive power. The character of the movement is very varied, and ranges from a slow undulatory motion to one of extreme rapidity. Many appear to progress in a definite direction. Others move continuously, first in one direction and then in another, and others again seem to hesitate before altering their course. They may either glide along smoothly or progress with a tremulous action. They appear to be able to avoid obstacles, and to FIG. 51. SPIROCH^TA FROM SEWAGE WATER, x 1200. set themselves free from objects with which they have accidentally come into contact. Vibrios have a peculiar serpentine movement, but other forms, such as the commonly-known Bacterium termo and segments of spirilla, such as comma-bacilli, revolve around their long axis as well as make distinct progression. The complete spirilla are charac- terised by the familiar corkscrew movement. With regard to cocci there is some doubt as to whether they are endowed with independent move- ment; any quivering or oscillation is generally regarded as only brownian or molecular. In some 1 56 BACTERIOLOGY. straight thread- forms, which are motile, the move- ment is very slow and vermicular in character, but in wavy threads, such as the Spirochcete plicatilis, there is not only an undulatory motion, with rapid progression across the field of the microscope, but if they are confined by more or less debris, they give very peculiar and characteristic spasmodic movements. The rod-forms of Proteus vulgaris exhibit very extraordinary movements on the surface of solid nutrient gelatine. Groups of rods may be observed to pass each other in opposite directions. Single individuals meet and progress side by side, or one or more individuals may part from a group and glide away independently. Occasionally a number of rods progress in single file. It is, however, difficult to believe that these movements can occur on a solid surface. The author is inclined to believe that there is an almost inappreciable layer of liquid on the surface of the gelatine, which is expressed after the gelatine sets. In tubes of nutrient agar-agar gelatinised obliquely and then kept upright the liquid so expressed collects at the bottom of the sloping surface. What the means are by which bacteria are en- dowed with the power of spontaneous movement and of progression may still be said to be unsettled. The author has watched the movement of long slender threads in sewage-contaminated water, which could only be explained by the inherent GENERAL MORPHOLOGY AND PHYSIOLOGY. 157 contractility of the protoplasmic contents ; for if any drawing or propelling organ existed in proportion to the length of the organism, it would probably have been visible. But in many cases the organism is provided with a vibratile lash or 9 ." FIG. 52. I. Coccus with flagellum. 2. Similar coccus dividing with two flagella 3. Colony of flagellated macrococci of Beggiatoa roseopersicina. 4. Short rod from the same Beggiatoa with flagella [all after Zopf]. 5. Bacillus with flagella [from a photograph by Koch]. 6. Bacillus subtilis [after Brefeld]. 7, 8. Short rod-forms of Beggiatoa roseopersicina with one flagellum [after Zopf]. 9. Very long rod of the same, with flagellum at both ends [after Warming]. 10. Vibrio, with double flagellum at each end [after Warming]. 1 1. Vibrio, with flagella [from a photograph by the author], 12. Spirillum with flagella [after a photograph by Koch]. 13. Spirillum with flagella [after Zopf]. 14. Spirillum with double flagella [after Zopf]. 15. Beggiatoa roseopersicina, with a triple flagellum at one end; and 16, with a double flagellum at both ends [after Warming]. flagellum at one end, or with one or more at both ends (Fig. 52). 158 BACTERIOLOGY. Some observers believe that the movement of cocci is due to the existence of a flagellum. In Bacterium termo the existence of a lash at either end was first determined by the researches of Dallinger and Drysdale. In motile bacilli, such as the hay bacillus and Bacillus ulna, and in vibrios and spirilla, the flagella can be readily recognised by expert microscopists with the employment of the best lenses, and, what is of equal importance, proper illumination. They are objects of extreme delicacy, and tenuity, and in stained preparations may be absent from retraction or injury. Koch succeeded in photographing them after staining with logwood, which turned them a brown colour. They may also be stained with the aniline dyes, for the author has observed them in vibrios in preparations stained with gentian violet, from which also they have been photographed, in spite of the violet colour, by the use of isochromatic dry plates. It is not certain whether the flagella are exten- sions of the cell-wall, or derived from the internal protoplasm. Van Tieghem holds the first view, and does not regard them as motile organs at all. Zopf, on the other hand, adheres to the second view, and moreover believes that they can be retracted within the cell-wall. Reproduction. Bacteria multiply by fission, and by processes which may be considered as representing fructification. The bacteria exhibiting the latter processes have been divided into two GENERAL MORPHOLOGY AND PHYSIOLOGY. '59 groups, distinguished by the formation of endo- spores in the one, and of arthrospores in the other. In the process of fission the cell first increases in size, and a transverse septum forms from the cell-wall, dividing the internal protoplasm into two equal parts ; these may separate and lead an independent existence, or remain linked together. In chains of FIG. 53. BACILLUS MEGATERIUM. a. A chain of rods, X 250. The rest X 600. b. Two active rods. d to /. Successive stages of spore-formation. h and /. Successive stages of germination. [After De Bary.] cocci the individual cells are easily visible and distinct, but in the thread-forms resulting from the linking together of rods, as in the anthrax bacillus, the composition of the thread is only demonstrated by the action of reagents. Endospore formation may be conveniently studied in Bacillus anthracis, Bacillus megaterium, or Bacil- lus subtilis. The protoplasm becomes granular, i6o BACTERIOLOGY. and at certain points in the thread a speck ap- pears, which gradually enlarges and develops into a circular or egg-shaped, sharply defined, highly refractive body. The spore grows at the expense of the protoplasm of the cell, which in time, to- gether with the cell-wall, entirely disappears, and the spore is set free. These phenomena are best seen in an immotile bacillus in a drop-cultivation FIG. 54. CLOSTRIDIUM BUTYRICUM, x 1020. B. Stages of spore-formation. C. Stages of germination. [After Prazmowski.] on a warm stage, the whole process may then be observed continuously from beginning to end. Spores may form in each link of the thread, so that a regular row results, or they may occur at irregular intervals. Spore-formation also occurs in free rods in the centre or at one end. Occasionally a spore develops at the extreme end, giving a bacillus the appearance of a drum-stick. The spore may be GENERAL MORPHOLOGY AND PHYSIOLOGY. l6l considerably wider, but is never longer than the parent cell. Arthrospore formation is illustrated in Leuco- nostoc mesenteroides. Certain elements in the chain of cocci, apparently not differing from the rest, FIG. 55- A THREAD OF BACILLUS ANTHRACIS WITH SPORES, IN A DROP-CULTIVATION, X 1400. become larger, with tougher walls, and more refrac- tive (Fig. 56). The remaining cells die, and these cells having acquired the properties of spores are set free, and can reproduce a new growth in any FIG. 56. LEUCONOSTOC MESENTEROIDES ; COCCI-CHAINS WITH ARTHROSPORES (after Van Tieghem and Cienkowski). fresh nourishing soil. That this occurs in all species which do not form endospores is at present only a supposition. Spores are invested by a thick membrane, which is believed to consist of two layers. To this they probably owe the property they possess of retaining vitality when desiccated, and of offering a greater ii 1 62 BACTERIOLOGY. resistance to the action of chemical reagents and heat than the parent cells. Spore-formation has been regarded by some as occurring when the nourishing soil is exhausted, thus providing for the perpetuation of the species. For instance, anthrax bacilli do not form spores in the living body, but when the animal dies it has been stated that development of spores takes place, and hence the danger of contaminating the soil if the body is disposed of by burial. Klein, however, has pointed out that if mice and guinea-pigs which have died of anthrax are kept unopened, the bacilli simply degenerate and ultimately disappear. Thus there is good reason for believing that spore- forma- tion is not due to exhaustion of the pabulum, but probably free access to oxygen constitutes an im- portant factor in inducing this condition. If we inoculate a potato with anthrax, copious spore-for- mation occurs, though we cannot consider that the nourishing soil has been exhausted. But we have in this case the surface of the potato freely exposed to the air in the damp chamber. In the same way, in cultivation on agar-agar solidified obliquely, so as to get a large surface, spore-formation readily takes place. Contamination of a burial-ground must result, therefore, from bodies in which a post- mortem examination has been made, by which the blood and organs have been freely exposed to the air, or from animals which have not been examined, owing to their hides being soiled with GENERAL MORPHOLOGY AND PHYSIOLOGY. 163 excretions, and with blood which issues from the mouth and nostrils before death. When spores are introduced into a suitable medium at a favourable temperature, they develop again into rods. The spore loses its sharp con- tour, and, at one pole or on one side, a pale process bursts through the membrane, gradually growing into a rod from which the empty capsule is thrown off (Figs. 53 and 54). Spores differ from the parent cells in their be- FIG. 57. SPORES OF BACILLUS AN- FIG. 58. SPORE-BEARING THREADS THRACIS, STAINED WITH GENTIAN OF BACILLUS ANTHRACIS, VIOLET, AFTER PASSING THE DOUBLE-STAINED WITH FUCH- COVER-GLASS TWELVE TlMES SINE AND METHYLENE BLUE, THROUGH THE FLAME, X I2OO. X I2OO. haviour to staining reagents. Like them, they can be stained with aniline dyes, but not by the ordinary processes. They require to be specially treated. This is probably due to the tough capsule, which must first be altered or softened by heat or strong acid, until it allows the stain to penetrate. Once stained, they again differ from the parent cells in resisting decolorisation ; this fact is taken advantage of to double-stain spore-bearing bacilli. In staining micro-organisms, the protoplasm is 1 64 BACTERIOLOGY. sometimes broken up into irregular segments or granules, as in many spirilla, and we may perhaps add the bacilli of tuberculosis and leprosy. The beaded appearance of the tubercle bacillus is well known. Some observers have regarded the beads, others the bright spaces between them, as spores. But spores in unstained preparations appear as glistening bodies with sharp contour, and do not stain at all, or very little, by the ordinary processes. It appears, therefore, very doubtful whether either the clear spaces or the beads are spores, espe- cially as the tubercle bacillus, when unstained, is a slightly curved hyaline rod, without any dif- ferentiation into granules. These considerations led the author to stain and examine tubercular sputum from various sources under careful illumination, and with such lenses as Powell and Lealand's ^ in. Horn. imm. The tubercle bacillus may then be frequently seen to consist of a very delicate sheath, holding together a number of deeply stained granules, for the most part round or cylindrical, with irregular contour, and differing considerably in size, while the light interspaces are seen to vary in form according to the shape of the granules. In some preparations more dis- tinct, and clearly ovoid, bodies may be observed which are sometimes terminal. They can be readily demonstrated by taking a photograph with a ^ in. Horn, imm., and subsequently enlarging the nega- tive to from 2,500 (Fig. 59) to 6,000 diameters. GENERAL MORPHOLOGY AND PHYSIOLOGY. 165 d\ -A It is not impossible that these ovoid bodies are spores, which, in their be- haviour towards staining reagents, thus form an exception to the general rule. But there can be little doubt that a tubercle bacillus consists, for the most part, of a very delicate FlG . 59 . -TUBERCLE BACILLI IN sheath, with protoplasmic SPUTUM ' x 2 5 ( from P hot - graphs). contents which have a great tendency to be broken up or coagulated into little segments or roundish granules, owing possibly to the treatment they are sub- jected to in making a microscopical LEPROSY BA- p re p arat i n - This, however, does not CILLI, FROM always occur, for the bacilli at times are A SECTION 1111 i i OF SKIN, n t beaded, but are stained in their en- x 1200. tirety. In the leprosy bacilli a similar appearance occurs. In stained sections the rods have a beaded appearance, but the intervals between the granules are sometimes very long, and occasion- ally the protoplasm appears to have collected only at the extreme ends FIG. 61. Of the rod (Fig. 60). Very probably GLANDERS BACILLI, . ' J r ' * FROM A SECTION the appearances in the case of the O F A GLANDERS bacillus of glanders (Fig. 61) and NODULE, x 1200. the bacterium of chicken-cholera (Figs. 62 and 63) may be similarly explained. 1 66 BACTERIOLOGY. The fact that tubercular sputum preserves its virulence for several months, even after desicca- tion, has been attributed to the formation of spores, and Babes has drawn attention to ovoid grains in old cultivations of the bacilli, which he succeeded in \___x staining red, while the bacilli FIG. 62. were sta i nec i bi ue> BACTERIUM OF CHICKEN- . . . CHOLERA, FROM BLOOD In his definition of spirilla OF^INFECTED HEN, x Zopf giv e s the spore-formation as absent or unknown. In comma-bacilli in sewage water, the author has often noted appearances very suggestive of refractive FIG. 63. FIG. 64. BACTERIUM OF CHICKEN-CHOLERA, COMMA-BACILLI IN SEWAGE FROM MUSCLE JUICE OF AN IN- WATER, STAINED WITH FECTED HEN, X 2500 (from a GENTIAN VIOLET, X 1200. photograph). spores (Fig. 64). The same also may be observed in vibrios, differing by their regular contour from the irregular spaces occasionally observed in stained preparations (Figs. 65 and 66). They are possibly only vacuoles. Respiration and Nutrition. Like all a-chlo- rophyllous vegetables, bacteria require for their nutrition oxygen, nitrogen, carbon, water, and certain mineral salts. Many require free access to oxygen, others can derive it from the oxidised GENERAL MORPHOLOGY AND PHYSIOLOGY. 167 compounds in the medium in which they grow. Pasteur divided bacteria into two great classes the aerobic and anaerobic ; and considered that the latter not only had no need for oxygen, but that its presence was actually deleterious. Though this view must be considerably modified, the terms are convenient, and are still retained. They are well illustrated by the bacillus of anthrax, and the bacillus of malignant oedema ; and a simple plan of demonstration has been employed by the author. A fragment of tissue from the spleen, for example, FIG. 65. FIG. 66. VIBRIOS IN WATER CONTAMINATED SPIRILLUM UNDULA, WITH SEWAGE, X 1200. X 1200. known to contain anthrax bacilli, is deposited with a sterilised inoculating needle, with the necessary precautions, on the surface of nutrient agar-agar in a test-tube ; another tube of nutrient agar-agar is liquefied, and when cooled down almost to the point of gelatinisation, a part is poured into the first tube, so that when it sets the piece of tissue is completely embedded. A piece of tissue from an animal suffering from malignant oedema is treated in the same way, and the tubes are placed in the incubator. If then we examine 1 68 BACTERIOLOGY. them after two or three days, we shall find no change in the anthrax tube ; the bacillus being eminently aerobic, no growth whatever has oc- curred. In the tube containing the bacilli of malignant cedema there will be a more or less characteristic cultivation. The nitrogen which is essential for building up their protoplasm can be obtained either from albumins, or from ammonia and its derivatives. That the albumins can be dispensed with was shown by Pasteur, who employed an artificial nourishing solution built upon a formula repre- senting the essential food constituents (p. 108). Carbon is derived from such substances as cane sugar, milk sugar, and glycerine, and, in some cases, by the splitting up of complex proteid bodies. Water is essential for their growth, but depriva- tion of water does not kill all bacteria. Desicca- tion on potato is employed for preserving some micro-organisms, as a new growth can be started, when required, by transferring some of the dried potato to fresh nourishing ground. Comma-bacilli, on the other hand, are destroyed by drying. Sugar, by abstracting water, prevents the development of micro-organisms in preserves. Mineral or inorganic substances, such as com- pounds of sodium and potassium, and different phosphates and sulphates, are necessary in small proportions. GENERAL MORPHOLOGY AND PHYSIOLOGY. 169 Circumstances affecting their growth. Nature of the Soil. Though we know the elements necessary, we are, nevertheless, as yet unable to provide a pabulum suitable for all kinds of bacteria. Thus we are quite unable to cul- tivate some species artificially. Others will only grow upon special media. Many grow upon nutri- ent gelatine ; but some species only if it be acid or alkaline respectively. Whether in the latter case this is due purely to the reaction or to the presence of the particular ingredients is an un- settled point. Though the comma-bacillus of Koch, like the majority of organisms, grows best on an alkaline medium, yet the surface of a potato is acid, and on this it is well known to flourish at the temperature of the blood. Effect of Temperature. In their behaviour to- wards temperature bacteria vary considerably, but still for the majority we may distinguish a maximum, optimum, and minimum temperature. Many grow best at the temperature of the blood, and hence the value of nutrient agar-agar, which is not liquefied at 37 C. The tubercle bacillus will only grow at a temperature varying between 30 and 4iC. On the other hand, many forms grow between the limits of 5 and 45 C. At these temperatures their functional activity is paralysed, but they are not destroyed, for by re- moval to favourable conditions they spring again to life. Bacteria seem to have a special power 1 70 BACTERIOLOGY. of resisting the effects of cold. It has been stated that comma-bacilli exposed to a tempera- ture of 10 for an hour, and bacilli of anthrax after exposure to a temperature of noC., still retained their vitality. Temperatures over 50 to 60 C. destroy most bacteria, but not their spores ; spores of anthrax retain their vitality after im- mersion in boiling water, but are destroyed by prolonged boiling. Roughly speaking, all patho- genic bacteria grow best at the temperature of the blood, and non-pathogenic bacteria at the ordinary temperature of the room. Effect of Movement. Bacteria probably grow best when left undisturbed. Violent agitation of a vessel in which they are growing certainly retards their growth, but a steady movement is stated not to affect it ; at any rate anthrax bacilli grow with enormous rapidity in the blood-vessels, in spite of the circulation. Effect of Compressed Air. Paul Bert maintained that a pressure of twenty-three to twenty-four atmospheres stopped all development of putre- factive bacteria. Oxygen, under a pressure of five or six atmospheres, is stated to stop their growth. Other observers have, however, obtained different results. Effect of Gases. Hydrogen and carbonic acid are stated to stop the movements of the motile bacteria. Chloroform is believed to arrest the changes brought about by the zymogenic species. GENERAL MORPHOLOGY AND PHYSIOLOGY. 171 Electricity. Cohn and Mendelsohn found that a constant galvanic current produced a deleterious effect owing to electrolysis. At the positive pole the liquid became distinctly acid, and at the negative pole distinctly alkaline. With a weak current there appeared to be no effect, two power- ful cells at the very least being necessary. Light. Downes has shown that sunlight is fatal to putrefactive bacteria. This is believed to be due to a process of induced hyper-oxidation, from which living organisms ordinarily are shielded by protective developments of the cell-wall, or of colouring matter, which cut off injurious rays. Duclaux has investigated the same subject, and observed that micrococci were more sensitive to sunlight than the spore-bearing bacilli. Engel- mann has described a bacterium whose movements cease in the dark, and Zopf states that in his cultures of Beggiatoa roseo-persicina the growth was much more strongly developed on the side of the vessel facing the light. Chemical Reagents. Many substances, such as carbolic acid, corrosive sublimate, chlorine, bromine, etc., have a marked effect upon the growth of bacteria. This will be more fully described in another chapter. In several cases the bacteria themselves secrete a substance which is injurious to their future development. Products of Growth. Bacteria may be grouped together according to the changes pro- 172 BACTERIOLOGY. duced in the media in which they grow. Thus we have pigment-forming, fermentative, putrefactive, and pathogenic bacteria. Chromogenic or pigment-forming bacteria elabo- rate during their growth definite colour stuffs. Such species are exemplified by Bacillus ianthi- nus, which produces a striking purple growth ; Bacillus pyocyaneus, which secretes pyocyanin, a substance which has been isolated and obtained in a crystalline form ; Micrococcus prodigiosus, which produces a pigment allied to fuchsine ; Beggiatoa roseo-persicina, which is characterised by the presence of bacterio-purpurin ; Sarcina lutea, Bacillus cyanogenus, and many others. Zymogenic or ferment bacteria produce their changes in non-nitrogenised media. Bacterium aceti, by its growth produces the acetic fermenta- tion in wine by which alcohol taking up atmo- spheric oxygen is converted into vinegar C 2 H 6 O + O 2 = C 2 H 4 O 2 + H 2 O. The fermentation of urine, by which urea is con- verted into carbonate of ammonia, can be brought about by several micro-organisms, but notably by the Bacterium urece. The change produced is represented by the following formula : = ( NH4 ) 2VC 3 ' Clostridium butyricum converts the salts of lac- tic acid into butyric acid, producing the butyric GENERAL MORPHOLOGY AND PHYSIOLOGY. 173 fermentation in solutions of starch, dextrine, and sugar. These bacteria are agents in the ripening of cheese, and the production of sauerkraut. Thus, in a solution neutralised with calcium carbonate : 2 [Ca(C 3 H 5 3 ) 2 ] + H 2 0=Ca(C 4 H 7 2 ) 2 +CaC0 3 + 3 CO 2 + H 8 . In the so-called viscous fermentation the Strep- tococcus viscosus produces a gummy substance in wines. According to Pasteur, the change may be thus represented : 25(C 12 H 22 O n ) + 25(H 2 O) = i2(C 12 H 20 O 10 ) + 2 4 (C 6 H 14 O 6 ) + i 2 (C0 2 )+ i 2 (H 2 0). And as another example may be mentioned the Bacillus acidi lactici, through whose agency sugar of milk is converted into lactic acid : C 12 H 24 12 = 4 (C 3 H 6 3 ). Saprogenic or putrefactive bacteria play a most important role in the economy of nature. They produce changes allied to fermentation in complex organic substances. The nitrification of soil has been attributed to their agency. Their action on proteids, according to Hoppe-Seyler, may be com- pared to digestion ; bodies like peptones are first produced, then leucin, tyrosin, and fatty acids ; lastly indol, phenol, sulphuretted hydrogen, am- monia, carbonic acid, and water. They abstract the elements they require, and the remainder enter into new combinations. Associated with the forma- tion of these substances are certain bodies, which have a poisonous effect when introduced into 1 74 BACTERIOLOGY. animals. These poisonous alkaloids, ptomaines, produce a septic poisoning, which must be dis- tinguished from septic infection. The effects of septic poisoning depend on the dose, whereas the effects of septic infection are, to a certain extent, independent of the dose. A small quantity of a septic poison may produce only transient effects, and a relatively large quantity may be necessary to produce vomiting, rigors, and death. Septic infection, on the other hand, may result equally from a small dose, because the poison introduced is a living organism which is capable of propaga- tion and multiplication. Our knowledge of these alkaloids is greatly attributable to the researches of Selmi, Gautier, and also Brieger and others. Brieger has isolated ptomaines from the human cadaver, putrid meat, fish, and cheese. These substances cadaverin, putrescin, saprin, peptotoxin, and many others vary in their toxic properties. Pathogenic bacteria are those which are genetically related to disease. Many organisms have been supposed to be pathogenic, or have been described in connection with diseases, which are only sapro- phytic associates. By the latter we mean organisms which feed upon dead organic matter. Such are many forms which are found on the skin, in the intestinal canal, and sometimes in the internal organs, especially the liver and kidneys ; the tissues have lost their vitality, and the organisms, through some lesion, have been carried into the circulation. GENERAL MORPHOLOGY AND PHYSIOLOGY. 175 That many organisms are causally related to disease, there is strong evidence in proof ; for no organism can be considered to be productive of disease unless it fulfils the conditions which have been laid down by Koch (p. 1 7). Great stress must be laid upon the importance of successive cultiva- tion through many generations, as the objection that a chemical virus may be carried over from the original source is thus overcome. Any hypo- thetical chemical poison carried over from one tube to another would, after a great number of such cultivations, be diluted to such an immense extent as to be inappreciable and absolutely inert. Though we may accept as a fact the existence of pathogenic organisms, we are not yet in a posi- tion to assert the means by which they produce their deleterious or fatal effects. Many theories have been propounded. It has been suggested that the organisms micrococci, for example may be compared to an invading army. The phagocytes arrayed against them endeavour to assimilate and destroy them, but perish themselves in the attempt. This might explain the breaking down of tissue, and the formation of local lesions, but does not assist us in understanding the fatal result in thirty-six to forty-eight hours produced by the inoculation of the bacilli of anthrax. Another view is that the invading army seizes upon the commis- sariat, appropriating the general pabulum, which 1 76 BACTERIOLOGY. is so essential to the life of the tissues. But this would hardly account for so acute and fatal a result as in anthrax, but would lead one to expect symptoms of inanition and gradual exhaustion. Moreover, against this theory we have the fact that death may result, for example, from anthrax, with the occasional presence of comparatively few bacilli ; and, again, the blood may teem with parasites such as the flagellated monads in well-nourished, healthy-looking rats, without apparently causing any symptoms whatever. In the same category may be placed the theory that eminently aerobic organisms seize upon the oxygen of the blood and produce death by asphyxia. Another explana- tion is afforded by the suggestion of interference with the functions of the lung and kidney by mecha- nical blocking of the capillaries. Here the same objection is met with in the case of anthrax, the same fatal result may occur with only a few bacilli, while other cases yield very beautiful sections, looking like injected preparations from the mapping out of the capillaries with the count- less crowds of bacilli (Plates XXIII. and XXIV). The most satisfactory explanation is probably afforded by analogy with the putrefactive bacteria. We have seen that they derive their necessary elements from complex organic substances, and accompanying the residue we find the presence of poisonous alkaloids. Do pathogenic bacteria act in the same way ? Does the anthrax bacillus GENERAL MORPHOLOGY AND PHYSIOLOGY. 177 produce a ptomaine anthracin, which in a certain dose produces death, independently of the number of bacilli, provided there are sufficient present to develop that dose ? Though this is possible, observers as yet have failed to extract from culti- vations of the anthrax bacillus any alkaloid with virulent properties. Lastly, it has been suggested that possibly a special ferment is secreted by the organisms, and that by the changes ultimately wrought by the action of this ferment, the symptoms and phe- nomena of disease arise. We have an analogy with this theory in the alkaline fermentation of urine by means of the Torula urece. By the researches of Musculus, and later of Sheridan Lea, it has been shown that a ferment is secreted by the cells which can be isolated in aqueous solution, and is capable of rapidly inducing an active fermentation of urea. Either of the two last theories assists us in under- standing how it is that in anthrax or in tuberculosis we may find the presence of only a few bacilli, or that, assuming both tetanus and hydrophobia to be due to microbes, we can have such a violent dis- turbance of the system produced by the presence of very few micro-organisms. We may conceive that different species of bacilli may vary greatly in their power of producing an alkaloid or secreting a ferment, just as the elaboration of pigment is much more marked in some species than in others ; thus it need not follow that the number of 12 178 BACTERIOLOGY. micro-organisms bears any relation to the viru- lence or activity of the substance they produce. There is, however, yet another factor in the pro- duction of disease. We know that in health we are proof against most of these micro-organisms ; if it were not so, we should all rapidly fall victims to the tubercle bacillus or some others, which we in health inhale with impunity. We know that a microbe may only cause a local lesion in one animal, and death in another. It is still more striking that the same micro-organism, as is the case with anthrax, may have no effect whatever upon certain species of animals, though it is deadly to others. Again, an animal naturally sus- ceptible to the effect of a pathogenic organism may be rendered proof against it. These matters will be discussed in a future chapter. Distribution of Bacteria. Bacteria are com- monly described as ubiquitous. They are ever present in the air, though not in such exaggerated numbers as is commonly supposed. In nutrient media exposed to the air one is often astonished at times at the comparatively few bacteria which develop in comparison to the amount of floating matter, such as mineral particles, scales, spores of fungi, and ddbris known to be present. In water they are also present in considerable numbers,, though of course varying according to the character of the water. Wherever there is putrefaction, they are present in vast numbers. In the soil, in GENERAL MORPHOLOGY AND PHYSIOLOGY. 179 sewage, in the intestines ; and in uncleanly persons especially, on the skin and between the teeth, various species may always be found, but in the healthy blood and healthy tissues bacteria are never present. In a previous chapter the method of examining the blood of living persons has been described, and there is, by this means, ample oppor- tunity for satisfying oneself that bacteria are never to be found in the blood in health. The organs removed from a perfectly healthy animal, with the necessary precautions, into sterilised media can be kept indefinitely without undergoing putrefaction, or giving any development of bacteria. This has been established by many observers, notably Cheyne and Hauser ; and the results of former observers to the contrary must be attributed to imperfect methods admitting of accidental con- tamination. CHAPTER IX. ANTISEPTICS AND DISINFECTANTS. IN the previous chapter several conditions were alluded to which affected the growth of bacteria, such as the nature of the nutrient soil, temperature, light, and electricity. The effect of certain chemi- cal substances, and of excessive heat and cold, was also mentioned ; but this constitutes a subject of such practical importance that it must be con- sidered more fully. Agents which retard the growth of bacteria are generally spoken of as antiseptics, as distinguished from disinfectants, which altogether destroy their vitality. Though chemical disinfectants, or germicides, when diluted, act as efficient antiseptics, the con- verse, that an antiseptic in a sufficiently concentrated form will act as a disinfectant, is not the case. The term " antiseptic," indeed, should be restricted to those substances or agents which arrest the changes bacteria produce, but which do not prevent their springing into activity when removed to favourable conditions. Thus excessive heat, which destroys ANTISEPTICS AND DISINFECTANTS. l8l bacteria and their spores, is a true disinfectant ; and excessive cold, which only benumbs them, retard- ing their development without killing them, is an antiseptic. Spores have a greater power of resisting the action of these various agents than the parent cells, and many species of micro-organisms differ from each other in their resisting power. An exact knowledge of the subject can, therefore, only be based upon investigations which will determine the effect of these agents upon pure cultivations of the different micro-organisms causally related to putrefaction and disease. In the latter case, especially, this is not possible in the present state of our knowledge. In some cases of communicable disease there is considerable doubt as to the etiological importance of the organisms which have been described ; in other cases no organisms have as yet been dis- covered, or the organisms cannot be artificially cultivated, or the disease is not reproduced by inoculation, so that there is no means of testing whether the agents have had any effect. One can, therefore, only draw general conclusions by selecting some well-known pathogenic and non-pathogenic micro-organisms, and considering the influence of chemicals, of hot air, and of steam upon them, as representing the effect upon the various contagia of disease and the causes of putrefaction. Such knowledge must necessarily prove of the greatest importance, to the sanitarian, who is 1 82 BACTERIOLOGY. concerned in preventing the spreading of disease and in the disposal of putrefactive matter, to the surgeon, who is anxious to exclude micro-organisms during surgical operations and to arrest the development in wounds of bacteria which have already gained an entrance, to the physician in the treatment of micro-parasitic diseases. The sanitarian and the surgeon must profit directly by such experiments, for in the disinfection of clothes and the sick-room by the one, and in the application of antiseptic dressings and lotions by the other, the micro-organisms are encoun- tered as in the test experiments apart from the living body. The physician, on the other hand, is principally concerned in dealing with micro-para- sites when circulating in the blood, or carrying on their destructive processes in the internal tissues. So far as our knowledge at present goes, the physician can avail himself but little of the effect of the direct application of the substances which have been found to retard or destroy the growth of the organisms in artificial cultivations, for the concentrated form in which they would have to be administered would prove as deleterious or as fatal to the host as to the parasites. Thus Koch has stated that to check the growth of the anthrax bacillus in man it would be necessary that there should be twelve grammes of iodine constantly in circulation ; and that the dose of quinine necessary to destroy the spirilla of relapsing fever would be ANTISEPTICS AND DISINFECTANTS. 183 from twelve to sixteen grammes. The retarding influence, however, of certain substances when diluted, and the fact that disinfectants are some- times equally efficacious in a diluted form when their application is prolonged, seem to indicate measures which may be adopted, in some cases, with chances of success, such as the inhalation of antiseptic vapours in phthisis. For the most part the physician must look rather to combating the effects of micro-organisms by restoring to its normal standard the lowered vitality which enabled the bacteria to get a footing. There is no wider field for research than the determination of the real effect of disinfectants and antiseptics. Painstaking and laborious as the researches are which have been hitherto made, the subject is so beset with fallacies, leading, in some cases, to totally erroneous conclu- sions, that it is not surprising that one meets on all sides with conflicting statements. The author has no intention of analysing these results, but a general idea will be given of the methods which have been employed, and for further details reference must be made to the original papers mentioned in the bibliography. Chemical Substances. It was customary to judge of the power of a disinfectant or antiseptic by adding it to some putrescent liquid. A small por- tion of the latter was, after a time, transferred to some suitable nourishing medium, and the efficacy 184 BACTERIOLOGY. of the substance estimated by the absence of cloudiness, odour, or other sign of development of bacteria in the inoculated fluid. Koch pointed out the errors that might arise in these experiments from accidental contamination, or from there being no evidence of the destruction of spores ; and we are indebted to him for a complete and careful series of observations with more exact methods. Instead of employing a mixture of bacteria, Koch's plan was to subject a pure cultivation of some well-known species with marked character- istics to the reagent to be tested. A small quantity was then transferred to fresh, nourishing soil, under favourable conditions, side by side with nutrient material inoculated from a cultivation without treatment with the disinfectant. The latter constituted a control test, which is most essential in all such experiments. To test the resistant power of bacteria which are easily destroyed, two species were selected, micrococcus prodigiosus, and the bacillus of blue pus. These were cultivated on potatoes, the surface of which was sliced off and dried. A fragment transferred to freshly prepared potato gave rise to a growth of the particular micro- organism ; but if after treatment with some reagent no growth occurred, the conclusion was drawn that the agent was efficacious in destroying the vitality of the bacteria. Anthrax bacilli in blood withdrawn from an animal just killed were taken to represent spore- ANTISEPTICS AND DISINFECTANTS. 185 less bacteria, while silk threads steeped in an artificial cultivation of the bacilli and dried, afforded a means of testing the vitality of spores. Even by employing pure cultivations on solid media, great precautions were necessary to avoid mistakes. If, for instance, a large quantity of the growth which had been subjected to some chemical solution were carried over to the fresh tube con- taining the nutrient medium, or if a silk thread, which had been dipped in a solution, were directly transferred to the new, soil, enough of the supposed disinfectant might be mechanically carried over to retard the development of the bacteria, though it was ineffectual in destroying them. From a growth not appearing, the conclusion might be drawn that the spores or the bacteria had been affected, and so a mistake occurs. To avoid this Koch made a point of transferring a minimum of the disinfected growth to as. large a cultivation area as possible, so that any chemical substance mechanically carried over, would be so diluted as to be inert. For the same reason threads, after withdrawal from the disinfecting solution, were rinsed in sterilised water, or weak alcohol, and then transplanted ; or, instead of judging from the development on nutrient gelatine, the effect of inoculation in a healthy animal was made the test. A few examples may be quoted in illustration. Silk threads, impregnated with anthrax spores, were placed in bottles containing carbolic acid of 1 86 BACTERIOLOGY. various strengths. A thread was removed from each on successive days, and transferred to nutrient gelatine, and the result noted. It was found that immersion of the thread in a 5 per cent, solution of carbolic acid was sufficient in two days to effect complete sterilisation, and seven days in a 3 per cent, solution was equally efficacious. Since for practical purposes a strength should be selected which would be effectual in twenty-four hours, Koch recommended that for general use, allowing for deterioration by keeping, a solution containing not less than 5 per cent, should be employed, and for complex fluids probably a still higher percentage would be necessary. In the case of sporeless bacilli the results were very different. Blood, containing the bacilli, from an animal just killed, was dried on threads, and after exposure for two minutes to a i per cent, solution, was completely sterilised. Fresh blood mixed with a i per cent, carbolic solution produced no effect on inoculation. If, on the other hand, the blood was mixed with a '5 per cent, solution, the virulence was not destroyed. The facility with which the bacilli are destroyed, compared with their spores, illustrates how easily errors may occur, if mere arrest of growth or loss of motility be regarded as a sign of the efficacy of disinfection. To test vapours, Koch exposed anthrax spores or the spores which occur in garden earth by sus- pending them over solutions, e.g., of bromine or ANTISEPTICS AND DISINFECTANTS. 187 chlorine in a closed vessel. After a time they were transferred to a nutrient medium to test their vitality. To test the power of sulphurous acid gas, the spores were spread about in a room in which the gas was generated by burning sulphur in the ordinary way for disinfecting a room. To test chemicals which might be recommended for disinfecting vans and railway carriages, spores were laid on boards which were then washed or sprayed, and the spores then transferred to the nutrient gelatine. By such simple methods Koch investigated a long list of chemical reagents, and according to these experiments the salts of mercury, and the chloride especially, proved most valuable. Where heat is not admissible, these compounds were therefore highly recommended, though their poi- sonous nature is a drawback to their indiscrimi- nate use. Koch states, for disinfecting a ship's bilge, where a 5 per cent, solution of carbolic acid must be left for forty-eight hours, a i in 1000 solution of mercuric chloride would only require a few minutes. There is, on the other hand, reason for doubting the efficacy of very dilute solutions ; for, though anthrax spores subjected to a i in 20,000 solution of mercuric chloride for ten minutes, and then washed in alcohol, gave no growth in nutrient gelatine, silk threads exposed for ten minutes to a i in 20,000 solution, or even i in 10,000, still proved fatal to mice. 1 88 BACTERIOLOGY. Herroun considers that the value of mercuric chloride as an antiseptic is much over-rated, as he has cultivated ordinary septic bacteria in albuminous filtrates, containing i in 2000. It is precipitated by albumins if used of greater strength, and is readily converted by the sulphur of albu- minous bodies into mercuric sulphide, a com- pound which has practically no antiseptic pro- perties. Sternberg has also made an elaborate series of experiments with regard to the action of germi- cides. In this case cultivations of well-known pathogenic organisms in liquid media were em- ployed. The supposed germicide was added to the liquid cultivation, and after two hours a fresh flask of sterilised culture was inoculated from the dis- infected cultivation, and placed in the incubator. In twenty- four to forty-eight hours, if the chemical were not efficient, there was evidence of a growth of bacteria. Blyth has investigated the disinfection of cultivations of Bacterium termo, of sewage, and typhoid excreta, and, in conjunction with Klein, the effect of well-known disinfectant materials on an- thrax spores. Miquel, Laws, and others have also contributed to our knowledge of the effect of anti- septics and disinfectants upon micro-organisms. In spite of all that has been done, there is room for many workers ; a great deal of ground must be gone over again to rectify discrepancies, examine conflicting results, and thus determine what ANTISEPTICS AND DISINFECTANTS. 189 observations may be relied upon for practical application. Hot Air and Steam. Koch, in conjunction with Wolfhiigel, also tested the value of hot air. A similar plan was adopted as in disinfection with chemicals. Bacteria and spores were subjected for a certain time to a known temperature in a hot- air chamber, and then were transferred to a nourishing soil, or animals were inoculated. Paper parcels, blankets, bags, and pillows, con- taining samples of micro-organisms wrapped up inside, were also placed in the hot-air chamber, to test the power of penetration of heat. The conclusions from such experiments were as follows : Sporeless micro-organisms at a little over 100 C. are destroyed in one hour and a half. Spores of bacilli require three hours at 140 C. If enclosed in pillows and blankets, exposure from three to four hours to 140 C. is necessary. Spores of fungi require one and a half hours at 110 C. to 115 C. Further experiments showed that at the tempera- ture necessary for the destruction of spores of bacilli almost all fabrics are more or less injured. Koch, in conjunction with Gaffky and Loffler, also investigated the effect of steam under pressure and at the atmospheric pressure. Rolls of flannel with anthrax spores or earth spores, and a thermometer wrapped up inside, were 1 90 BACTERIOLOGY . subjected to steam, and the results compared with the effect obtained with hot air. Thus in hot air four hours' exposure to a temperature of 130 C. to 140 C. brought the temperature inside the roll to 85 C., and the spores were not injured ; on the other hand, ex- posure to steam under pressure at 120 C. for one and a half hours, raised the internal temperature to 117 C. and killed the spores. By such experiments the superior penetrative power of steam-heat was established. To test steam-heat at the atmospheric pressure, water was boiled in a glass flask with its neck prolonged by means of a glass tube, the tempera- ture in which was found to be uniform throughout Anthrax and earth spores placed in the tube were found to be unable to withstand steam at 100 C. even for a few minutes. It was, therefore, concluded that disinfection by steam at atmospheric pressure was superior to hot air from its greater efficiency, and to steam under pressure from the simplicity of the necessary apparatus. Parsons and Klein made some experiments which were more in favour of dry heat than the above. These observers state that anthrax bacilli are destroyed by an exposure of five minutes to from 1 00 C. to 103 C., and that anthrax spores are de- stroyed in four hours at 104 C. ; or in one hour at j 1 8 C. Guinea-pigs inoculated with tuberculous pus which had been exposed for five minutes to 104 C., ANTISEPTICS AND DISINFECTANTS. remained unaffected. They concluded that as none of the infectious diseases, for which disinfecting measures are in practice commonly applied, are known to depend upon the presence of bacilli in a spore-bearing condition, their contagia are not likely to retain their activity after being heated for an hour to 105 C. (220 F.). In experiments with steam, the results were in accordance with those already given, and complete penetration of an object by steam-heat for more than five minutes was deemed sufficient. They also arrived at the same result as in Koch's experiments, that steam-chambers are preferable to those in which dry heat is employed, though it must be borne in mind that some articles, such as leather, are injured by exposure to steam. CHAPTER X. IMMUNITY. THE condition of being insusceptible to an infective disease may be either natural or acquired. In the description of the pathogenic organisms several examples of natural immunity will be en- countered. The bacillus of septicaemia, so fatal to house mice, has been shown to have no effect upon field mice. The bacillus of anthrax is innocuous to cats, white rats, and, it is commonly asserted, to adult dogs and asses. The bacterium of rabbit septicaemia is equally inert in dogs, rats, and guinea-pigs. The immunity may be as in these cases complete, or only partial. Ordinary sheep are very easily affected w r ith anthrax, but Algerian sheep only succumb to large doses of the virus. Natural immunity may not only be characteristic of certain species, but it may occur in certain individuals of a susceptible species. The same occurs in man, for certain individuals, though equally exposed during an epidemic of small-pox, may escape where others readily fall victims to the disease. IMMUNITY. 193 Acquired immunity is illustrated by the protec- tion afforded by one attack of the exanthemata against subsequent attacks. Thus one attack of measles or small-pox, as a rule, affords complete protection. A knowledge of the immunity result- ing in the latter case led to the introduction of inoculation of small-pox as a protection against natural small-pox. Immunity may be acquired by acclimatization, for the inhabitants of tropical climates are less susceptible to the diseases of the country, malarial fevers for instance, than strangers. In civilised communities also there appears to be a degree of acquired immunity, for the infectious diseases introduced among savages or isolated communities have assumed the most virulent properties. The immunity acquired by protective inocula- tion constitutes, in connection with the study of pathogenic micro-organisms, a subject of pre- eminent interest and importance. Pasteur, in his researches upon fowl-cholera, observed that after non-fatal cases the disease either did not recur, or the severity of a subsequent attack was in inverse proportion to the severity of the first attack. It occurred to him to endeavour to obtain the virus of this disease in a form which would provoke a mild attack of the disease, and thus give protection against the virulent form. This attenuation or mitigation of the virus was 194 BACTERIOLOGY. successfully attained in the following manner: Cultivations of the microbe, in chicken-broth, were allowed to remain with a lapse of several months between the carrying on of successive cultivations in fresh media. The new generations which were then obtained were found to have diminished in virulence, and ultimately a virus was obtained which produced only a slight disorder; on re- covery the animal was found to be proof against inoculation with virulent matter. The explanation given by Pasteur of this change was, that prolonged contact with the oxygen of the air was the influence which diminished the virulence, and he endeavoured to prove this by showing that if broth were in- oculated in tubes which could be sealed up, so that only a small quantity of air was accessible to the microbe, the virulence of the cultures was retained. Toussaint investigated the possibility of attenuat- ing the virus of anthrax. Sheep injected with 3 ccm. of defibrinated blood, containing anthrax bacilli, which had been exposed to 55 C. for ten minutes, recovered, and were afterwards insuscep- tible. Pasteur subsequently argued that this method did not admit of practical application ; difficulties would arise in dealing with infective blood in quantity, and artificial cultivations started from this blood could not be relied upon, as they proved sometimes as virulent as ever. Pasteur endeavoured to apply the same method IMMUNITY. 195 for obtaining an attenuated virus of anthrax, as he had successfully employed in chicken-cholera. A difficulty was soon encountered, for in cultiva- tions of the bacillus with free access of air spore- formation readily takes place, and the spores are well known to have an extraordinary power of retaining their virulence. Pasteur found that the bacilli ceased to develop at 45 C., and he believed that spore-formation ceased at 42 43 C., the bacilli continuing to develop by fission only. The cultivations were, therefore, kept at this tempera- ture, and at the end of eight days the bacilli were found to have lost their virulence, and were quite inert when inoculated in guinea-pigs, sheep, or rabbits. This total destruction was, however, preceded by a gradual mitigation, so that a virus could be obtained, by taking it at the right time, which only gave a mild disease, and afforded subsequent protection. At Melun, in 1881, the protective inoculation against anthrax was put to a practical test. Sheep and oxen were inoculated with the mitigated virus, and then with a virulent form ; at the same time other sheep and oxen were inoculated with the virulent form without previous vaccination as a control experiment. The unprotected sheep died without exception ; the unprotected oxen suffered from cedematous swellings at the seat of inocu- lation, and a rise of temperature; but all the protected animals remained healthy. 1 9 6 BACTERIOLOGY. As a result of these experiments an idea arose that by preventive inoculation with attenuated virus all communicable diseases would in time be eradicated; but this does not follow, for ail com- municable diseases do not confer immunity after a first attack, and in some cases the very reverse is believed to occur. Thus erysipelas of the face leads to an increased liability to subsequent attacks of the same disease. Again, the occurrence of one disease is stated to induce a liability to others ; small-pox and typhoid fever are regarded as predisposing to tuberculosis ; so that the principle of preventive inoculation does not apply in these cases, and its effect would probably tend rather to deleterious results than otherwise. Even with regard to the prevention of anthrax, Pasteur's researches were opposed and criticised. Koch investigated the subject, and came to the con- clusion that the process did not admit of practical application, chiefly on the ground that as immunity only lasted a year, the losses from the vaccination process would be as great or even greater than from the spontaneous disease ; further, there was danger in disseminating a vaccine of the strength required to be effectual. Chauveau proved that the attenuation was due to the temperature, and not to the prolonged effect of oxygen. By keeping cultivations at 42 43 C. in vacuo, the virulence was found to disappear in twenty- four hours, and by Zceeping cultivations at a low temperature with free IMMUNITY. 197 access of air the virulence was retained. Chauveau considered, therefore, not only that oxygen was not the agent, but that the mitigation was much more easily effected in its absence. In spite of these adverse criticisms, these researches never- theless confirmed the principle of Pasteur's con- clusion, that immunity could be induced by experimental measures, and further showed that he had considerably advanced the method by which this could be effected. Chauveau succeeded also in attenuating the virus by a modification of Toussaint's method. Sterilised broth was inoculated with the bacilli, and placed in the incubator at 42 43 C. After the lapse of twenty hours it was removed to another incubator at 47 C. According to the time of exposure to this increased temperature, the mitigation varied in de- gree. Thus inoculation with the virus, before it was exposed to 47 C., was fatal to guinea-pigs; but after one hour at 47 C. the virulence was diminished, and, though ultimately fatal, life was prolonged ; after two hours' exposure at 47 C. only half the animals died ; and after three hours' exposure they recovered and were rendered refractory to sub- sequent inoculation. Attenuation of the virus has also been induced by chemical means. Chamberland . and Roux stated that a fresh growth started from a cultiva- tion of bacilli which had been subjected for twenty- nine days to ^Q of carbolic acid was found tu> BACTERIOLOGY. be inert in guinea-pigs and rabbits. Bichromate of potash added to a cultivation in the proportion f T2TToo ToW ave after three days* a new growth, which killed rabbits, guinea-pigs, and half the sheep inoculated; after ten days, rabbits and guinea-pigs, but not sheep ; and after a longer time even guinea-pigs were unaffected. In other diseases similar results have been obtained. Arloing, Cornevin, and Thomas found that by inoculating a small quantity of the virus of symp- tomatic anthrax anywhere in the subcutaneous connective tissue, or a moderate quantity at the root of the tail, and even by intravenous injection, immunity was obtained from a virulent dose. In swine-erysipelas, Pasteur and Thuillier ob- tained attenuated virus upon quite another principle They discovered that by passing the virus through pigeons the virulence was increased, but by passing it through rabbits it was progressively diminished. Thus a virus was obtained from the rabbit, which produced only a mild disease in pigs, and after recovery complete immunity. Similarly in rabies Pasteur finds that passage of the virus through various animals considerably modifies its properties. By inoculating a monkey from a rabid dog, and then passing the virus through other monkeys, the virulence is diminished ; but by inoculating a rabbit from the dog, and passing the virus from rabbit to rabbit, the virulence is increased. More recently IMMUNITY. 199 Pasteur has employed another method of attenuat- ing the virus of rabies. The spinal cord of inoculated rabbits is removed with all possible precautions, and portions a few centimetres in length are suspended in flasks in which the air is dried by fragments of potash. By this process the virulence is found to gradually diminish and finally disappear. Animals inoculated with portions of these cords, after suspension for a certain time, are rendered refractory to inoculation with virulent cords. Having rendered dogs, which had been previously bitten, free from the supervention of symptoms of hydrophobia by means of protective inoculation, Pasteur proceeded to apply the same treatment to persons bitten by rabid animals, with results which tend to the belief that a prophylactic for rabies has been found, though this must still be considered to be sub judice. The question as to what constitutes immunity is a vexed one. Raulin has shown that Aspergillus niger develops a substance which is prejudicial to its own growth in the absence of iron salts in the nutrient soil. Pasteur has suggested that in rabies side by side with the living and organised substance there is some other substance which has, as in Raulin's experiment, the power of arresting the growth of the first substance. If we accept the theory of arrest by some chemical substance, we must suppose that in the acquired immunity afforded by 2OO BACTERIOLOGY. one attack of an infectious disease this chemical substance is secreted, and, remaining in the system, opposes the onset of the micro-organism at a future time. In the natural immunity of certain species and individuals we must suppose that this chemical substance is normally present. Another theory is, that the micro-organisms assimilate the elements which they require for their nutrition from the blood and tissues, and render the soil impoverished or otherwise unsuitable for the development of the same micro-organisms here- after; this condition may be permanent, or the chemical constitution of the tissues may be restored to normal, when immunity ceases. If, however, we explain acquired immunity by the result of the growth of a previous invasion of micro-organisms, we are still confronted with the difficulty of explain- ing natural immunity. A third theory is that the tissues are endowed with some power of vital resistance to the develop- ment of micro-organisms, similar to the vital resistance to the coagulation of the blood, which is supposed to exist in the lining membrane of the healthy blood-vessel ; that in some species and indi- viduals this exists to a high degree, and hence their natural immunity But this does not explain how one attack confers immunity from a subsequent one. One would expect that the vital resistance would invariably be lowered by a previous attack, and increased liability be the constant result. IMMUNITY. 2OI Lastly, that leucocytes appear to have the power of destroying bacteria in some cases, has been demonstrated by the researches of Metschnikoff, If anthrax bacilli are inoculated in the frog, the white blood- cells are observed to incorporate and destroy them until they entirely disappear, and the animal is not affected. But if the animal, after inoculation, is kept at a high temperature, the bacilli increase so rapidly that they gain the upper handover the leucocytes, and the animal succumbs. In septicaemia of mice the white blood-cells are attacked and disintegrated by the bacilli in a similar way. It is difficult, however, to accept any explanation of immunity from these observations, to suppose, for example, that immunity depends upon the micro-organisms being unable to cope with the leucocytes in certain species. It is difficult to conceive that the leucocytes in the blood and tissues in the field mouse are differently constituted from those in the house mouse, so that they form an effectual barrier in the one case, though so readily destroyed in the other. PART III. SYSTEMATIC AND DESCRIPTIVE, WITH SPECIAL MICROSCOPICAL METHODS. CHAPTER XL CLASSIFICATION OF BACTERIA. IN reviewing the history of the various classifica- tions which have from time to time been proposed, we shall see that the gradual improvements in the means of studying such minute objects, the methods of cultivating them artificially, and of studying their chemistry and physiology, and the ever-increasing revelations of the microscope, have resulted in establishing these microscopic objects as members of the vegetable kingdom, ranking among the lowest forms of fungi. While enabling us to settle their position as a whole, these improved methods have further given us so great an insight into the life-history of individual forms, that, with regard to the division into genera and species, we are, up to the present time, still in a position of doubt and uncertainty. Miiller, in 1773, was the first to suggest a classi- fication. He established two genera, Monas and Vibrio, and grouped them with the Infitsoria. In 206 BACTERIOLOGY. 1824 Bory de Saint Vincent also attempted a classi- fication ; but it was not until Ehrenberg in 1838, and Dujardin in 1841, worked at the subject, that a scientific distinction of species was attempted. Ehrenberg described four genera : I. Bacterium . . filaments straight, rigid. II. Vibrio . . filaments snake-like, flexible. III. Spirillum . . filaments spiral, rigid. IV. Spirochaete . . filaments spiral, flexible. Dujardin united Spirillum and Spirochcete, and classed them thus : I. Bacterium . . filaments rigid, vacillating. II. Vibrio V/_ . filaments flexible, undulatory. III. Spirillum . . filaments spiral, rotatory. Up to that time, bacteria were still considered as Infusoria; but the year 1853 marked the com- mencement of a new era in their history, for Robin then pointed out the affinity of the Bacteria and Vibrios to Leptothrix. Davaine, in 1859, still more definitely insisted that the Vibrios were vegetables, and that they were in fact allied to the Alga. Since that time a flood of light has poured in upon this subject through the writings of Hoff- mann, Pasteur, Cohn, Rabenhorst, Hallier, Billroth, Warming, Nageli, Magnin, Marchand, Sternberg, Van Tieghem, Koch, Fliigge, De Bary, Zopf, CLASSIFICATION OF BACTERIA. 207 Cornil, Babes, and many other workers in the recent widespread revival of bacteriological research. Of all these writers we are most indebted to Cohn,* not only on account of his researches, which extended over very many years, but also for his system of classification, which has since been almost universally adopted. In his first classification, published in 1872, Cohn considered the Bacteria as a distinct group be- longing to the Algce, and divisible into four tribes, including six genera : I. Sphaerobacteria . globules (Micrococcus). II. Microbacteria . short rods (Bacterium). III. Desmobacteria . long rods (Bacillus and Vibrio). IV. Spirobacteria . spirals (Spirochaete and Spirillum). Cohn noted, in spite of placing them with the Algcz, that the absence of chlorophyll connected the Bacteria to Fungi, and we find Nageli subsequently adopting this view, and employing the term Schi- zomycetes. Billroth, in 1874, disputed the division into species, and considered that all the forms described by Cohn were but developmental forms of one micro- organism, Coccobacteria septica. In the following year Cohn answered the criticism of Billroth, and produced a second classification, in which he still * Cohn, Beitrdge zur Biologic der Pflanzen, 1872, et seq. 2O8 BACTERIOLOGY. maintained that distinct genera and species existed. The genera Cohn considered to be distinguished by definite differences in shape, which were adhered to throughout life, while some special feature, as a difference in size or physiological action, or some minute difference in form, determined the various species. Cohn illustrated, by his well-known com- parison of a sweet and a bitter almond, the appearances of which are similar, but the proper- ties very different, that a distinction into species might depend upon a difference in physiological action only. Others strongly support Conn's views. By cultivating various micro-organisms through several generations, we conclude that a micrococcus cannot be transformed into a bacterium, or a bac- terium into a bacillus or spirillum. There is also no evidence to show that a bacillus can change its nature, and be converted from a harmless into a pathogenic form, as asserted by Biichner.* The second classification of Cohn (1875) only differed from the first in that, instead of keeping the bacteria as a separate group, he placed them, from their closerelation ship with the Ihyccckrc- macece, under a new group, the Schizophytes, and added the genera Leptothrix Beggiatoa, Crenothrix, Sarcina, ASCOCOMIS, Streptococcus, Myconostoc, and Streptothrix. Nageli maintained that Bacteria were allied to * Biichner, Ueber d. experim. Erzeugung d. Milzbrandconta- giums aits d. Heupilzen. CLASSIFICATION OF BACTERIA. 2O9 Yeasts, and should be included in the class of Fungi. In fact, he divided the fungi-producing decomposition into : Mucorini . . . "> . moulds Saccharomycetes . FL . yeasts Schizomycetes ... . . fission-fungi (This last class comprising bacteria.) Fliigge,* following Rabenhorst, maintained the term Schizomycetes, and divided them as shown in the table on the following pages : > * Fliigge, Fermente und Mikrofiarasiten. 1883. 210 BACTERIOLOGY. ^ . ^ ^ ^0 o5 -C 'o ' *o > ~ H "^ U U .s CO 1 6 . "3 x 1 tS S c co o p C; .S | 1 ^ ^ .s CO 5 in o s g U VH 13 U 1 t-H r piOAo jo punoj CLASSIFICATION OF BACTERIA. 211 . ^ 8 - ^ =..si:u! lit DQ KJ oq ^r c/y \Streptothrix. [ Clathrothrir. Myconostoc, : ^.i c r~^" , . ,. ;. ; I> H JU , -;.. ^ ^ "S c ^ iS 3 S . O '^"uTJ *"* ^ . . tf c -S bi c o -M b^3 '+3 ., S '5 C J3 9 .2 *s -^ 9 CO iJ TD .S CO iJ . S -1 S 1 H S x'& C .2 'c5 JSS >: - U< c r^ ^ C 1 c 53 ^ " 73 JS p* 6 13 nJ 2 bjo . S 5S 6 J3 iii ill 3 . C ^ j^ rf* 3 ^ ^ 2 .S o | c 1 ll 8 1 1 g J^i ^ ^ -5 . ^j .2 5 > u, 2 E '" u slil ' il 'C >r d CO rt rmation of arthros 'ructification unkn dogenous spores . P i N o N o o SJN HH ^3 M ^3 H H .G > o c H Y ' r ^7^ M ^ 0) CO ' 0) ^ a^ ^-^ fc^-' CO OJ "^ co.'tJ .i2 w T C 'c3 1 1 1 CO S 1 CU bx C ^o ,G '^ rt C^ . r^3 rt ,Q CU T3 *Q o^t 6- ' 1 .S G .2 H 5-1 Ixo ^ co g g bO C Oj | 0) bo 1 I 1 5-i ^CU 13 g threads. | ^ co & _C nj ,Q T3 '^3 S*^3 *53 cti o >^ .2 G OJ S C T3 rt qH JJH '-M rt :rew-like flexible < < < c h I C/5 H in v v ^ CO CO | CO B i 2 1 8 BACTERIOLOGY . It has already been mentioned that the produc- tion of arthrospores is only established in a very few species. Therefore, we are hardly justified in assuming that all bacteria, the spore-formation of which is quite unknown, are to be included with those in which this kind of fructification has been observed, and consequently to distinguish genera on the same grounds may be considered, to say the least, somewhat premature. In Baumgarten's classi- fication the genus bacterium is dispensed with, and the genera divided into two groups, the mono- morphic and the pleomorphic. GROUP I. MONOMORPHIC. Genera. Coccus. Bacillus. Spirillum. GROUP II. PLEOMORPHIC. Genera. Spirulina. Leptothrix. Cladothrix. Fliigge also, in his recent classification, includes the genus bacterium in the genus bacillus. The new classification differs also from the original one in the grouping together of the different species according to the character and behaviour of the colonies in nutrient gelatine. The abolition, in Fliigge's and Baumgarten's classification, of the genus bacterium is no doubt owing to confusion having arisen from the distinction between a CLASSIFICATION OF BACTERIA. 219 bacterium and a bacillus, being a question of length. Observers differed as to whether a rod of a certain length ought to be considered a bac- terium or a bacillus. To meet this difficulty a rough- and-ready rule was suggested, viz., that a rod less than twice its breadth in length should be considered as a bacterium, and otherwise a bacillus. But this purely arbitrary division was inadequate, from the fact that a rod at one stage of its growth or under certain FIG. 68. BACTERIUM PNEUMONIA CROUPOS^E, x 1500 (after Zopf). conditions might, as i far as length went, truly be a bacterium, and under other circumstances be of such a length as to entitle its being considered a bacillus. We avoid such confusion if we follow Zopf, and acknowledge as a difference between a bacterium and a bacillus the presence or absence of that form of spore-formation now distinguished as endogenous spore-formation. We can then most conveniently retain this generic term, to include that group of rod-forms in which this spore-formation is as yet unknown ; moreover, we shall find that by so 22O BACTERIOLOGY. doing, with one or two exceptions, we get collected together those short rod-forms (Fig. 68), which appear to link the simple cocci to the spore-bearing rods or bacilli. This must surely lead to less confusion than FIG. 69. EMMERICH'S BACTERIUM, x 700 (after Emmerich). regarding all rod-forms as bacilli, and massing them together into one genus. For by those who adopt the latter plan, not only are very short rods with rounded ends included as bacilli, e.g., Bacillus FIG. 70. COLONIES ON NUTRIENT GELATINE, x 60. Neapolitanus or Emmerich's bacterium (Fig. 69), but even cells which are described as ovoid are also regarded as bacilli, as in Loffler's so-called Bacillus parvus ovatus. CLASSIFICATION OF BACTERIA. 221 The grouping together of the different species according to the character of the colonies in nutrient gelatine (Figs. 70, 71) is also of question- able advisability. These characters can hardly FIG. 71. COLONIES ON NUTRIENT AGAR-AGAR, x 60. be considered to be of sufficient importance, or indeed in many cases to be sufficiently constant, to serve by themselves for this purpose. In many cases a slight variation in the composition of the FIG. 72. COLONY OF BACILLUS ANTHRACIS, x 60. From a cover-glass impression-preparation, stained with gentian-violet. nutrient medium may considerably affect the ap- pearances of the colonies. At the same time, the appearances are very characteristic of cer- tain species of bacteria (Fig. 72), and in other 222 BACTERIOLOGY. cases the characters of the colonies, together with the characters of the growth in test-tubes, assist us in distinguishing species which are morpho- logically similar, as in the case of the comma bacilli of Finkler and of Koch. The classification here given will be found to be a convenient form for arranging the micro- organisms for reference, and it may lead the investigator to work upon the same lines as Zopf, and by tracing the life-history of individual forms in pure cultivations, either to extend the work of establishing protean species or to restrict the FIG. 73. BACTERIUM OF RABBIT SEPTICAEMIA. doctrine of pleomorphism to a very few forms. For though the author adheres to the lines of classification proposed by Zopf, he is not prepared to accept his teachings in their entirety ; thus, to embrace all described species, and to be consistent with the author's views, it has been necessary not only to add to Zopf s classification, but in many cases to modify his arrangement of species. For instance, Zopf regards the bacterium of rabbit septicaemia (Fig. 73) as a micrococcus. Of some species alteration in the nomenclature is justified by necessity. Any arrangement at present can only be con- CLASSIFICATION OF BACTERIA. 223 sidered provisional, and, therefore, that arrangement which is of most practical assistance, and which leads to a clear description of the important characteristics on which the final classification will depend, must be considered to be the best. For example, if we abolish the genus bacterium, any rod-form may be at once classed as a bacillus ; on the other hand, in the plan here adopted, we must determine the presence or absence of endogenous spore-formation before we can decide whether it be a bacterium or a bacillus. This necessarily leads to a more thorough study of their life-history. In the systematic de- scription which follows, stress is laid upon the morphological appearances of bacteria, upon the absence or presence of spore-formation, and upon the appearances under cultivation, in addition to other characteristics, such as the changes produced by their growth. The determination of species rests upon the accumulated evidence afforded by a thorough knowledge of their life-history. The form of the organism, the changes it effects, and the microscopical appearances under cultivation must be collectively taken into account. CHAPTER XII. SYSTEMATIC AND DESCRIPTIVE. THE Schizomycetes, Spaltpilze, or Fission-fungi have already been described as divisible, according to Zopf, into four groups: Coccacece, Bacteriacece, Lep- totrichecz, and Cladotrichece. They comprise the following genera and species : GROUP I. COCCACE^:. Genus I. Streptococcus (Chain-cocci). Division in one or more directions. Individual cocci remain united together to form chains. Genus II. Merismopedia (Plate-cocci). Division in two directions, forming lamellae or plates. Genus III. Sarcina (Packet-cocci). Division in three directions, forming colonies in cubes or packets. Genus IV. Micrococcus (Mass-cocci). Division in one direction, cocci after division remain aggre- gated in irregular clusters, or singly, or in pairs or in chains of three or four elements. Genus V. Ascococcus (Pellicle-cocci). Like micro- coccus, but the cocci grow in characteristic gelatinous pellicles. SYSTEMATIC AND DESCRIPTIVE. 225 Genus I. Streptococcus, Streptococcus pyogenes, Rosenbach. Cocci occurring singly, in chains, and in zoogloea. The individual cocci are small spherical cells, with a special tendency after fission for the resulting elements to remain attached to each other, forming chains or rosaries. There may be a few, three or more elements, linked together, or a great number, forming straight, serpentine, and twisted chains. Preparations, stained preferably by the method of Gram (Plate XIX.), should be studied minutely with the best lenses and the best method of illu- mination in order to make out all the minute details. The individual elements composing the chains will be found to vary considerably in size ; here and there in a preparation will be found a chain com- posed of excessively small elements, in another part the elements are all on a larger scale, and again in another part the elements will be peculiarly con- spicuous on account of their size. Very character- istic appearances result from the fact that the elements enlarge and divide both longitudinally and transversely, and, indeed, the largest elements, for the most part, clearly show a division in two direc- tions, resulting in the formation of tetrads. So great is the diversity in the size of the elements of some of the chains, that one might imagine that 15 226 BACTERIOLOGY. there was more than one kind of streptococcus present in a preparation, until on examining some of the longest chains it is observed that various sizes are represented in different lengths of the same chain. In addition to the forms resulting from the fission of the elements, there are here and there in a chain, and sometimes terminally, larger elements, which are spherical, spindle-shaped, or in the form of a lemon. In the length of the chains, as in the size of the individual elements, there is usually a great diversity. In some cases they are composed of only a few, three or four elements, or in others eight, ten, or twenty. Here and there an exquisite rosary will extend in a straight line across the field of the microscope, or be twisted, curved, or serpen- tine ; in some preparations twisted or entangled strands are observed which are composed of several hundred elements. Such chains will be found to be much thicker in one part than another. Another characteristic appearance is produced by separation of the elements resulting from fission in the long direction of the chain, by which lateral twigs or branches are formed. Another character, which is very striking, may be seen when the individuals in a chain have become separated ; an unstained or faintly stained membrane may be found bridging across the interval. This will become still more visible in some of the preparations contrast- stained with eosin. Cultivations. In plate-cultivations the appear- SYSTEMATIC AND DESCRIPTIVE. 22 7 ances of the colonies are not very striking. They appear to the naked eye after three or four days as extremely minute, greyish-white, translucent dots, which under the microscope have a slightly yellowish-brown colour. They are finely granular and well defined. They do not liquefy the gelatine, and after several weeks do not exceed the size of a pin's head. If the surface of nutrient gelatine solidified ob- liquely be traced over once or twice with a platinum needle bent at the extremity into a little hook charged with the cocci, a ribbon-shaped film de- velops in two or three days. This film is composed of minute, greyish -white, translucent dots or droplets, which can be more easily recognised with the aid of a pocket lens. According to the number of organ- isms sown on the jelly, the dots or colonies may be completely isolated, or form a more or less continuous film. The film by reflected light has an iridescent appearance like mother-of-pearl, but has a bluish or bluish-grey tint by transmitted light, and with a pocket lens appears distinctly brownish. The gela- tine is not liquefied, and even after several weeks the cultivation is limited to the inoculated area, and the individual colonies are not larger than pins' heads. In gelatine-cultivations of the same age, but kept in the incubator at i8C, the colonies get irregular in form, especially at the margin of the film, and give the growth an arborescent, fringed, or serrated appear- ance. Cultivated on the oblique surface of nutrient 228 BACTERIOLOGY. agar-agar at 37 C. the growth is very similar, form- ing a film composed of minute dot-like colonies like grains of sand. But the film appears less trans- parent, is whiter, and the colonies have a greater tendency to get irregular in form. If inoculated with one tracing of the needle the growth is scanty, but tends to get thicker in the centre than towards the margins, which may have a terraced appearance. Inoculated in the depth of gelatine, there appears after a day or two at 1 8 C. a thread-like growth along the track of the inoculating needle. This delicate growth is found on examination with a pocket lens to consist of a linear series of extremely minute granules. In a few days more the beads or granules become more marked, but even after weeks the cultivation only appears like a string of minute white, compact, globular masses or grains. In broth at 37 C- the cocci in twenty-four hours create a turbidity, and gradually develop beautiful chains varying in length according to the age of the culti- vations. Even in forty-eight hours there may be chains of eight, ten, twenty, or a hundred elements. After a few days the growth settles down at the bottom of the tube in the form of a white deposit, while the supernatant liquid becomes clear again. Inoculated subcutaneously in the ear of rabbits, they produce in two days an inflammatory thickening with erysipelatous redness without suppuration. They occur in pus and in diseases associated with septic complication. PLATE 19 1'aizmqp 228 2. edTH.KJ.eywa ,136, Ger Street. SYSTEMATIC AND DESCRIPTIVE. 229 The diseases and conditions in which streptococci have been found will be enumerated. In some cases the strep- tococci are identical with the first named, and in others there is need for further investigation ; some being regarded as varieties ', and others as distinct species. Abscesses. In abscesses a chain forming micro- coccus was first described by Ogston. It was later studied by the methods of cultivation introduced by Koch and named by Rosenbach Streptococcus pyogenes. According to Fliigge, after subcutaneous inoculation of mice with a small quantity of a cultivation, there is no result in 80 per cent, of the animals experimented on. Sometimes there is limited pus-formation at the seat of inoculation, sometimes the animals die without any very striking pathological appearances. Rosenbach examined six cases of pyaemia. From Case i cultivations of Streptococcus pyogenes were ob- tained from the blood of the patient during life. The blood was stroked over the surface of the culture medium, and in two tubes the Staphylococcus pyogenes aureus was also present. In Case 2, with suppurative pleuritis, the pleura was tapped during life, and cultiva- tions of Streptococcus pyogenes were obtained. In Case 3 streptococci were found in the metastases in the kidneys and in the other suppurations that were examined. In Case 4 the pleura was opened during life, and Streptococcus pyogenes and Staphylococcus pyogenes aureus were isolated from the fluid which escaped. In Case 5 pure cultiva- tions of Streptococcus pyogenes were obtained from pus from the knee, which was punctured during life. This case was one of erysipelas and pyaemia after removal of a carcinoma of the breast. In Case 6, a case of whitlow with metastatic abscesses, the patient recovered, and no streptococci were found. Thus in six cases of 2 30 BACTERIOLOGY. metastatic pyaemia Streptococcus pyogenes was found five times, partly in the blood and partly in the metastatic deposits, and twice in company with Staphylococcus pyo- genes aureus. Baumgarten, also, found the Streptococcus pyogenes in the internal organs in pyaemic cases, and Eiselsberg found Streptococcus pyogenes in company with Staphy- lococcus pyogenes aureus in the blood of cases of septicaemia. Erysipelas. In erysipelas Fehleisen isolated a streptococcus and described the appearances on culti- vation. Fehleisen regarded the organism as quite dis- tinctive, with special characters on cultivation, and claimed that it produced erysipelas when inoculated in the human subject. Rosenbach pointed out the extremely close resemblance in every respect to the Streptococcus pyogenes, though he described an apparent difference on cultivation on nutrient agar-agar. Other observers maintain that these organisms cannot be distinguished with certainty by either their morphological appearances or by their characters on cultivation. More- over, the marked differences which have been described after inoculation were not obtained by those who re- peated the experiments. Passet found that the results of inoculating Streptococciis pyogenes in the rabbit's ear induced a very similar condition to the result of inocu- lation of Streptococcus erysipelatis ; and both organisms, by subcutaneous inoculation, and by inoculation in the cornea of rabbits and other animals, induced results without any constant difference. Passet showed that the inoculation of the cornea produced the same form of keratitis. Hoffa and Hajek described minute differences at the seat of inoculation, but Biondi and Eiselsberg repeated the experiments, and failed to establish the alleged differences. Baumgarten also investigated this subject, and failed to prove any essential difference, and, indeed, found much more often than might have SYSTEMATIC AND DESCRIPTIVE. 231 been expected from the publications of previous authors that no marked result at all was obtained on inocu- lation ; and he concluded that Streptococctis pyogenes and Streptococcus erysipelatis in their form, cultivation, and their effects on animals were identical. Passet, Biondi, Eiselsberg, Baumgarten, and Frankel have definitely accepted the identity of the streptococcus associated with suppuration, and the streptococcus associated with erysipelas. Spreading Gangrene. From a case of spreading gangrene, which was identical with Ogston's erysipelatoid wound gangrene, and regarded by him as the most intense and dangerous form of erysipelas, Rosenbach obtained pure cultivations of a streptococcus by incising the skin of the limb, and inoculating tubes from the turbid reddish fluid which escaped. That the streptococcus was identical with Streptococcus pyogenes was ascertained by comparison with a cultivation derived from pus, of the mode of growth, and of the effect on animals. Surgical Fever. Eiselsberg proved the presence of a streptococcus in the blood of several cases of surgical fever in Billroth's clinic. The organism was identified by cultivation with Streptococcus pyogenes. Diphtheria. In three cases of typical diphtheria Loffler found a streptococcus. He isolated it by culti- vation, found that it was similar in form, characters on cultivation, and effects after inoculation, to Fehleisen's streptococcus of erysipelas. Loffler was not inclined to regard them as identical, because Fehleisen never found his cocci in the blood-vessels. Fliigge named the organism Streptococcus articulorum, and states that, after subcutaneous inoculation or injection of a cultivation in mice, a large proportion of the animals die, and in the sections of the spleen and other organs the streptococci are again seen. Baumgarten investigated the same sub- ject, and decided that the streptococcus was identical with Streptococcus pyogenes. 232 BACTERIOLOGY. Puerperal Fever. Frankel isolated a strepto- coccus from puerperal fever, which he at first called Streptococcus puerperalis, but subsequently identified with Streptococcus pyogenes. Winkel obtained a pure culti- vation of a streptococcus from the blood of the heart in a case of puerpera peritonitis. It produced erysi- pelatous redness when inoculated in the rabbit's ear, and in form and in cultivation was similar to the strep- tococcus in erysipelas. Gushing found Streptococcus pyogenes associated with puerperal infection. The cocci were found in endometritis diphtheritica as well as in secondary puerperal inflammation. These obser- vations were still further confirmed by Baumgarten, and Bumm isolated the same organism in puerperal mastitis. Scarlet Fever. The occurrence of a streptococcus in certain cases of scarlet fever has been observed by several investigators Crooke, Loffler, Babes, Heubner, and Bahrdt, and notably by Frankel and Freudenberg. Crooke, in cases of scarlet fever with severely affected throat, found bacilli, cocci, and streptococci in the organs of the throat, and cocci in the internal organs. Crooke left it an open question whether these cocci were the specific organisms of scarlet fever, or to be regarded as diphtheritic or septic associates. He inclined on clinical grounds to the latter view. Loffler, in cases of scarlatinal diphtheria, found the same chain-forming micrococcus which he had found in typical diphtheria. Babes was able to constantly prove in inflammatory products secondary to scarlatina the presence of a strepto- coccus greatly resembling that in pus. Heubner and Bahrdt, in a fatal case of scarlet fever in a boy, complicated with suppuration of the finger- and knee-joints and with pericarditis, found a streptococcus identical in form, from his description, with Streptococcus pyogenes. Cultivations were not made. The secondary SYSTEMATIC AND DESCRIPTIVE. 233 infection started from diphtheritically affected tonsils, which were followed by retro-pharyngeal abscesses. Frankel and Freudenberg examined for micro-organisms three cases of scarlatina with well-marked affection of the throat. In all three cases they obtained cultivations of cocci from the submaxillary lymphatic glands, spleen, liver, and kidney. These cocci could in no way be dis- tinguished from Streptococcus pyogenes derived from pus, nor from the undoubtedly identical streptococcus which one of them (A. Frankel) had already repeatedly culti- vated in large numbers from puerperal affections. In two of the cases Streptococcus pyogenes was the only organism present, and in all three cases it was far in excess of other colonies which developed. The organisms were also found in sections of the organs by microscopical examination. The identity of this streptococcus with Streptococcus pyogenes and Streptococcus puerperalis was established by comparison of their macroscopical and microscopical appearances in cultivations on nutrient agar-agar, nutrient gelatine, and in broth, both at the ordinary and at higher temperatures, and also by experi- ments on animals. They concluded that it could be stated with certainty that the organisms in question did not stand in causal relation to scarlet fever. They considered that special methods of microscopical and biological research were apparently needed for demon- strating the true scarlet fever contagium, which probably was especially present in the skin. They considered that the presence of the streptococcus was due to a secondary infection, to which the door was opened by the lesions of the throat a view which was supported by the fact that the organisms were found in the submaxillary lymphatic glands. They preferred to use the term " secondary " to " complicated " or " combined " infection, because this expresses the fact that by the effect of the scarlatinal virus the soil is rendered suitable for this ubiquitous microbe when it has once gained an entrance. 234 BACTERIOLOGY. Klein found the streptococcus in five out of eleven cases of scarlet fever, twice in association with other cocci, and three times alone, and regarded it as the contagium of scarlet fever. The cases from which the organism was ob- tained were all cases with ulcerated throat. An analysis of these cases is shown in the following table : Name of patient. Condition of tonsils. Source of blood. Micro-organisms isolated. Death or recovery. I Lilian Fuller, Severely Finger Streptococcus Ultimately aged 5 ulcerated recovered. /Staphylococcus 2 Kate Fuller, aged 2 Much ulcerated n 1 pyogenes aureus < Liquefying micro- j coccus Died of pyaemia. 3 Henry Lampson, aged 8 Ulcerated \Streptococcus / Staphylococcus \Streptococcus |- Recovered. 4 (a woman), aged 40 Much ulcerated Arm None [Not stated.] ^ (a girl), li JJ jj aged 19 6 Blanche Moody, Ulcerated Finger Streptococcus Recovered. aged 15 7 Ellen Warr, Much || None [Not stated.] aged 22 ulcerated 8 Robert Hughes, Ulcerated )f jj aged 8 9 Florence Glossop, Heart Streptococcus Died. aged 2i 10 Edward Fenton, ,, None aged 3 ii R. Boucher, Advanced jj ,, aged 20 months ulceration But the evidence is sufficient to show that in any disease accompanied with a lesion of the skin or mucous membrane, in which the blood and tissues are profoundly affected by the virus of that disease, septic micro- organisms gain an entrance into the circulation, and may not only escape destruction, but may gain the upper hand and set up destructive processes. And it 'is interesting to observe in connection with this, that the streptococcus was isolated in all cases at or about the SYSTEMATIC AND DESCRIPTIVE. 235 time at which the temperature was highest ; in other words, at a time when the tissue resistance to an invasion of micro-organisms would be lowered. The occurrence of streptococci, in certain cases of diphtheria, scarlet fever, cow-pox, small-pox, measles, typhoid fever, must be regarded as a secondary result associated with suppu- ration or with septic or pyaemic complication ; in fact, with the exception of negative evidence, bacteriology has not assisted us in the least in the determination of the real nature of the morbific agent or actual contagium of scarlet fever. Small-pOX. Hlava has established the presence of Streptococcus pyogenes in the pustules of variola, and Garre found streptococci in the internal organs in a case of variola hsemorrhagica. In a fatal case of variola complicated with pemphigus, Garre found a streptococcus in the pemphigus vesicles. Whether it was identical with Streptococcus erysipelatis Garre left an open question. Foot-and-mouth Disease. In 1868 Brown figured a streptococcus in the milk of cows affected with foot-and-mouth disease. From the vesicles of this disease in sheep, Klein isolated a streptococcus which he regarded as the contagium of the disease. From Klein's description of its morphological features and characters on cultivation, it closely corresponds with Streptococcus pyogenes. Baumgarten regards Klein's alleged con- tagium as most probably the Streptococcus pyogenes. Against the fact of this organism being the virus of this disease, and in favour of its being a septic organism, we have the results of inoculation experiments. A great number of subcutaneous inoculations in sheep were without any result. One of several guinea-pigs fed with the organism had an abscess, another an ulcer. In sheep fed with the organism, two out of nine, after a number of repeated administrations, developed a disease, regarded by Klein as foot-and-mouth disease. There is the 236 BACTERIOLOGY. possibility of two fallacies one that the disease, if really foot-and-mouth disease, may have occurred incidentally; the other, that the result which occurred may have been a spurious form of foot-and-mouth disease. The control experiment of exposing a number of sheep with these two sheep to see if the disease would spread was wanting. Cattle Plague. Semner cultivated streptococci from the blood and lymphatic glands of a sheep suffering from cattle plague. A calf inoculated with a cultivation was stated to have died in seven days from cattle plague. The cocci were stated to lose their virulence by successive cultivation, and the weakened cultivation to protect against the virulent disease. It is extremely likely that this was another manifestation of septic streptococci, and that the animal died very probably of septic infection. Klein is also of opinion that the specific nature of these micro- organisms cannot be considered to be established. Strangles. Schutz has described a streptococcus as the contagium of glanders. Contagious Mammitis. Nocard has cultivated a streptococcus in contagious mammitis which he regards as specific. Yellow Fever. Babes observed the presence of streptococci in the vessels of the kidney and liver in yellow fever. Cultivation experiments are wanting. It was probably secondary infection with Streptococcus pyogenes. Bilious Fever. Babes, in a case of fievre bilieuse typhoide, found masses of streptococci filling the vessels of the liver, kidney, and spleen. Probably another in- stance of secondary infection with Streptococcus pyogenes. Measles. From the blood and from inflammatory post-products in measles Babes isolated a streptococcus, which he describes as closely resembling the Streptococcus pyogenes. Ulcerative Endocarditis. Wyssokowitsch found cocci in the internal organs in ulcerative endocarditis, and produced the disease in animals after injury to the valves SYSTEMATIC AND DESCRIPTIVE. 237 by injection of Streptococcus pyogenes and other organisms. Wechselbaum, by microscopical research and by cultiva- tion experiments, proved the presence of Streptococcus pyogenes in acute verrucous endocarditis. Baumgarten confirmed this. He found Streptococcus pyogenes alone in one case and accompanied by Staphylococcus aureus in another. Typhoid Fever. Senger found a streptococcus in a case of typhoid with secondary infection. This was probably Streptococcus pyogenes. Dunin found the well- known pyogenic organisms in post-typhoid suppuration, mostly as staphylococci, but sometimes streptococci. Pneumonia. Wechselbaum found a Streptococcus pneumonia, which resembled Streptococcus pyogenes and erysipelatis morphologically and on cultivation. It was found in twenty-one cases of pneumonia by microscopical research, and cultivated in nineteen. It had no effect on the rabbit's ear. Baumgarten, nevertheless, regards this as Streptococcus pyogenes, and is of opinion that it is only a matter of further research to establish that view. Neumann found streptococci by microscopical research in the lungs, and by cultivation isolated a streptococcus in pneumonia after typhoid. It corresponds with Wech- selbaum's streptococcus in not affecting the rabbit's ear. Kmpyema. Rosenbach obtained a pure cultivation of Streptococcus pyogenes in a case of empyema. Wechsel- baum, in two cases, also established its presence. Broncho-pneumonia. Thaon found a strep- tococcus in the lungs of children in fatal cases of broncho-pneumonia, complicating measles, diphtheria, and whooping-cough. It was regarded as identical with the streptococcus isolated by Loffler from diphtheria. Frankel discovered a streptococcus in the lungs of a case of true croup complicated with broncho-pneumonia, and by culti- vation established its identity with Streptococcus pyogenes. Progressive Tissue Necrosis in Mice. Koch produced a disease in mice by subcutaneous injection of BACTERIOLOGY. putrid blood. In tissue sections a chain coccus was found, and Baumgarten is of opinion that it is very probably identical with the Streptococcus pyogenes ; but cultivations are still wanting. FIG. 74. STREPTOCOCCUS OF PROGRESSIVE TISSUE NECROSIS IN MICE. (a) Necrotic cartilage cells, and (d) Chains in masses; (c) Chains isolated [after Koch]. Anthrax. Charrin found cocci in rabbits, examined some hours after death from anthrax. These, when isolated, produced death in rabbits from septicaemia with- out suppuration. Chains composed of from fifteen to twenty elements were found in all the organs. This was probably another instance of Streptococcus pyogenes. Syphilis. Kassowitz and Hochsinger found the presence of a streptococcus in the tissues and internal organs, and especially in the blood-vessels, in fatal cases of congenital syphilis. These observers regarded their discovery as having an important bearing on the etiology of syphilis, but Kolisko pointed out that it was only the result of septic infection with presence of Streptococcus pyogenes^ as had already been established in scarlet fever. Cerebro-spinal Meningitis. From the menin- geal exudation of a case of apparently idiopathic cerebro- meningitis, Banti found by Koch's methods the presence of Streptococcus pyogenes and Staphylococcus aureus and SYSTEMATIC AND DESCRIPTIVE. 239 albus. The cocci probably entered through an abscess of the jejunum. Blepharadenitis and Dacryocystis. Wid- mark isolated by cultivations Streptococcus pyogenes and other organisms from cases of blepharadenitis and phleg- monous dacryocystis. In phlegmonous dacryocystis Widmark found Streptococcus pyogenes almost exclusively. Leukaemia. Flligge cultivated a streptococcus from necrotic patches in the spleen of a fatal case of leukaemia. Cultures corresponded very closely with Streptococcus pyogenes. Inoculation in the ears of rabbits produced similar results to Streptococcus pyogenes or erysipelatis. Fliigge calls it Streptococcus pyogenes malignus, but con- cludes that it is probably identical with the streptococcus from pus. Soil. Nicolaier, and later Guarneri, isolated a strep- tococcus from soil. Microscopically it could not be distinguished from other streptococci. Baumgarten is of opinion that it is neither in form nor in cultivation to be distinguished with certainty from Streptococcus pyogenes. Air. Emmerich succeeded in proving the presence of streptococci in the air of a hospital where erysipelas had broken out. These cocci, in their form, their characters on cultivation, and in inoculation results, were identified with the Streptococcus erysipelatis. Putrefaction. Baumgarten points out that strep- tococci are found in the most various substances under- going putrefaction. EXAMINATION AND CULTIVATION OF STREPTOCOCCI. Cover-glass preparations can be stained with the watery solutions of the aniline dyes. In some cases very beautiful preparations can be obtained by using Neelsen's solution, and removing excess of stain by rinsing in alcohol. To examine pus, milk, or broth, take an ordi- nary platinum inoculating needle bent at the extremity 240 BACTERIOLOGY. into a booklet. Dip it into the liquid to be examined, and spread it on a cover-glass into as thin a film as possible ; the preparation is treated in the ordinary way, that is to say, the film is allowed to dry, and the cover is taken up with forceps, and passed three times through the flame with its prepared side uppermost. Gram's Method of Eosin. In this way the strep- tococci are stained blue, and stand out in marked contrast to the rest of the preparation. Use freshly pre- pared solution. Float the cover-glasses on the solution for ten minutes to half an hour, then transfer them to iodine-potassic-iodide solution, until they assume the colour of a tea leaf ; then immerse them in alcohol until they are decolourised ; dip them in an alcoholic solution of eosin for a few moments, and then transfer them to clove oil to clarify the film ; to remove the clove oil gently press the cover between two layers of clean filter paper, then mount in xylol balsam (Plate XIX.). A good method for cultivating streptococci is to employ a sterilised looped platinum wire, and to spread a droplet, for example, of pus or blood over the surface of nutrient agar-agar solidified obliquely. The tubes are then placed in the incubator at 37 C. ; the streptococci will appear in the course of two or three days in the form of minute dotted colonies. If present alone, and in considerable quantities, the inoculated surface will exhibit a pure cultivation consisting of a number of such colonies, whilst a flocculent mass is observed in the liquid which collects at the bottom of the agar-agar tubes ; this flocculent mass will be found to be composed of chains. From such a tube inoculate a number of the small flasks employed in Pasteur's laboratory for cultivations in liquids. In this way a number of pure cultivations in milk and broth are established, which can be readily examined from time to time. From a pure cultivation in broth or agar-agar tubes of nutrient gelatine can be inoculated. Cover-glass- preparations from the growths on solid media can be SYSTEMATIC AND DESCRIPTIVE. 241 made in the usual way, and stained with either a watery solution of fuchsine or gentian violet ; but to stain pre- parations made from milk or broth, or from the liquid in agar-agar tubes, use the method of Gram ; the stain will then be removed, except from the streptococci, and very beautiful preparations result. Genus II. Merismopedia. SPECIES. ASSOCIATED WITH DISEASE : Merismopedia gonorrhoeae . . Pathogenic in man. ,.. f Saprophytic in man. Micrococcustetragonus . . j Pa L>genic in animals. Diplococcus albicans tardissimus . Saprophytic in man. UNASSOCIATED WITH DISEASE : Micrococcus citreus conglomeratus \ Micrococcus subflavus . . . >-Simple saprophytes. Micrococcus albicans amplus . .J Merismopedia gonorrhcese (Coccus of Gonor- rhoea]. Cocci 0*83 IJL in diam., singly, in pairs, in tetrads, and zooglcea groups. They are found in gonorrhceal pus adhering to the pus corpuscles and epithelial scales. Artificial cultivations have been carried out,* and it has been claimed that the pathogenic character of the cocci has been esta- blished by inoculation. Micrococcus tetragonus. Cocci about i //, in diam., in groups of four (tetrads), surrounded by a hyaline capsule. They are found in the sputa of phthisical patients and in the walls of tubercular cavities. In a test-tube of nutrient gelatine they form an irregular white growth, more especially in the upper part of the needle track * Bockhart, Sitzungsberichte der Phys. Med. Gesell. Wiirzburg. 1882. 16 242 BACTERIOLOGY. (Plate V., Fig. i). On the sloping surface of nutrient agar-agar thick, whitish, heaped-up masses develop. Guinea-pigs and mice inoculated with a minute quantity of a pure cultivation die in from two to ten days, and the groups of the characteristic tetrads may be found in the capillaries through- out the body, especially in the spleen, lung, and kidney (Plate II., Fig. i). Double infection can be produced by inoculating a mouse with a pure cultivation of Bacillus anthracis two or three days after inoculation with Micrococcus tetragonus. On examination after death, the capillaries of the lungs, liver, and kidney are filled with both anthrax bacilli and masses of tetrads* (Plate XXIV., Fig. 2). Micrococcus citreus conglomeratus, Bumm. Cocci i '5 //-in diam., similar to gonococci. They form lemon-yellow colonies on plate-cultivations. Isolated from blennorrhceic pus and from dust from the air. Diplococcus albicans tardissimus, Bumm. Cocci morphologically identical with gonococci. They grow extraordinarily slowly on gelatine. They were iso- lated from urethral pus. MicroCOCCUS SubflavuS, Bumm. Cocci morpho- logically resembling gonococci. Cultivated on nutrient gelatine, they form whitish dots which become gradually greyish and then yellow in colour, and confluent. They were observed in lochial discharges and vaginal secretions. Micrococcus albicans amplus, Bumm. Cocci in pairs and tetrads similar to gonococci, but considerably larger. Found in vaginal secretions. * The Author, Lancet, 1885. SYSTEMATIC AND DESCRIPTIVE. 243 Genus HI. Sarcina. UNASSOCIATED WITH DISEASE : Sarcina lutea . Sarcina aurantiaca . Sarcina ventriculi . Sarcina intestinalis Sarcina urinse , J \ Sarcina litoralis Sarcina Reitenbachii Sarcina hyalina Sarcina alba . >Chromogenic saprophytes. Simple saprophytes. Sarcina lutea. Cocci singly, in pairs, in tetrads, and in packets. A single individual in a tetrad may be divided into two, or into four, so that a tetrad within a tetrad results. Cultivated in nutrient agar-agar in a test-tube, they form a colourless growth along the track of the needle, and a bright canary-yellow layer upon the surface, where they have access to the air (Plate VII., Fig. i ; Plate IX., Fig. i). In plate-cultivations the colonies are round, slightly granular in appearance, and yellow. Cultivated in a test-tube containing nutrient gelatine, they grow rapidly ; the gelatine becoming liquid, the yellow growth forms a wad about the middle of the tube (Plate VI., Fig. 2), or, liquefying the whole of the gelatine, subsides to the bottom of the test-tube. Cultivated on sterilised potatoes, they form a yellow layer (Plate XV., Fig. i). In drop-cultures in bouillon the subdivision into tetrads within tetrads and formation of groups of 8, 1 6, and 24 can be studied (Plate I., Fig. 7). In- oculation of mice produces negative results. The cocci are occasionally present in the air. 244 BACTERIOLOGY. Sarcina aurantiaca. Cocci singly, in pairs, in tetrads, and in packets. They form small orange-yellow colonies on plate-cultivations, and in test-tubes slowly liquefy the gelatine along the whole needle track, forming on the surface an orange-yellow growth. On potatoes they slowly develop the same pigment. Sarcina ventriculi, Goodsir.* Cocci reach- ing 4 p in diam., united in groups of four, or multiples of four, producing cubes or packets with rounded-off corners. Contents of the cells are greenish or yellowish-red. They occur in the stomach of man and animals in health and disease, and were first detected in vomit. Sarcina intestinalis, Zopf.f Cocci in groups of four or eight. Very regular in form ; never in the large packets which occur in Sarcina ventriculi. They are found in the intestinal canal, especially the caecum, of poultry, particularly fowls and turkeys. Sarcina urinse, Welcker. Very small cocci, i'2 p, in diam., united in families of 8 to 64. Observed in the bladder. Sarcina litoralis, Oersted. Cocci 1-2 2 ^ in diam., bound together in 4 to 8 families, which, in their turn, may unite and include as many as 64 tetrads. Plasma colourless ; in each cell I 4 sulphur granules. Discovered in sea-water containing putrefying matter. Sarcina Reitenbachii, Caspary. Cocci about 1-5 to 2*5 p, in diam., at the time of division lengthened to 4 p. Mostly united together from 4 to 8 in number ; occasionally 16 or more. Colourless cell-wall, lined with * Goodsir, Edinburgh Med. and Surg. Journal. 1842. t Zopf, Die S$altpilze. 1885. SYSTEMATIC AND DESCRIPTIVE. 245 rose-red layer of plasma. Found on rotting water- plants. Sarcina hyalina, Kutzing. Cocci round, 2-5 ^ in diam., almost colourless. United in families of 4 to 24 cells, reaching I 5 ju, in diam. In marshes. Sarcina alba. - Small cocci. They form small white colonies on nutrient gelatine. In test-tube culti- vations they grow slightly along the needle track, but are heaped up on the surface without liquefying the gelatine. They are present in the air. Gen us IV, Micrococcus. SPECIES. ASSOCIATED WITH DISEASE : In man In animals In plants yellow Micrococcus pyogenes aureus Micrococcus pyogenes albus . Micrococcus pyogenes citreus Micrococcus cereus albus Micrococcus cereus flavus . Micrococcus in scarlatina . . Micrococcus in measles . Micrococcus in whooping-cough . Micrococcus in haemophilia neona- torum Micrococcus in typhus . Micrococcus in acute atrophy Micrococcus in dental caries . Micrococcus in gangrene ... , Micrococcus pyogenes tenuis . Micrococcus in rabies Micrococcus of septicaemia in rabbits .... Micrococcus of pyaemia in rabbits . Micrococcus of progressive suppu- ration in rabbits . Micrococcus parvus ovatus . Micrococcus of pyaemia in mice Micrococcus perniciosus . . . Micrococcus bombycis . Micrococcus insectorum Micrococcus amylivorus . ''"' ' }pyogenic in man (?). Pathogenic in animals. > Non-pathogenic. Possibly only saprophytic. Pathogenic (?). Pathogenic. Pathogenic (?). Pathogenic (?). 246 BACTERIOLOGY. /Chromogenic saprophytes. .Simple saprophytes. UNASSOCIATED WITH DISEASE : Micrococcus cyaneus Micrococcus aurantiacus Micrococcus chlorinus Micrococcus violaceus Micrococcus luteus. Micrococcus rosaceus Micrococcus haematodes Micrococcus candidus Micrococcus candicans Micrococcus foetidus Micrococcus crepusculum Micrococcus cinnabareus Micrococcus flavus liquefaciens Micrococcus flavus tardigradus Micrococcus versicolor Micrococcus viticulosus Micrococcus lacteus favi ormis Micrococcus fulvus Micrococcus viscosus Micrococcus coronatus Micrococcus radiatus Micrococcus flavus desidens Micrococcus pyogenes aureus. (Staphylo- coccus pyogenes aureus, Rosenbach. Yellow coccus in pus.* Coccus of acute infectious osteomyelitis). Cocci singly, in pairs, very short chains, and irregular masses. Cultivated on nutrient agar-agar an orange- yellow culture develops, looking like a streak made with oil paintt (Plate IX., Fig. 2). Cultivated in a test-tube of nutrient gelatine, the gelatine is rapidly liquefied, and the growth subsides as an orange- yellow sediment. On potatoes and blood serum a similar orange-yellow culture grows luxuriantly. The micro-organisms injected into the pleura or knee of a rabbit produce, as a rule, a fatal result on the following day ; but if it survives longer, it eventually dies of severe phlegmon. If injected * Ogston, Brit. Med. Journ. f Rosenbach. 1881. SYSTEMATIC AND DESCRIPTIVE. 247 into the knee of a dog, suppuration occurs, fol- lowed by disintegration of the joint. The cocci do not cause any septic odour in pus, nor does any gas develop. Albumen is converted by their action into peptones. They occur in the pus of boils and in the abscesses of pyaemia, puerperal fever, and acute osteomyelitis. Injected into the peritoneal cavity of animals, they set up peritonitis, and introduced into the jugular vein they produce septicaemia and death. When a small quantity of a cultivation was introduced into the jugular vein after previous fracture or contusion of the bones of the leg, the animal died in about ten days, and abscesses were found in and around the bones, and in some cases in the lungs and kidneys. Similar cocci were found in the blood and pus of the animals.* Micrococcus pyogenes albus (Staphylococcus pyogenes albus, Rosenbach). Cocci microscopically indistinguishable from the above. In cultivations also they resemble the Micrococcus pyogenes aureus, but the growth consists of opaque white masses. They liquefy nutrient gelatine rapidly, and subside to the bottom as a white sediment. They are also similar to the above-mentioned in their pathogenic action. Pure cultivations of the organism were obtained from a case of acute suppuration of the knee-joint. Micrococcus pyogenes citreus (Staphylo- * Becker, Deiitsche Med. Wochenschr. Nov., 1883. 248 BACTERIOLOGY. coccus pyogenes citreus, Passet).* Cocci singly, in pairs, very short chains, and irregular masses. If cultivated on nutrient gelatine or nutrient agar-agar, a sulphur or lemon-yellow growth develops (Plate X., Fig. 3). When inoculated under the skin of mice, guinea-pigs, or rabbits, an abscess forms after a few days, from which a fresh cultivation of the micro-organism can be obtained. They are frequently present in pus. Micrococcus cereus albus (Staphyloccocus cereus albus, Passet).* Cocci, morphologically simi- lar to the above, but distinguished by forming on nutrient gelatine a white, slightly shining layer, like drops of stearine or wax, with somewhat thickened, irregular edge. The needle track de- velops into a greyish- white, granular thread. In plate-cultivations, on the first day, white points are observed, which spread themselves out on the sur- face to spots of i 2 mm. When cultivated on blood serum a greyish-white, slightly shining streak develops, and on potatoes the cocci form a layer which is similarly coloured. (Plate IV., Fig. 3, XI. Fig. 3.) Micrococcus cereus flavus (Staphylococcus cereus flavus, Passet).* Cocci which also occur in pus. If cultivated in nutrient jelly, the growth, which is at first white, becomes lemon-yellow, somewhat darker in colour than Micrococcus pyogenes citreus. Microscopically Micrococcus cereus flavus corresponds with Micrococcus cereus albus, and they both form * Passet, Fortschritte der Medicin, Jan. I5th and Feb. ist, 1885. SYSTEMATIC AND DESCRIPTIVE. 249 zooglcea of medium-sized cocci (diam. n6 p.). In- oculation experiments with both kinds gave negative results. Among the micro-organisms present in pus a coccus has been described as occurring occasionally which is almost identical with Bacterium pneumonia croupos(, long and "55 /x, broad, singly, in pairs, chains, or zooglcea. They were detected in the digestive organs of the chinck-bug (Blissus leucopterus) when suffering from a certain contagious disease. Micrococcus amylivorus, Burrill. Oval cells, I 1*4 ju, long, / fj, broad, singly, in pairs, and rarely in fours, never in chains, are found embedded in an abundant mucilage which is very soluble in water. They have been described as producing the so-called " fire blight " of the pear tree and other plants. Micrococcus cyaneus, Cobn<(Bact**uintm cyaneum, Schroter). Elliptical cells, growing upon cooked potato, and producing a blue colour. In nutrient solutions they form zooglcea, at first colourless, then bluish-green, and finally intense blue. Micrococcus aurantiacus, Schroter. Cocci, oval, 1*5 jut in diam., singly or in pairs, or in zooglcea. They occur as orange-yellow spots which coalesce into patches. A golden-yellow pellicle develops when they are cultivated * Wolff, Vir -chow's Archil). 1883. 254 BACTERIOLOGY. in nutrient liquids. The colouring matter is soluble in water. They were observed on boiled potatoes and white of egg. Micrococcus chlorinus, Cohn. Cocci occur in the form of a finely granular zooglcea, causing a yellowish- green or sap-green layer on boiled eggs and nourishing solutions. The colouring matter is soluble in water, and is decolorised by acids. Micrococcus violaceus, Schroter. Cocci or elliptical cells, described as uniting into violet-blue gela- tinous spots, which again unite to form larger patches. They were observed on boiled potatoes exposed to the air. Micrococcus luteus, Schroter. Cocci similar in size to the above, elliptical, with highly refractive cell contents. They form yellow drops of I 3 mm. diam. on boiled potato, and a thick, wrinkled, yellow skin on nutrient liquids. The colouring matter is insoluble in water, and unchanged by sulphuric acid or alkalies. MicroCOCCUS rosacCUS. Cocci forming pink colonies, and a rose-coloured growth on the surface of nutrient agar-agar. Observed contaminating an old cul- tivation (Plate X., Fig. 2). Micrococcus hsematodes, Zopf. Cocci, which, cultivated on boiled white of egg in a damp chamber in the incubator, form a red layer. The reaction of the colouring matter is similar to that produced by Micrococcus prodigiosus. They have been observed in human sweat, especially from the axilla, colouring the surrounding parts and the linen an intense brick or blood-red colour.^ Micrococcus candidus, Cohn. Cocci forming snow-white points and spots, upon slices of cooked potato. Possibly identical with the following : Micrococcus candicans, Flugge. Cocci which collect in masses. In plate-cultivations they form in two or three days milk-white colonies. Cultivated in test- tubes they form a white nail-shaped cultivation. They were isolated from contaminated plate-cultivations. * Babes, " Vom Rothen SchweiSs," Biol. Centrabl., Bd. 2. 1882. SYSTEMATIC AND DESCRIPTIVE. 255 MicrOGOCCUS foetidus, Rosenbach. Small oval cocci. Cultivated in agar-agar they develop gas-bubbles and a foetid odour. Isolated from carious teeth. Possibly closely allied to, if not identical with, the following : MicroCOCCUS crepUSCulum, Cohn (Monus crepus- culum, Ehrenberg. Mikrokokken in faulenden Substraten, Fliigge). Round or short oval cells, scarcely 2 //, in diam. ; singly or in zooglcea. They occur in various infusions and putrefying fluids in company with Bacterium termo. Micrococcus cinnabareus, Fliigge. Large cocci occurring in twos, threes, and fours. In plate- cultivations they grow very slowly, forming punctiform colonies, in colour bright red at first, and afterwards reddish-brown. In test-tube cultivations they form on the surface of the gelatine a heaped-up, red-coloured growth. Found contaminating old cultivations. Micrococcus flavus liquefaciens, Fliigge. Cocci, diplococci, and zooglcea. On plate-cultivations they form yellowish colonies, and in test-tubes yellowish beads, which become confluent and rapidly liquefy the gelatine. Micrococcus flavus tardigradus, Fliigge. Cocci forming chrome-yellow colonies. Cultivated in test- tubes they form yellowish beads in the needle track, which remain isolated, and do not liquefy the gelatine. Isolated from contaminated cultures. Micrococcus versicolor, Fliigge. Small cocci, which form iridescent colonies. In test-tubes they grow in the form of yellowish beads, and develop an iridescent layer on the surface. Micrococcus viticulosus, Fliigge. Oval cocci, I '2 /A in length, and I p, in width. In plate-cultivations the colonies differ when embedded in the nutrient medium and when growing on the surface. The characteristic appearance consists of a delicate network, which is visible also in test-tube cultivations in the track of the needle. On the surface of the medium they form a viscous layer. They were isolated from contaminated cultivations. 256 BACTERIOLOGY. Micrococcus lacteus faviformis, Bumm and Bockart. Cocci 1*25 /x, in diam. Cultivated in gelatine they form milk-white confluent colonies, and preparations made from the cultivations have a characteristic honeycomb appearance. Isolated from vaginal secretions, and from sputum. Micrococcus fulvus, Cohn. Cocci round 1-5 p, in diam., frequently in pairs. They form rusty-red conical drops of a firm consistency, and about '5 mm. diam., on horse-dung. Micrococcus viscOSUS, Pasteur. Globular cells '2 p, in diam., singly or in chains. These and allied forms have been considered to be the cause of mucoid fermentation in wine and beer* (vin filant, biere malade). MicroCOCCUS COronatUS (Streptococcus coronatus, Fliigge). Cocci I ^u, in diam., singly, in short chains, and in zooglcea. In plate-cultivations the colonies have a characteristic halo, formed by the liquefaction of the gelatine around the colony. Isolated from the air. MicroCOCCUS radiatus (Streptococcus radiatus, Fliigge). Cocci less than I JJL in diam., singly, and in short chains. They rapidly form whitish colonies with a yellowish-green sheen. They liquefy the gelatine, the colonies sinking down, and after one or two days develop- ing a circlet of rays. A peculiar ray-like appearance is characteristic also of the growth in test-tubes. Isolated from contaminated plate-cultivations. Micrococcus flavus desidens (Streptococcus flavus desidens, Fliigge). Cocci, diplococci, and short chains. They form yellowish-white colonies, which gradually sink down in the gelatine. In test-tubes they form china-white, confluent masses in the tract of the needle, and on the surface a yellowish-brown slimy layer. Isolated from contaminated plate-cultivations. * Pasteur, Etudes sur le Vin; sur la Biere. 1866 ; 1876. SYSTEMATIC AND DESCRIPTIVE. 257 Gemis V. Asco coccus. SPECIES. UNASSOCIATED WITH DISEASE : Ascococcus Billrothii . Zymogenic saprophyte. Ascococcus Billrothii. Small globular cocci, united into characteristic colonies. They form on the surface of nourishing fluids a cream-like skin, divisible into an enormous number of globular or oval families. Each family is surrounded by a thick capsule of cartilaginous consistency. In a FIG. 76. Ascococcus BILLROTHII [after Cohn]. solution containing acid tartrate of ammonia the fungi generate butyric acid, and change the origin- ally acid fluid into an alkaline one. They were first observed on putrid broth, and later on or- dinary nourishing solutions ; they also readily develop upon damp slices of boiled roots, carrots, beetroots, etc. 17 258 BACTERIOLOGY. METHODS OF STAINING COCCI. Cocci stain well with watery solutions of gentian-violet, methyl-violet, fuchsine, methylene blue, and bismarck- brown. For examining cocci in liquids such as pus or blood, or in cultivations in solid media, a little of the material should be spread out on a cover-glass (page 65), and stained with a drop or two of a watery solution of fuchsine or methyl-violet. The former is especially recom- mended for staining Merismopedia gonorrhcecz. For a zooglcea, or pellicle of micrococci, Klein recom- mends transference bodily to a watch-glass containing the dye, leaving it there till deeply tinted, then taking it out with a needle, washing in water, and then in alcohol till excess of colour is removed. It must then be transferred to a glass slide, spread well out, and a drop of clove-oil placed on it ; after a minute or two the clove-oil is drained off, a drop of Canada balsam added, and covered with a cover-glass.* Cocci in the tissues may be stained by immersing the sections in an aqueous solution of gentian-violet, or in aniline-gentian-violet solution, then rinsing in water, decolorising in alcohol, treating with clove-oil, and pre- serving in balsam (p. 76) ; or, after washing with alcohol, they may be rinsed with water, and stained for half an hour with Weigert's picrocarmine. From this they are again removed to water, then to alcohol, clove-oil, and Canada balsam. The method of Gram is much more satisfactory (p. 76, Plate II., Figs, i and 2). Sections should be examined with and without a contrast stain. The after-stain most com- monly employed is eosin. The sections after the process of decolorisation should be placed in a weak alcoholic solution of eosin (two or three drops of a concentrated alcoholic solution added to a watch-glassful of alcohol), till stained a delicate pink. They are then rinsed in * Klein, Micro-organisms and Disease. 1885. SYSTEMATIC AND DESCRIPTIVE. 259 fresh alcohol, treated with clove-oil, and preserved in Canada balsam. Sections containing cocci of osteomyelitis may be after- stained 'with weak solution of vesuvin. Safranine and picro- lithium-carmine may also be used as contrast stains (p. 78). Nuclear stains, such as carmine, haematoxylin, may also be employed. Sections may be left one minute in Gre- nadier's solution, then washed out in weakly acidulated alcohol (2 1000) ; and finally treated in the usual way, with alcohol, oil of cloves, and balsam. Sections containing Micrococcus tetragonus are best stained with Gram's method and eosin (Plate XXIV., Fig. 2), but they may also be treated by the method of Friedlander, to demonstrate their capsules (p. 263). To stain the cocci of rabbit-septicamia in the tissues, place the sections twenty-four hours in Loffler's solution, wash in water faintly acidulated with acetic acid, then treat with alcohol, oil of cloves, and balsam. GROUP II. BACTERIACE^:. Genus I. Bacterium. Cocci and rods, or only rods, which are joined together to form threads. Spore- formation absent or unknown. Genus II. Spirillum. Threads screw-form, made up of rods (long or short) only, or of rods and cocci. Spore-formation absent or unknown. Genus III. Leuconostoc. Cocci and rods. Spore- formation present in cocci. Genus IV. Bacillus. Cocci and rods, or rods only, forming straight or twisted threads. Spore- formation present either in rods or cocci. Genus V. Vibrio. Threads screw-form in long or short links. Spore-formation present. Genus VI. Clostridium. Same as bacillus, but spore- formation takes place in characteristically en- larged rods. 260 BACTERIOLOGY. Genus I. Bacterium. SPECIES. ASSOCIATED WITH DISEASE : 'Bacterium pneumoniae crouposae Bacterium pseudo-pneumonicum . In man . / Bacterium Neapolitanum Bacterium in rhinoscleroma . Bacterium in diphtheria Bacterium saprogenes . 'Bacterium decalvans 'Bacterium in diphtheria of calves . Bacterium of diphtheria of pigeons Bacterium choleras gallinarum Bacterium septicum agrigenum Bacterium of septicaemia in rabbits Bacterium of Davaine's septicaemia Tn animals ( Bac terium septicum sputigenum . Bacterium crassum sputigenum . Bacterium pneumonicum agile Bacterium oxytocum perniciosum . Bacterium cavicida Bacterium coli commune . ,. Bacterium lactis aerogenes . Panhistophyton ovatum ' . . In plants . Bacterium hyacinthi UNASSOCIATED WITH DISEASE: Bacterium synxanthum . Bacterium indicum Bacterium rubrum . . ..,,.. Bacterium prodigiosum . .. Bacterium luteum ." ; *'..-. Bacterium violaceum . ..- . w Bacterium brunneum Bacterium fluorescens putidum Bacterium fluorescens liquefaciens^ Bacterium ureae . . ^ . Bacterium aceti Bacterium Pasteurianum Bacterium liodermos . ; v Bacterium multipediculum . Bacterium ramosum liquefaciens Bacterium Zopfii . . Bacterium merismopedioides . Bacterium Pfliigeri Bacterium photometricum . Bacterium litoreum ;'"_ Bacterium fusiforme . . Bacterium navicula Proteus vulgaris . Proteus mirabilis . Proteus Zenkeri . v ;: Bacterium termo Bacterium lineola . Pathogenic (?) ; possibly only saprophytic in man, pathogenic in animals. Saprophytic in man, patho- genic in animals. Pathogenic in man (?). Saprophytic in man (?), pathogenic in animals. Saprophytic in man, patho- genic in animals. Saprophytic. Pathogenic (?). Pathogenic. Pathogenic (?), ^Chromogenic saprophytes. Zymogenic saprophytes. 'Simple saprophytes. SYSTEMATIC AND DESCRIPTIVE. 26l Bacterium pneumoniae crouposae (Pneumo- coccus, Friedlander). Cocci ellipsoidal and round, singly, or in pairs (diplococci), rods and thread- forms. The cell-membrane thickens, and develops into a gelatinous capsule, which is round if the coccus is single, and ellipsoidal if the cocci occur in pairs or in rod-forms (Fig. 77, Plate I., Fig* 5). Cultivated in a test- tube of nutrient gelatine they grow along the needle track in the form of a round- headed nail (Plate V., Fig. 2), without liquefaction FIG. 77. BACTERIUM PNEUMONIA CROUPOSAE, FROM PLEURAL CAVITY OF A MOUSE, x 1500. A, B. Thread-forms. C, D, E. Short rod -forms. G. Diplococci. H. Cocci. I. Streptococci. [After Zopf.] of the gelatine. The cocci when artificially culti- vated have no capsule, but it again appears after their injection into animals. The cocci can also be cultivated on blood serum and on boiled potatoes. They occur in pneumonic exudation.* Inoculation of dogs with a cultivation of the cocci occasionally gave positive results ; but in rabbits no results * Friedlander, Fortschr. d. Med. 1883. 262 BACTERIOLOGY. followed. Guinea-pigs proved to be susceptible in some cases ; but thirty-two mice, after injection of a cultivation diffused in sterilised water into the lungs, died without exception. The lungs were red and solid, and contained the cocci, which were also present in the blood, and in enormous numbers in the pleural exudation. Inhalation experiments by spraying the cocci diffused in water into mouse cages succeeded in producing pneumonia and pleurisy in three out of ten mice. The nail-shaped cultivation is not always produced, nor are these conclusions accepted by all investigators.* METHODS OF STAINING THE BACTERIA OF PNEUMONIA. (Pneumonic- Coccen, Friedlander.) Cover-glass preparations (p. 65) of pneumonic sputum or exudation may be treated as follows : (a) Stain by the method of Gram, and after-stain with eosin (p. 76). (b) Treat with acetic acid, then stain with gentian- violet or bismarck-brown. Examine in distilled water, or dry and preserve in Canada balsam. (c) Float them on weak solutions of the aniline dyes twenty-four hours ; differentiation between coccus and capsule is thus obtained. (d) Stain with osmic acid ; the contour of the capsules is brought out. Sections of pneumonic lung should be stained by (a) Method of Gram. (b) Method of Friedlander. This method is employed to demonstrate the capsules in tissue sections. It consists * Klein, Micro-organisms and Disease. 1885. SYSTEMATIC AND DESCRIPTIVE. 263 in placing the sections twenty-four hours in the following solution : Fuchsine ..... i Distilled water . . . .100 Alcohol . . . t0 /' J V V> 5 Glacial acetic acid . . . 2 They are then rinsed with alcohol, transferred for a couple of minutes to a 2 per cent, solution of acetic acid, and in the usual way treated with alcohol and oil of cloves, and preserved in Canada balsam. Bacterium pseudo-pneumonicum (Bacillus pseudo-pneumonicus, Passet). Cocci round, oval, and occasionally elongated, similar to the bacterium of pneumonia. The oval forms are '87 //, in width, and i 1 6 //, in length. The colonies on plates appear in twenty-four hours as white dots ; in test-tubes the growth develops as a greyish-white layer. If injected into the pleura they set up pleuritis, and into the abdomen peritonitis, in mice, rats, and guinea-pigs. Subcutaneous inoculation produces septicaemia in mice, and abscesses in rats, guinea- pigs, and rabbits. Inhalation experiments gave no results. They were isolated from pus. Bacteria Neapolitanum (Bacillus Neapolitan, Emmerich). Short rods with rounded ends. In width 9 /i (Fig. 78). They form circular colonies, which later become irregular, granular, strongly refractive, and of a yellowish-brown colour. By introducing a large quantity into small animals changes were produced in the intestines with an analogy to the post-mortem appearances of cholera. They are probably identical with bacteria found in healthy 264 BACTERIOLOGY. faeces. They were isolated from some cases of cholera at Naples. Bacterium of Rhinoscleroma (Bacillus of Rhino scleroma, Cornil and Alvarez*). Cocci and cu FIG. 78. BACTERIUM NEAPOLITANUM, x 700. (a) From intestinal contents in a case of cholera ; (b) From peritoneal fluid of an inoculated guinea- pig [after Emmerich]. short rods, 1*5 3 p. in length, '5 '8 JJL thick. Deeply coloured points or granules may occur in the course of the rods when stained, but it is very doubtful whether these can be considered as spores. The bacteria are encapsuled, the capsule being round when enclosing a coccus, and ovoid when enclosing FIG. 79. BACTERIA OF RHINOSCLEROMA, x 1400. Encapsuled cocci, diplo- cocci, and short and long rod-forms [after Cornil]. a rod (Fig. 79). The capsule is composed of a tough resisting substance ; two or more capsules may unite by fusion, enclosing two or three, or a great number of rods. The bacilli were observed in sections of a tumour, rhinoscleroma, which develops * Cornil and Babes, Les Bacteries. 1885. SYSTEMATIC AND DESCRIPTIVE. 265 on the lip and on the nasal and pharyngo-laryngeal regions. METHOD OF STAINING THE BACILLUS OF RHINO- SCLEROMA. Method of Cornil and Alvarez : - Sections are immersed in a solution of methyl-violet (B) for twenty-four to forty-eight hours, with or without the addition of aniline-water ; are then decolorised after treat- ment with the solution of iodine in iodide of potassium. If the sections are left to decolorise in alcohol for forty- eight hours, the capsule is rendered visible. Bacterium in diphtheria of man (Bacillus of diphtheria, Loffler). Rods about the same length as the tubercle bacillus, but about twice as thick ; the longer ones consist of single individuals linked together. Spores not observed. They were cultivated in a mixture consisting of three parts of calf's or lamb's blood serum, to which was added one part of neutralised veal broth, containing i per cent, peptone, i per cent, grape sugar, \\ per cent, common salt. Cultivated in 5 per cent, gelatine at 20 22 C, the rods developed into irregular involution-forms. In- oculation gave doubtful results. The bacillus was isolated from diphtheritic membrane.* They were particularly noticeable in those typical cases characterised by a thick false membrane, extending over the fauces, larynx, and trachea. They occupied * Loftier, Mittheil. a.d. K. Gesundheitsamte (Microfiarasites in Disease, New Syd. Society). 266 BACTERIOLOGY. the deeper layers below the masses of bacteria which are found on the surface, such as the streptococci already described (p. 231). METHOD OF STAINING THE BACTERIA IN DIPHTHERIA. LoffleSs Method: Sections are placed in Loffler's solution for a few minutes, and excess of stain removed by \ per cent, solution of acetic acid. They are then treated with alcohol and cedar-oil, and mounted in Canada balsam. Bacterium saprogenes (Bacillus saprogenes No. 3, Rosenbach). Rods isolated from the putrid marrow of a case of compound fracture. Cultivated on nutrient agar- agar, an ash-grey, almost liquid culture is developed, with a strong characteristic odour of putrefaction. Injected into the knee-joint or abdomen of a rabbit, an opaque, yellowish-green infiltration resulted (vide Bacillus sapro- genes, p. 346). Bacterium decalvans, Thin. Cocci, singly or in pairs, r6 fji in length. Observed in the roots of the hair in cases of Alopecia areata. Bacterium in diphtheria of calves (Bacillus vitulorum, Loffler). Rods about five or six times as long as wide, mostly united in long threads. A piece of tissue placed on blood serum developed a white layer composed of the bacteria. Successive generations were not obtainable. Mice inoculated directly from the calf died of a characteristic illness, and the same long bacteria were again found in the inoculated animals accompanying widespread infil- tration, starting from the point of inoculation. In- oculation of guinea-pigs and rabbits gave doubtful SYSTEMATIC AND DESCRIPTIVE. 267 results. The bacteria were found in the deeper stratum of the diphtheritic patches. Bacterium of diphtheria of pigeons (Bacillus columbarum, Loffler). Short rods with rounded ends, mostly in irregular masses. In plate-cultiva- tions on nutrient gelatine they formed whitish patches on the surface, and compact, ball-like masses when embedded in the gelatine. They were also cultivated on blood serum and potatoes. Subcutaneous inoculations in pigeons with a pure cultivation produced local inflammation and necrosis ; inoculation in the mucous membrane of the mouth gave the appearances of the original disease. Other animals were only locally affected, except mice, in which characteristic symptoms and death resulted. They were isolated from the diphtheritic exudations in pigeons, and in sections were found in the vessels of the lungs and liver. Bacterium cholerae gallinarum (Micrococcus cholera gallinarum, Zopf. Bacterium of Fowl-cholera. Microbe du chole'ra despoules). Cocci 2 3 ft in diam., short rods staining deeply at either pole, and longer beaded rods (Figs. 80, 81). In the tissues they appear mostly as rods 2 3 p, in length and 5 p in diam., with their extremities stained more deeply than their middle* (vide p. 165). When cultivated by introducing a drop of the infected blood into sterile chicken broth, a number of round bodies, * Cornil and Babes, Les Bacteries. 268 BACTERIOLOGY. undergoing rapid movement and as a rule united as diplococci, or elongated and contracted in the middle, appear in the broth, which is at first slightly milky, but becomes limpid, and the microbes at the same time pass into a finely granular state. From this, FIG, 80. BACTERIUM OF CHICKEN CHOLERA; BLOOD OF INOCULATED HEN, X 1200. however, fresh cultures can still be started. Culti- vated in a test-tube of nutrient gelatine, after from three days to a week there develops along the needle track a fine, almost imperceptible, greyish thread FIG. 81. BACTERIUM OF CHICKEN CHOLERA, FROM MUSCLE JUICE OF. INFECTED HEN, X 2500 [from a Photograph]. without liquefaction of the gelatine (Plate IV., Fig. 2). The growth is exceedingly scanty, even after several weeks. Fowls suffering from the disease usually die very rapidly. In the less acute cases they are somnolent, weak in their legs, and their wings trail. They suffer from diarrhoea, and pass into a state of sopor and die. The micro-organisms are found in large SYSTEMATIC AND DESCRIPTIVE. 269 numbers in the blood and organs after death, and in the intestinal discharges. A drop of the broth injected into the connective tissue in the region of the pectoral muscles causes the death of the fowl the following day, with charac- teristic pathological changes.* If a culture be kept for some time, and a fowl be then inoculated with it, instead of death only local changes are produced, and the fowl is protected against the action of a virulent culture ; thus affording an example of so- called mitigation of the virus.\ The microbe is serobic, and its toxic effect has been supposed to be due to the abstraction of oxygen from the blood producing asphyxia. Bacterium septicum agrigenum (Bacillus sep- ticus agrigenus, Nicolaier). Cells morphologically similar to the microbe of chicken cholera. The colonies on plate- cultivations have a yellowish-brown centre, with a greyish- yellow zone. In test-tube cultivations the appearances are not characteristic. They are pathogenic in mice and in rabbits. The organs show no characteristic post-mortem appearances, but the bacteria abound in the blood. They were isolated from earth. Bacterium of septicaemia in rabbits (Bacillus cuniculicida, Koch). Short rods, slightly pointed at both ends ; in width '6 p, 7 //,, in length i -4 //,. They stain deeply at the ends, leaving an uncoloured * Cornil, " Observ. Hist, sur les Lesions des Muscles determinees par 1'injection du Microbe du Cholera des Ponies " (Archives de Physiologic. 1882) ; Cornil and Babes, Les Bacteries. 1885. t Pasteur, " Sur le Cholera des Poules," Comfit. Rendus. 1880. 270 BACTERIOLOGY. interval in the middle (Fig. 82), an appearance which must be distinguished from a diplococcus or figure of eight. Two or more bacteria may be linked together in a chain. They may be cultivated in bouillon, blood serum, and nutrient gelatine. In plate-cultivations of the latter, they produce dot- like colonies, and in test-tubes little spherical masses in the needle track, and a layer on the free surface. The smallest quantity inoculated subcu- taneously or in the cornea of a rabbit produces a rise of temperature and laboured breathing after 10 12 hoars, and death in 16 20 hours. The FIG. 82. BACTERIUM OF RABBIT SEPTICAEMIA; BLOOD OF SPARROW, X 700 [after Koch]. spleen and lymphatic glands are found to be en- larged, and the lungs congested, but no extravasa- tions, and no peritonitis. In the blood the charac- teristic rods abound, and in sections they are found in the vessels and capillaries. Mice and birds are very susceptible ; guinea-pigs and white rats have an immunity. The disease was produced by in- oculating rabbits with contaminated water (River Panke) and with putrid meat infusion. Bacterium of Davaine's septicaemia. Rods similar to the bacteria described by Koch. They were also found in the blood of rabbits suffering SYSTEMATIC AND DESCRIPTIVE. 271 from septicaemia, which, however, differed from Koch's septicaemia in that guinea-pigs were sus- ceptible and pigeons immune. Bacterium septicum sputigenum (Microbe de salive, Pasteur. Micrococcus Pasteuri, Sternberg.* Bacillus septicus sputigenus, Frankel). Cocci oval, singly, in pairs, and in chains ; often lanceolate or rod-shaped ; encapsuled. They grow well in broth, and on agar-agar at 30 to 35 C. On the solid media they form a superficial, nearly transparent deposit of gelatinous consistence. They are patho- genic in rabbits, producing typical " sputum septicaemia." Fowls and dogs have an immunity, and guinea-pigs are less susceptible than rabbits. Mice die within forty-eight hours after being inocu- lated. The blood of an infected rabbit just dead is more potent than a liquid culture or than saliva containing the coccus. An animal which recovers after an injection of saliva is stated to be protected from the potent virus. The pathogenic power is modified by cultivation at a temperature between 39*5 and 40*5. The organism has been supposed to be intimately associated with croupous pneu- monia, but any exact relation cannot be considered as established. The organism differs from the Bacterium pneumonia crouposcz in that it is patho- genic in rabbits, it can be directly isolated from rusty sputum, and it requires a temperature for * Steinberg, Stud. BioL Lab., John Hopkins, Univ. u, No. 2, 1882. Journal Royal Microscop. Society. 1886. 272 BACTERIOLOGY. its growth at which nutrient gelatine is liquefied. The cocci were first observed in the blood of a rabbit inoculated with healthy saliva, and again found in a rabbit which died after inoculation with the saliva of a child suffering from rabies. Later they were isolated from the blood of rabbits in- oculated with the buccal secretions of different individuals, and were found to be constantly present in the rusty sputum of pneumonic patients. Bacterium crassum sputigenum, Kreibohm. Short thick rods with rounded ends. Colonies on plate-cultivations appear as clear grey-white points, which ultimately form greyish slimy drops. In test-tubes they develop very quickly a nail- shaped growth. They are fatal to mice, and after death are found in the blood, and in sections, more especially in the capillaries of the liver. Rabbits die of septicaemia after intravenous injection. A large quantity of a cultivation injected into the cir- culation sets up fatal gastro-enteritis in rabbits and dogs in 3 10 hours. They were isolated from sputum. Bacterium pneumonicum agile (Bacillus pneumonicus agilis, Schou.). Short thick rods, or almost elliptical cells, often two to four linked together. They form dark granular colonies, which after twenty-four hours commence to liquefy the gelatine ; a movement is then visible in the centre of the colony, and an appearance of circum- ferential rays results. They grow also on blood SYSTEMATIC AND DESCRIPTIVE. 273 serum, bouillon, and potatoes. Cultures injected through the chest wall, or into the trachea, or ad- ministered by inhalation, set up pneumonia. They were isolated from pneumonic lungs of rabbit. Bacterium oxytocum perniciosum (Bacillus oxytocus perniciosus, Wyssokowitsch). Short rods with rounded ends, somewhat shorter and thicker than the bacterium of sour milk. They form yellowish colonies, and in test-tubes develop a nail-shaped growth. Culti- vated in milk they produced curdling, and an acid reaction. They were sometimes pathogenic in rabbits. Isolated from sour milk. Bacterium cavicida (Bacillus cavicida, Brieger). Very small rods, about twice as long as broad. They form colonies in the form of whitish concentric rings. On potatoes they develop dirty yellow tufts. They are very fatal to guinea-pigs. Isolated from human faeces. Bacterium coli commune, Escherich. Short slightly curved rods, 1*5 ju, in length, '3 '4 //, thick, colonies yellowish and granular. They develop a white scum on agar-agar and blood serum. Fatal to guinea-pigs and rabbits, when inoculated intravenously. Isolated from faeces of infants fed exclusively on mother's milk. Bacterium lactis aerogenes, Escherich. Short rods with rounded ends, 1*4 2 JJL long, *5 //- wide. Culti- vations in gelatine resemble the bacterium of pneumonia. They produce fermentation in milk and in solution of grape-sugar. Pathogenic effects similar to the above. Isolated from the same source. Panhistophyton ovatum, Lebert (Nosema bom- bycis, Micrococcus ovatus. Corpuscles du ver a soie). Shining oval cocci, 2 3 ^ long, 2 ^ wide, singly and in pairs, or masses ;* or rods, 2*5 //, thick, and twice as long.f They multiply by subdivision. They were experimentally proved * Fliigge, Fermente und Mikro-Parasiten. 1883. f Zopf, Die Sfialtyilze. 18 274 BACTERIOLOGY. to be the cause of pebrine^ gattine, maladie des corpuscles or Flecksucht; and were discovered in the organs of diseased silkworms, as well as in the pupae, moths, and eggs. Bacterium hyacinth!, Wakker. Cells resembling Bacterium termo. Observed in the yellow slime of diseased hyacinth bulbs. Bacterium synxanthum, Ehrenberg (Bacterium xanthinum. Bacterium of yellow milk}. Cocci */ i JK, in length, and rod-forms.* They produce a yellow colour in boiled milk, which at first becomes acid, and then strongly alkaline. They also occur on boiled potatoes, carrots, etc., where they form small lemon-yellow masses. The colouring matter soluble in water, insoluble in ether and alcohol, unchanged by alkalies, decolorised by acids. It is similar to yellow aniline colours both spectro- scopically and in ordinary reactions. Bacterium indicum (Micrococcus indicus, Koch. Bacillus indicus, Fliigge). Very short rods with rounded ends. In plate-cultivations on nutrient agar-agar, the colonies have a scarlet tint. They are round, ovoid, or spindle-shaped, and have characteristic granular margins (Fig. 83). Grown upon nutrient agar-agar in a test-tube, the appear- ances are very characteristic. In a pure cultivation a brilliant, vermilion-coloured reticulated pellicle develops on the surface (Plate VIII., Fig. i). In the track of the needle beneath the surface no pigment is formed (Plate VII., Fig. 2). Cultivated in nutrient gelatine they liquefy the medium, and colour it crim- son. The growth, of a darker crimson hue, subsides to the bottom of the tube. Upon sterilised potato they form a vermilion layer (Plate XVI., Fig. 2). * Zopf, Die Spaltyilze. SYSTEMATIC AND DESCRIPTIVE. 275 Bacterium rubrum, Frank. Minute motile rods, singly, in twos, and fours. They were observed on boiled rice, where they develop a brick-red pigment. Bacterium prodigiosum (Micrococcus prodigi- osus, Bacillus prodigiosus, " Blood-rain" " Bleeding host"}. Very short rods with rounded ends, and thread-forms, -5 i ^ in width, forming at first rose-red, and then blood-red zooglcea. They grow luxuriantly when cultivated on sterilised potatoes (Plate XIV., Fig. i), and on the sloping surface of FIG. 83. BACTERIUM INDICUM ; COLONIES ON NUTRIENT AGAR-AGAR, X 60. nutrientagar-agar(PlateVIIL, Fig. 3). They appear occasionally on bread, boiled rice, and starch-paste, and more rarely on boiled white of egg and meat. Milk sometimes becomes coloured blood-red by the growth of this fungus, an appearance formerly attributed to a disease of the cow. In Paris, in 1843, the fungus was peculiarly prevalent, attacking especially the bread produced in the military bakehouses. The cells themselves are colourless. The colour- ing matter resembles fuchsine ; it is insoluble in water, but soluble in alcohol. The addition of 276 BACTERIOLOGY. acids changes it to a carmine red, and of alkalies to a yellow colour. Bacterium luteum (Bacillus luteus, Flugge). Short immotile rods. Colonies irregular in form, appear brownish under a low power, but macroscopically yellow. In test-tube cultivations they form a yellow growth with- out liquefying the gelatine. They occur contaminating plate-cultivations. Bacterium violaceum, Bergonzini. Cells similar to Bacterium termo, '6 I JJL thick, 2 3 /x long. They occur on white of egg, forming a violet pigment. Bacterium brunneum, Schroter. Motile rods, producing a brown colour. They were observed on a rotting infusion of maize. Bacterium fluorescens putidum (Bacillus fluorescens putidus^ Flugge). Short rods with rounded ends ; motile ; spore-formation not known. They form small dark colonies with a greenish sheen and penetrating odour. In test-tubes they produce a pale-grey turbidity, and after three days colour the medium with a greenish tinge spreading down from above. On potatoes they rapidly develop a brownish layer. They occur on decom- posing substances, producing a greenish coloration. Bacterium fluorescens liquefaciens (Bacillus fluorescens liquefaciens, Flugge). Short rods with rounded ends. Colonies on plates develop an iridescence around them. In test-tubes a similar iridescent sheen is produced. On potatoes they develop a brownish layer. Bacterium ureae (Micrococcus urece, Cohn). Cocci 1*25 2 p in diam., singly or in chains, and rods. The rods split up by division into chains of cocci, and the latter are finally set free. The cocci increase further by subdivision, and a jelly-like membrane develops around them. Masses of cocci SYSTEMATIC AND DESCRIPTIVE. 277 exist in the form of irregular or roundish lumps. Cultivations, after twenty-four hours, consist exclu- sively of rods ; after forty-eight hours, of cocci chains ; and in fourteen days, of zoogloea ; the cocci transplanted into fresh nourishing solution again grow into rods. These observations point to the existence of a pleomorphic species, Bacterium urece ; and the former nomenclature, Micrococcus urece > must be regarded as untenable. They are aerobic ; occurring in urine they set up ammoniacal fermenta- tion, converting urea into carbonate of ammonia.* Rods, 2 p, long and i JJL wide, have been isolated from stale urine (Bacillus urece, Leube), which also most energetically cause the ammoniacal fermentation of urine. Bacterium aceti. Cocci, short rods, long rods, leptothrix-forms, and zoogloea. Cocci and short rods may occur in the same thread. The long rods and threads may develop irregular swellings, so-called involution-forms, which have a thickened membrane and a grey colour. The effect of the action of this microbe is to oxidise alcohol in wine and other fruit juices into vinegar. The masses of zoogloea united together form a membranous layer which must not be mistaken for the pellicle formed by Saccharomyces mycoderma. The latter prepares the medium for the action of the Bacterium aceti. Bacterium Pasteurianum, Hansen. Morpho- logically similar to Bacterium aceti, but the cells contain * Zoph, Die Spaltyilze. 1885. 278 BACTERIOLOGY. a starch-like substance, which is turned blue by iodine. They occur in beer-wort. Bacterium liodermos (Bacillus liodermos, Fltigge. Potato bacterium}. Short rods with rounded ends, motile. On plate-cultivations the colonies appear as small white pellicles floating on liquefied gelatine. In test-tubes the gelatine is liquefied, and the growth sinks down in floccu- lent masses. They occur on potatoes, forming a smooth shining layer, which ultimately becomes crumpled. Bacterium multipediculum (Bacillus multipedi- culus^ Fliigge). Long slender rods. They form peculiar insect-like colonies on plate-cultivations. In test-tubes the appearance is less characteristic. They occur on potatoes, forming a dirty yellow growth. Bacterium ramosum liquefaciens (Bacillus ramosus liquefaciens, Fliigge). Rods, slowly motile. They form characteristic colonies on plate-cultivations. The colonies gradually sink down, forming a well-marked funnel with later an appearance of concentric rings. In test-tubes the funnel-shaped liquefaction sends ofF rays into the surrounding gelatine. They occur occasionally, contaminating cultivations. Bacterium Zopfii, Kurth. Cocci, i 1-25 /x, in diameter ; rods and threads. Cultivated in a streak on nutrient gelatine spread out on a glass slide, a peculiar development takes place. In twenty-four hours after inoculation threads have developed ; in forty-eight hours windings of the threads are observed, and in six days the threads have broken up into cocci (Fig. 84). They were observed in the intestine of fowls, especially in the contents of the vermiform appendix. Inoculation of rabbits was followed by negative results. Identical with Bacillus figurans (Crookshank). SYSTEMATIC AND DESCRIPTIVE. 279 Bacterium merismopedioides, Zopf. Forms threads i 1*5^ in thickness ; these subdivide into long rods, short rods, and finally into cocci. The cocci divide first in one and subsequently in two directions, forming characteristic groups, which FIG. 84. BACTERIUM ZOPFII. SUCCESSIVE CHANGES IN THE SAME THREAD, X 740 : (a) A thread-form, (6) breaking up into rod-forms, (c) into cocci [after Kurth]. appear like merismopedia. These groups may eventually consist of 64 x 64 cells or more, and ultimately form zooglcea. The cocci develop again into rods and threads. They were observed in water containing putrefying substances (River Panke Berlin).* Zopf, Die Spaltpilze. 1885. 280 BACTERIOLOGY. Bacterium Pfliigeri, Ludwig. Large, round cocci, mostly in zooglcea, and thread-forms composed of rods. They can be cultivated on boiled white of egg and potatoes. They were observed to produce phosphor- escence in putrid fish and meat. Gelatine not liquefied. Bacterium photometricum, Engelmann. Cells slightly reddish in colour, motile. The movements are stated to depend on light. Bacterium litoreum, Warming. Cells ellipsoidal, 2 6 ju, long, 1*2 2*4 p, wide, occur singly in sea-water, never as chains or zooglcea. Bacterium fusiforme, Warming. Cells spindle- shaped, with pointed ends, 2-5 p long and -5 '8 p. thick. Observed as a spongy layer on the surface of sea-water. Bacterium navicula, Reinke and Berthold. Cells spindle-form or ellipsoidal, including motile and non-motile forms. They have one or more dark spots, which may be coloured blue by iodine. They have been observed in rotting potatoes. Proteus vulgaris. This and the two following species have been isolated* from putrefying meat infusion, and are stated to be intimately connected with the process of putrefaction. In the history of their development coccoid, bacterioid, spindle- form, spirulinar, and involution forms have been described. In Proteus vulgaris the bacteria vary in size ; some measure 4 p, in length, and are almost as broad as long, and others vary from "94 1*25 p, long and "42 "63 p wide. They are actively motile, and cultivated on nutrient gelatine they convert it into a turbid, greyish-white liquid. If cultivated in a capsule containing 5 per cent, of nutrient gelatine, a few hours after inoculation the most characteristic * Hauser, Ueber Fdulniss-Bacterien. 1885. SYSTEMATIC AND DESCRIPTIVE. 28 1 movements of the individual bacilli are observed on the surface of the nutrient gelatine, although at this early stage no superficial liquefaction can be detected. Probably the movements depend upon the existence of a thin liquid layer, as they are not observed if the nutrient medium contains 10 per cent, of gelatine. Proteus mirabilis. Cocci -4 /u, -9 //,. They occur singly and in zooglcea, and sometimes in tetrads, pairs, chains, or as short rods in twos resembling Bacterium termo ; in fact, in all con- ceivable transition forms. Cultivated on nutrient gelatine they form a thick, whitish layer in con- centric circles, which in time liquefies the medium. Similar movements are observed in capsule-cultiva- tions as in Proteus vulgaris. Proteus Zenkeri. Cocci, -4 p, in twos like Bacterium termo, and short rods 1*65 p. long. Cul- tivated on nutrient gelatine no liquefaction results, but a thick, whitish-grey layer is formed. The bacilli are motile, and the same phenomena are observed on the solid medium as in the other forms. In cover-glass impressions most varied groupings of the bacilli are seen, and also developmental and involution-forms. The two following forms are only provisionally re- garded as distinct species. They are both probably phase-forms of protean species. Bacterium termo, Dujardin. Short cylindrical 282 BACTERIOLOGY. or oblong cells, 1*5 jj, long, *5 7 broad, generally occur- ring as dumb-bells. The cells have dark contents, invested by a thick membrane, and are provided with flagella, to which the characteristic movements are due (Plate I., Fig. 8). They are associated with putrefaction, invariably appearing in decomposing albuminous substances and liquids. A growth can be readily started by placing a piece of meat in water in a warm place. Cultivated in broth, they produce a turbidity, and on sterilised potatoes a slimy grey layer. Bacterium lineola. Cells 3-8 p 5-2 //, long, I '5 ft wide. They occur singly or in pairs, occasionally in zooglcea, but never in chains. The cells are provided with flagella, and contain strongly refringent contents. They resemble Bacterium termo in form and in movement, but are considerably larger. They occur in well water and stagnant water, and form slimy heaps on rotting potatoes, and zooglcea and pellicles on various infusions. Cultivated on nutrient agar-agar they form a semi-trans- parent growth (Plate X., Fig. i). Genus //. Spirillum. SPECIES. ASSOCIATED WITH DISEASE : /Spirillum Obermeieri In man / Spirillum cholerae Asiaticae ^Spiri Spirillum Finkleri T i / Spirillum sputigenum In animals .{ S J irillum tyrogenum UNASSOCIATED WITH DISEASE : Spirillum rubrum Spirillum plicatile Spirillum serpens Spirillum tenue Spirillum undula Spirillum volutans Spirillum Rosenbergii Spirillum attenuatum Spirillum leucomelaneum . Pathogenic. j" Pathogenic in man (?), pos- .-! sibly only saprophytic. Pa- \ thogenic in animals. /Saprophytic in man. Patho- '\ genie in animals. ./Saprophytic. Pathogenic in \ animals. Chromogenic saprophyte. Simple saprophytes. SYSTEMATIC AND DESCRIPTIVE. 283 Spirillum Obermeieri (Spirochcete Obermeieri, Cohn. Spirillum of Relapsing Fever]. Threads similar to the Spirillum plicatile. In length mostly 1 6 40 p., with screw- curves regular (Plate I., Fig. 19). They move very rapidly, and exhibit peculiar wave-like undulations. They have been observed in the blood of patients suffering from relapsing fever,* but never in the secretions. They only occur during the relapses, and are absent during the non- febrile intervals. Their number is variable, but usually is strikingly great. Outside the body, in blood serum and 50 per cent, salt solution, the threads preserve their movements. From analogy to the Spirillum plicatile it is presumed that these threads are composed of articulated rods and cocci. Monkeys have been inoculated with success from man,t but inoculations of mice, rabbits, sheep, and pigs give negative results. The spirilla were found in the blood of the in- oculated monkeys in great numbers, and also in the brain, lung, liver, kidney, spleen, and skin ; and are believed to be the cause of the disease. METHODS OF STAINING THE SPIRILLUM OBERMEIERI. In cover-glass preparations of blood the spirilla stain strongly with fuchsine, methyl-violet, gentian-violet, or bismarck-brown. In sections, brown aniline stains have been recommended. * Obermeier, Med. Centralb. 1873. + Carter, Lancet. 1879 and 1880. Koch, Cohris Beitrage. 284 BACTERIOLOGY. Spirillum cholerse Asiaticae (Comma-bacillus, Koch). Curved rods, spirilla, and threads (Plate I., Fig. 1 8). The curved rods or commas are about half the length of a tubercle-bacillus. They occur isolated, or attached to each other, forming S -shaped organisms or longer screw-forms ; the latter resem- bling the spirilla of relapsing fever. Finally they may develop into spirilliform threads. In old cul- tivations threads are found with bulgings or irregu- larities, which are called involution-forms (Plate I., FIG. 85. COVER-GLASS PREPARATION OF THE EDGE OF A DROP OF MEAT INFUSION, containing a pure cultivation of comma-bacilli, with (a) spirilli- form threads, X 600 [after Koch]. Fig. 35).* The commas are actively motile ; their movements and development into spirilla may be studied in drop-cultivations (Fig. 85). In plate- cultivations, at a temperature of from 16 20 C., the colonies develop as little specks (Fig. 86), which begin to be visible after about twenty-four hours. Examined with a low-power, and a small diaphragm, these colonies have the following characteristics. They appear as little masses, granular, and of a very faintly yellowish-red tinge, which have liquefied * Compare also Van Ermengem, Recherches sur le Microbe du Choi. Asiat. 1885. SYSTEMATIC AND DESCRIPTIVE. 285 the gelatine, and sunk down to the bottom of the resulting excavations (Fig. 87). In test-tubes of slightly alkaline nutrient gelatine (10 per cent.), the appearance of the growth / (X, FIG. 86. COLONIES OF COMMA-BACILLI ON NUTRIENT GELATINE, NATURAL SIZE [after Koch], is very striking. It commences to be visible in about twenty-four hours. Liquefaction sets in very slowly, commencing at the top of the needle track around an enclosed bubble of air, and form- ing a funnel continuous with the lower part of the FIG. 87. COLONIES OF KOCH'S COMMA- BACILLI, X 60 ; from a nutrient-gelatine plate-cultivation. growth (Plate. IV., Fig. i) ; the latter preserves for several days its resemblance to a white thread (Figs. 93 and 94).* In about eight days, however, liquefaction takes place along the whole of the needle track. On a sloping surface of agar-agar the cultivation * From Remarks on Comma-bacillus of Koch. Lancet. 1885. 286 BACTERIOLOGY. develops as a white, semi-transparent layer, with well- defined margin. In potato-cultivations the microbe will only grow at the temperature of the blood (37 C.), forming a slightly brown, transparent layer. Inoculation of a cultivation of the bacillus in the duodenum of guinea-pigs, with * and without f 9 ttlj Pi&v- Fig. 88. Fig. 89. FIG. 88. COVER-GLASS PREPARATION FROM. THE CONTENTS OF A CHOLERA INTESTINE, X 600. (a) .Remains of the epithelial cells; (b) Comma- bacillus; (c) Group of comma-bacilli [after Koch], FIG. 89. COVER-GLASS PREPARATION OF CHOLERA DEJECTA IN DAMP LINEN (two days old), X 600. Great proliferation of the bacilli with spirilla (a) [after Koch]. ligation of the bile-duct, has given positive results. More recently these results have been confirmed by the following method: Five ccm. of a 5 per * Nicati et Rietsch, Com. a r Academic de Medecine. 1884. f Van Ermeng-em, Le Microbe du Cholera Asiatique. 1885. SYSTEMATIC AND DESCRIPTIVE. 287 cent, solution of potash were injected into the stomach of a guinea-pig, and twenty minutes after TO ccm. of a cultivation of comma-bacilli diffused in broth were similarly introduced. Simultaneously with the latter, an injection of tincture of opium was made into the abdominal cavity, in the propor- FIG. 90. SECTION OF THE Mucous MEMBRANE OF A CHOLERA INTESTINE, X 600. A tubular gland (a) is divided transversely ; in its interior (6) t ;A and between the epithelium and the basement membrane (c) are numerous comma-bacilli [after Koch]. tion of i ccm. for every 200 grammes' weight of the animal. Those who have had success with inoculation experiments maintain that choleraic symptoms were produced without any trace of peritonitis or putrid infection, and that the comma- bacilli of Koch were again found in the intestinal contents, and fresh cultivations established. On the other hand, these results have been 288 BACTERIOLOGY. disputed, the fatal effects of the inoculation attri- buted to septicaemic poisoning, and the proliferation of the bacilli considered to be dependent upon an abnormal condition of the intestines induced by the injection of tincture of opium.* It is, however, very Fig. 91. Fig. 92. Fig. 93. Fig. 94. PURE CULTIVATIONS IN NUTRIENT GELATINE. Fig. 91. Tinkler's bacillus, twenty-four hours old. Fig. 92. two days old. . Fig. 93. Koch's cholera-bacillus, twenty-four hours old. Fig. 94. two days old. probable that these organisms, like several others which have been isolated from intestinal discharges, are truly pathogenic in the lower animals. The comma-bacilli were found in the superficial necrosed * Klein, Brit. Med. Journal, and Micro-organisms and Disease. 1885. Lankester, Nature, xxxi. ; Nineteenth Century. July, 1885. Klein and Gibbes, "An Inquiry into the Etiology of Asiatic Cholera." Bluebook,i%%$. SYSTEMATIC AND DESCRIPTIVE. 289 layer of the intestine, in the mucous flakes and liquid contents of the intestinal canal of cases of Asiatic cholera* (Figs. 88, 90). It is stated that they were also detected in a -^ tank which contained the water v ^ ffi, -^ ( supply to a neighbourhood l/% ^ ^ S where cholera cases occurred ; / I v C ^ ^J but comma-shaped organisms 1 f ^ \^^ "" are commonly present in ^ - .. FIG. 95. COMMA -SHAPED Sewage - Contaminated Water ORGANISMS WITH OTHER /T ^. x r i .1-1. BACTERIA IN SEWAGE- (Flg. 95). The COmma-baCllll CONTAMINATED WATER, are aerobic, and their develop- ment is arrested by deprivation of oxygen. They are destroyed by drying and the presence of various antiseptic substances. They are distinguished from all other comma-shaped organisms by the test of cultivation. The entirely different results obtained * At a meeting of the Physiological Society, May i5th, 1886, at Cambridge, a preliminary communication was made upon the investi- gations in Spain referred to in the first edition of this work. The observations made by Roy, Brown, and Sherrington rather tend, in the opinion of the author, to confirm Koch's views. Comma-bacilli were found to be present, in some cases, in enormous numbers, and the frequency of their occurrence led these observers to believe that they must bear some relation to the disease. At the same time, as they failed to find them in all cases, they regarded the existence of a causal relation as not proven. They failed to find the Naples bacterium or the small straight bacillus noted by Klein ; but they drew attention to certain peculiar mycelium-like threads in the mucous membrane of the intestines. These organisms, however, judging from a preparation stained with methylene-blue which was exhibited at the meeting, appeared to the author to much more closely resemble some of the involution-forms of the comma-bacillus, filaments a masses globuleuses, figured by Van Ermengen, than anything else he had seen. Yet assuming these peculiar structures to belong as described to some species of Chytridiacese, it is very 19 BACTERIOLOGY. in the case of the comma-bacilli of cholera nostras (Figs. 91 to 94), renders a thorough acquaintance with these bacilli of the greatest importance as an aid in diagnosis. METHODS OF STAINING THE COMMA-BACILLI OF KOCH. In cover-glass preparations they may be well stained in the ordinary way with an aqueous solution of methyl-violet or fuchsine, or by the rapid method, without passing through the flame (p. 67, Babes' method). Nicati and Rietsctis method* A small quantity of the stools or of the scraping of the intestinal mucous membrane is spread out on a glass slide and dried, then steeped during some seconds in sublimate solution or in osmic acid (i 100). It is then stained by immersion in fuchsine-aniline solution ( i or 2 grammes of Bale fuchsine dissolved in a saturated aqueous solution of aniline), washed, dried, and mounted in Canada balsam. In sections of the intestine their presence may be -demonstrated by (a) Koch's method, ,f Sections of the intestine, which must be well hardened doubtful whether they can be considered to have any significance. Methylene-blue has been employed by Koch and others, including the author, for staining sections of the intestine from cholera cases, and had they been constantly present it is hardly possible that such striking objects could have been overlooked. Again, we must bear in mind that hyphomycetous fungi occasionally have been found to occur saprophytically in the intestinal canal as well as in the lungs, external auditory meatus, and elsewhere. We must wait, before expressing a more definite opinion, until the report of these observers is published in full. * Brit. Med. Journal, Sept., 1885. t Berliner Klinische Woch., No. 31. SYSTEMATIC AND DESCRIPTIVE. 29 1 in absolute alcohol, are left for twenty-four hours in a strong watery solution of methylene-blue, or for a shorter time if the colour solution is warmed. Then treated in the usual way. (ft) Babes' method* Sections, preferably from a recent case of cholera, and made as soon as possible after death, are left for twenty-four hours in a watery solution of fuchsine (fabrique de Bale), then washed in distilled water faintly acidulated with acetic acid, or in sublimate solution (i 1000), passed rapidly through alcohol and oil of cloves, dried with filter paper, and pre- served in Canada balsam. Spirillum Finkler- Prior (Comma-bacillus in Cholera nostras). Curved rods thicker than the comma-bacillus of Koch, and spirilla. The colonies on plate-cultivations (Plates XII and XIII.) are very much larger than those of the comma-bacillus of Koch of the same age. They have the faintest yellowish-brown tinge, a well-defined border, and a distinctly granular appearance. They liquefy nutrient gelatine very rapidly, so that the first plate of a series is, as a rule, completely liquefied on the day following inoculation, and the second plate in two or three days more. In a test-tube cultivation in nutrient gelatine the appearances are especially characteristic ; the gelatine is very rapidly liquefied along the whole track of the needle, so that the cultivation resembles a conical sack, or the finger of a glove turned inside out * Cornil and Babes, Les Bacteries, p. 458. 1885. 292 BACTERIOLOGY. (Figs. 96 and 97). On a sloping surface of nutrient agar-agar a white moist layer forms very quickly. On potatoes they grow at the ordinary tem- perature of the air, producing a brownish layer and corrosion of the surface of the potato. They were discovered in the evacuations of cases of cholera nostras, and were claimed at first to be identical with the comma-bacillus of Koch. By the test of cultivation they are now ascertained to be distinct. They also have been shown to be pathogenic.* Spirillum spu- tigenum, Lewis. Curved rods very similar to the comma-bacilli of Koch. Many have failed with repeated attempts to cultivate these bacilli, and, therefore, maintain that they are quite distinct biologically from the spirilla * Finkler and Prior, Erganzungshefte zum Centralblatt fur Allgemeine Gesundheitspflege, Erster Band. 1885. Fig. 96. Fig. 97. PURE CULTIVATIONS OF THE SPIRILLUM FINKLER-PRIOR IN NUTRIENT GELATINE. Fig. 96. Fig. 97- In twenty-four hours. In thirty-six hours. SYSTEMATIC AND DESCRIPTIVE. 293 associated with Asiatic cholera. Klein asserts that they can be cultivated in an acid nutrient gelatine, and that they are identical with Koch's comma-bacilli in their mode of growth. This has not FIG. 58. SPIRILLUM SPUTIGENUM. Occurring ,with spirochaeta denticola, leptothrix-filaments, micrococci, and bacteria in a scraping from a carious tooth, X 1200. been confirmed. They occur with other bacteria in saliva, and in scrapings from carious teeth (Fig. 98). Spirillum tyrogenum, Deneke. Curved rods, slightly smaller than Koch's comma-bacilli, with a great tendency to form long spirillar threads FIG. 99. SPIRILLUM TYROGENUM. From a cultivation in nutrient gelatine, X 12.00. (Fig. 99). The colonies on plate-cultivations are sharply defined, and of a greenish-brown colour After a time they liquefy the gelatine, but the liquefaction is much more marked than in colonies of Koch's commas of the same age, though not so rapid as in the case of the commas of cholera nostras. In test-tubes of nutrient gelatine a turbid 294 BACTERIOLOGY. liquefaction occurs along the needle track, and on the surface of nutrient agar-agar a yellowish-white layer develops. Inoculation of potatoes gives no result. Administration of the bacilli by the mouth, in the manner employed for testing the pathogenic effect of Koch's bacilli, produced a fatal result in a few cases ; on the other hand, injection into the duodenum failed entirely. The pathogenic proper- ties may be, therefore, considered as not yet established. They were isolated from old cheese. Spirillum rubrum, Esmarck. Curved rods, spirilla, and spirochetse. They are actively motile. FIG. 100. SPIRILLUM PLICATILE (Marsh-spirochsete). From sewage- contaminated water, x 1200. The growth on artificial nutrient media is extremely slow. In test-tube cultivations they grow along the track of the needle, forming a filament of a wine- red colour, and without causing liquefaction. The growth on the surface of the gelatine is colourless. In broth-cultivations long spirillar threads are formed. They were isolated from the putrid tissues of a mouse. Spirillum plicatile, Ehrenberg (Marsh-spiro- chcete). Thin threads, 2-25 //, in breadth, with numerous narrow windings, no 125 /x, long, occur- ring also in spirulinar forms. The threads have SYSTEMATIC AND DESCRIPTIVE. 295 primary and secondary windings ; the former are in each example of equal size, but the latter are often irregular ; their ends are cut off bluntly, and they exhibit rapid movement. They occur abundantly in marsh-water in summer, and can be obtained by allowing algae to decompose in water (Fig. 100). On cultivation the threads break up into long rods, short rods, and finally cocci. This change is ren- dered visible by making cover-glass preparations, and staining with aniline dyes. FIG. 101. SPIRILLUM UNDULA, X 1500. The following may be provisionally described as distinct species > though they are probably the spiral phase-forms of protean species* Spirillum serpens, Muller ( Vibrio serpens]. Threads n 2& p. long, '8 1*1 p. thick, with three or four windings. They are actively motile, often united into chains, or forming swarms, and are abundant in stagnant liquids. Spirillum tenue. Very thin threads, with at least i, usually 2 5 spirals. Height of a single screw is 2 3 p., and the length of spiral, therefore, 4 15 p.. They are very swiftly motile, and often occur in felted dense swarms in vegetable infusions. Spirillum undula. Threads r 11-4 p. thick, 912 /xlong; spirals 4*5 p. high ; each thread has i 3 spirals. They are actively motile, and possess at each end a flagellum. They occur in various infusions (Fig. 101). Spirillum volutans, Ehrenberg. Threads 1-52 p. thick, 25 30 p, long ; tapering towards their extremities, which are rounded off. They possess dark granular contents. Each thread has 2| 3^ windings or spirals, whose height is 9 13 p.. They have a flagellum at each end, and are sometimes motile, sometimes not. They are found in various infusions and water of marshes. 296 BACTERIOLOGY. Spirillum Rosenbergii. Threads with i 1 windings; 412 jj. long; 1-5 2-6 p. thick. They are colourless, but the contents include strongly refractive sulphur granules. Also spirals 67-5 p in height, which are actively motile, are found in brackish water. Spirillum attenuatum, Warming. Threads much attenuated at the ends, which consist usually of three spirals. The middle spiral is about ii p. high, and 6 /x in diameter ; and the end ones 10 n high, and 2 \i. in diameter. They are found in brackish water. Spirillum leucomelaneum, Koch. A rare form observed in water covering rotting algae. Dark and glass-like spaces alternate in the spirillum, resulting from a regular arrangement of the dark granular contents. Genus III. Leuconostoc. SPECIES. UNASSOCIATED WITH DISEASE: Leuconostoc mesenteroides . . Zymogenic saprophyte. Leuconostoc mesenteroides, Cienkowski (Gomme de sucrerie, Froschlaichpilz, Frogspawii jungus). Cells singly, in chains, and in zooglcea, surrounded by a thick gelatinous envelope (Fig. 102). The life-history has been very thoroughly investigated.^ The spores, r8 2 p. in diameter, are of a round or ellipsoidal form, with thick mem- brane and shining contents. The outer membrane- layer bursts, and a middle lamella oozes out, and forms a thick gelatinous envelope, while the inner layer remains adherent to the plasma. Thus the spore-germination leads to the formation of a * Cienkowski, Die Gallertbildungen d. Zuckerrubensaftes, 1878 ; and Van Tieghem, " Sur la Gomme de sucrerie," Ann. Sc. Nat. 1879. SYSTEMATIC AND DESCRIPTIVE. 297 coccus with a gelatinous envelope. The coccus then elongates into a short rod-form, and the gelatinous envelope becomes ellipsoidal. The rod divides into two cocci, and each of these lengthens into a rod and divides. By repetition of this process FIG. 102. LEUCONOSTOC MESENTEROIDES. i. Spores. 2. Spores after germination, showing gelatinous envelope. 3> 4> 5> 6. Increase by division. 7. Glomerular form of zooglrea. 8. Section of an old mass of zooglcea. 9. Cocci chains with arthro- spores [after Tieghem and Cienkowski]. a chain of cocci results, encased in a cylindrical or ellipsoidal envelope. The chains increase in length, become twisted up, and eventually fall apart into pieces of various lengths. In nourish- ing liquids a great number of little masses are formed, which adhere together, and produce 298 BACTERIOLOGY. pseudo - parenchymatous structures. These latter may join together, forming still larger agglomera- tions. The masses of zoogloea are of almost a cartilaginous consistency, and admit of sections being made with a razor. After a long time the envelope liquefies, and the cocci are set free ; the latter introduced into fresh nourishing media de- velop new colonies. In the chains some of the cocci become enlarged without changing their form. These acquire the properties of spores, and are called arthrospores (p. 161). This micro-organism occurs occasionally in beet-root juice and the molasses of sugar-makers, forming large gelatinous masses resembling frog- spawn. The vegetation is so rapid, that forty-nine hectolitres of molasses, containing 10 per cent, of sugar, were converted within twelve hours into a gelatinous mass ; consequently, it is a formidable enemy of the sugar manufacturers. SYSTEMATIC AND DESCRIPTIVE. 299 Genus IV. Bacillus. SPECIES. ASSOCIATED WITH DISEASE: /Bacillus leprae Bacillus in syphilis In man Bacillus typhosus Bacillus malariae .... Bacillus of choleraic diarrhoea from meat-poisoning Bacillus pyrogenes fcetidus . Bacillus in septicaemia in man Bacillus in gangrenous septi- caemia . (Bacillus tuberculosis . : _ .Bacillus anthracis ^Bacillus mallei . . . . Bacillus malignant oedema . in ctiiiiimia^ Bacillus of septicaemia of mice . Bacillus of ulcerative stomatitis. in the calf . Bacillus in swine-typhoid . Bacillus of swine-erysipelas Bacillus in tetanus Bacillus alvei .... UNASSOCIATED WITH DISEASE : Bacillus pyocyaneus . Bacillus ianthinus Bacillus cyanogenus Bacillus acidi lactici . Bacillus Fitzianus Bacillus subtilis . Bacillus ulna Bacillus tumescens Bacillus megaterium . Bacillus figurans . Bacillus mycoides Bacillus tremulus. Bacillus of jequirity Bacillus caucasicus Bacillus dysodes . Bacillus Hansenii Bacillus erythrosporus Bacillus septicus . Bacillus saprogenes Bacillus fcetidus . Bacillus putrificus coli Bacillus coprogenes fostidus Bacillus aerophilus Bacillus mesentericus fuscus Bacillus mesentericus vulgatus Bacillus phosphorescens Pathogenic. Pathogenic (?) ; possibly only saprophytic. i> Pathogenic in animals. Pathogenic. Saprophytic in man, patho- genic in animals. Saprophytic. Pathogenic. . j-Chromogenic saprophytes. ' l-Zymogenic saprophytes. Simple saprophytes. Bacillus leprae, Hansen Fine slender rods, 4 6 //, long, and less than i p. wide, occasionally 3OO BACTERIOLOGY. pointed at both ends, some clearly motile, and others not. In tissue sections they have a beaded appear- ance (Fig. 103). Spore-formation has been de- scribed. They have been cultivated artificially on blood serum and glycerine-agar-agar. Inoculation experiments on monkeys and other animals have failed to produce the disease ; though in cats and rabbits there have been indications of success.* The bacilli occur in enormous numbers in tubercular leprosy in the nodules of the skin (Plate XX., FIG. 130. LEPROSY BACILLI FROM A SECTION OF SKIN, x 1200. Figs, i and 2), and of the mucous membrane oi the mouth, palate, larynx, etc.f They occur also in the liver, spleen, testicles, lymphatic glands, and kidneys (Plate III., Fig. 2); and in the interstitial tissue of the nerves in anaesthetic leprosy. They probably spread by the lymphatics, and are not found in the blood. In their behaviour to staining reactions they are similar to the bacillus of tubercle, except that they stain much more readily. METHODS OF STAINING THE BACILLUS OF LEPROSY. Cover-glass preparations may be made in the ordinary way, or by a special method, which consists in clamping a * Damsch, Virchoixfs Archiv, Bd. 92, Heft i. t Thin, Med. Chir. Trans. Lond., 1883 ; Brit. Med. Journal, No. 129, 1884, and Steven, Brit. Med. Journal, No. 1281, 1885. London.Pabliahedbv H. SYSTEMATIC AND DESCRIPTIVE. 30! nodule with a pile-clamp, until a state of anaemia of the tissue is produced. On pricking with a needle or sharp knife a drop of clear fluid exudes, from which cover-glass preparations may be made.* Cover-glass preparations and sections may be stained by Ehrlich's method (p. 78), or with Neelsen's solution and decolorising with dilute sulphuric acid, or sections by the following process : Method of Babes.^ Preparations are stained in a solution of rosaniline hydrochlorate in aniline-water. Decolorise in 33 per cent, hydrochloric acid, and after-stain with methylene-blue. Bacillus in syphilis, Lustgarten.J Rods re- sembling the bacilli of leprosy and tuberculosis, 3 4 //, long, '8 ft thick. Two or more colourless, ovoid points in the course of the rod are visible with a high power ; it is thought that they are possibly spores. The bacilli are always found in the interior of nucleated cells which are more than double the size of leucocytes. They have been observed in the discharge of the primary lesion, and in hereditary affections of tertiary gummata. Some observers state that an identical bacillus is found in normal secretions, and others || have described a bacillus associated with specific lesions, which is stated to differ from the above in its behaviour towards staining reagents. METHOD OF STAINING THE BACILLUS OF SYPHILIS. Method of Lustgarten : Sections are placed for from twelve to twenty-four hours in the following solution, at the ordinary temperature of * Manson, Lancet. 1884. t Babes, Comfit. Rend, de I'Acad. d. Sc. 1883. \ Lustgarten, Die Syphilisbacillen. Mit4Tafeln. 1885. Alvarez et Tavel, " Recherches sur le Bacille de Lustgarten," Archiv de Phys. Norm, et Path.> 17 ; Klemperer, " Ueber Syphilis und Smegma Bacillen," Deutsche Med. Woch. 1885. || Eves and Lingard, Lancet, April loth, 1886. 302 BACTERIOLOGY. the room, and finally the solution is warmed for two hours at 60 C : Concentrated alcoholic solution of gentian-violet 1 1 Aniline water . . '. ' ', ;f ' : i ! . 100 The sections are then placed for a few minutes in absolute alcohol, and from this transferred to 1*5 per cent, solution of permanganate of potash. After ten minutes they are immersed for a moment in a pure concentrated solution of sulphurous acid. If the section is not com- pletely decolorised, immersion in the alcohol and in the acid bath must be repeated three or four times. The sections are finally dehydrated with absolute alcohol, cleared with clove-oil, and mounted in Canada balsam. Method of De Giacomi : Cover-glass preparations are stained with hot solution of fuchsine containing a few drops of perchloride of iron. They are then decolorised in strong perchloride of iron and after-stained with vesuvin or bismarck-brown. Method of Doutrelepont and Schiltz : Sections are stained in a weak aqueous solution of gentian-violet and after-stained with safranin. Bacillus typhosus, Eberth (Bacillus in typhoid fever). Rods, '2 //, broad, and forming filaments up to 50 /x long ; * or f rods, short, rounded at their ends, and occasionally constricted in the middle ; some exhibiting spore-formation. These bacilli have been observed in inflamed Peyer's glands, in the spleen, mesenteric glands, and the lungs in fatal cases of typhoid fever. More recently J a bacillus has been cultivated on several plates of * Kleb's A rch. f. Experimental Pathol. 1 880. t Eberth, Vir chow's Archiv, Bd. 83. J Gaffky, MittheiL a. d. K. Gesundheitsamte. 1884. SYSTEMATIC AND DESCRIPTIVE. 303 gelatine which were inoculated from different spleens. After twenty-four hours the course of the inoculation streak became visible, and in forty-eight hours a dis- tinct whitish growth had developed. With a low power this was found to consist of numerous colo- nies of a yellow-brownish colour. The gelatine was not liquefied. The rods varied in length (Fig. 104), were capable of development into threads, FIG. 104. BACILLUS TYPHOSUS FROM A POTATO-CULTIVATION, X 1500. and were motile. They can be cultivated on pota- toes at 37 C. They grow well also on blood serum, forming a whitish-grey, somewhat transparent layer.. Spore-formation occurs at the ends of the rods. It is stated that inoculation experiments have been made in some cases with success.* METHODS OF STAINING THE BACILLUS OF TYPHOID FEVER. To stain the bacilli in the tissues the method of Gram can be employed, or the sections may be left for twenty- four hours in methylene-blue. Koch recommends bis- marck-brown. To colour the spores cover-glass prepara- tions and sections must be left for several days in the fuchsine solution employed in the method of Ehrlich (p. 78) ; or the solution may be warmed, and in the case of cover-glasses, even raised to boiling-point. They * Fraenkel and Simmonds, Die Atiolog. bedeutung des Typhus- bacillus. 1886. 304 BACTERIOLOGY are then decolorised with nitric acid, and after-stained with methylene-blue. Bacillus malariae, Klebs (Bacillus in intermittent fever}. Rods, 2 7 /x, long, which grow into twisted threads. Spore-formation takes place in the centre, or at either end (Plate I., Fig. 14). Inoculated in rabbits they were stated to produce a febrile disorder analogous to malarial fever,* and in the spleen and marrow the threads and spores of the bacilli were found in abundance. Bacilli with end-spores have been discovered also in the blood of patients suffering from malaria, t The bacilli were first described as present in the soil of the Roman Campagna. According to more recent observations, more importance is to be attached to peculiar amoeboid bodies and motile filaments present in the blood of cases of malaria (see Appendix D). Bacillus of choleraic diarrhoea from meat- poisoning, Klein.J Rods from 3 9 /x, in length, I '3 //, wide, rounded at their extremities, singly or in chains of two. Spore-formation occurs, the spores being I /x, thick, oval, and situated in the centre or at the end of the rod. Feeding with the bacilli and inoculation produced positive results. At the autopsy, pneumonia, peri- tonitis, pleuritis, enlargement of the liver and spleen, and haemorrhage were observed, and bacilli were present in the blood and exudations of the animal. They oc- curred in the blood and juices, and especially in the glomeruli of the kidneys, of several fatal cases of choleraic diarrhoea. * Klebs and Tommasi Crudeli, Archiv f. Experimental Pathol. 1879. t Marchiafava, ibid. \ Klein, p. 87. SYSTEMATIC AND DESCRIPTIVE. 305 Bacillus pyogenes foetidus, Passet. Small rods, with rounded ends of about 1*45 p. in length, and -58 p, in width ; often in twos, or linked together in chains. They are motile, and spore-formation occurs. When cultivated in nutrient gelatine, a greyish, veil-like growth forms on the surface. In plate-cultivations white points appear after twenty-four hours, which develop into greyish spots, and these enlarging coalesce into a layer. In nutrient agar-agar the cultivation resembles the growth on gelatine. On blood serum a moderately thick greyish- white streak develops, and on sterilised potato an abundant, shining, brownish culture. From all these media a putrid odour emanates, but no smell is detected from a culti- vation in milk. Inoculated into mice and guinea-pigs abscesses are produced or death from septicaemia results. They were isolated from putrid pus. Bacillus in septicaemia of man, Klein.* Rods singly or in chains, I 2-5 p long, '3 -5 //, wide, which were observed in the blood-vessels of the swollen lym- phatic glands. They are possibly identical with the following : Bacillus in gangrenous septicaemia, Ar- loing and Chauveau. Short rods, possessing spores, were observed around wounds in gangrenous septicaemia, and considered to be the cause of the gangrene. Bacillus tuberculosis, Koch.f Rods, 2 4 p* and occasionally 8 p. long, very thin, and rounded at the ends. They are straight or curved, and frequently beaded (Fig. 105), and occur singly in pairs, or in bundles. They are found in the cells of tubercles, especially in the interior of giant cells. In the latter they are often accompanied * Klein, Micro-organisms and Disease. 1885. t Koch, Berl. Klin. Woch., No. 15, 1882 ; and Mittheil. aus dem Kaiserlich. Gesundheitsamte, "^Etiologie der Tuberkulose." 20 o O6 BACTERIOLOGY. with grains which exhibit the same colour reaction (Plate XXL, Fig. i). They are non-motile. Spore-formation has been described (see p. 164). A good medium for cultivation is solid blood serum of cow or sheep, with or without the addition of gelatine ; and the most favourable tem- perature for their development is 37 38 C. The growth takes place very slowly, and only between the temperatures of 30 and 41 C. In about eight PIG. 105. BACILLUS TUBERCULOSIS, FROM TUBERCULAR SPUTUM, STAINED BY EHRLICH'S METHOD, X 2500. From Photographs. or ten days the growth appears as little whitish or yellowish scales and grains (Plate XVI 1 1., Fig. i). The bacillus can also be cultivated in a glass capsule on blood serum, and the appearances of the growth studied under the microscope. The scales or pellicles are then seen to be made up of colonies of a perfectly characteristic appearance, which may be still further studied by making a cover-glass impression (p. 69, and Plate XVIII., Fig. 4). They are then seen to be composed of bacilli, arranged more or less with their long axis SYSTEMATIC AND DESCRIPTIVE. 307 corresponding with that of the colony itself, and with an appreciable interval between the individual bacilli. The colonies themselves appear as fine curved lines, the smallest being mostly S-shaped. Longer colonies have serpentine twistings and bendings, which often recall the curves of fancy lettering. The ends of the lines run to sharp points, but the middle of the growth is spindle-formed. The youngest colonies are extremely delicate and narrow, but the older colonies increase in size, are thicker across, and, blending with each other, gradually obliterate the characteristic appearances ; a lamellated growth results, which increases, and gives the appearance to the naked eye of the scale or pellicle already described. The blood serum is not liquefied unless putrefactive bacteria contaminate the culture. A fresh tube can be inoculated with one of these little scales, and a new generation started. The scales gradually increase in size, and consist entirely of bacilli. In about three to four weeks the cultiva- tion ceases to increase, and it is then necessary to inoculate a fresh tube. Glycerine-agar-agar is even a better medium than blood serum (Fig. 106). A relatively small portion of a cultivation inocu- lated into the subcutaneous tissue, into the peritoneal or pleural cavity, into the anterior chamber of the eye, or directly into the blood stream, produces after three or more weeks artificial tuberculosis in guinea-pigs and rabbits. Dogs and cats can also be infected by experimental inoculation. 308 BACTERIOLOGY. The appearances observed at the autopsy are, swollen lymphatic glands in the neighbourhood of the inoculation, followed by softening and abscess ; enlargement of the spleen and liver, with for- mation of caseous tubercles ; tuberculosis of the lungs, bronchial glands, and peritoneum. After inoculation of the eye, grey tubercles appear on the iris, and undergo enlarge- ment and caseation, followed by tuberculosis of the eyeball and organs generally. The ba- cilli appear to be the direct cause of tuberculosis, and the presence of the bacillus in the sputum of patients is regarded as a distinctive sign of the ex- istence of this disease. The detection of the bacillus has, con- sequently, become a test which is daily applied by physicians in forming clinical diagnoses. The bacilli are found in all tubercular growths of man, FIG. 1 06. From a photograph of a monkeys, cattle (Perlsuckt\ birds, cultivation of the tubercle- . bacillus on giycerine-agar- and many other animals, and agar; six months old. n Q{ tuberculosis, in rabbits, guinea-pigs, cats, etc. (Plate XXL, Fig. 2). In man the bacillus can be detected in the tissues, in the sputum, in the blood, and in the urine.* * Babes, Centralbl.f. d.Med. Wtssensch., 1883, p. 145. PLATE 21 fajcwg p. 308. Fig 2. Zdqar Cryficskank fa.itpm&. London.Ribli3kedbv H.Jt.Lewi3,436,Gaver Streeb . olca Day IfSon PLATE 22. foil plaute 21. Figl. Fig 2. Londo^Puhlialiedby H.KLevria,136.Goww Street . Yincenl Bnoka.Day i,Son..'UtK . SYSTEMATIC AND DESCRIPTIVE. 309 Tuberculosis may also be produced by inhalation and feeding experiments (p. 137). The channels of infection in man are also most probably the pulmonary or intestinal mucous membranes. The possibility of inoculation of skin wounds is open to doubt. The bacilli or their spores are inhaled from the air, or taken in with food. As a relatively high temperature is required for their growth, they cannot thrive outside the animal body in cold climates. Morphologically identical bacilli have also been observed, but very sparsely, in sections of lupus. METHODS OF STAINING THE TUBERCLE BACILLUS. Numerous methods have been recommended for stain- ing the bacillus tuberculosis, each of which will be given in detail. Neelsens method is preferred by the author for both sections and cover-glass preparations. Koctis original method. Cover-glass preparations or sections are laid in Koch's solution (No. 23,^.) for twenty- four hours, or for one hour if the solution is warmed to 40 C. Rinse in water ; immerse in a watery solution of vesuvin for two minutes ; rinse again in water, and examine ; or, after rinsing in water, treat with alcohol, clove-oil, and Canada balsam. Ehrlichs method. Cover-glass preparations are allowed to float in a watch-glass, containing a solution of gentian- violet or fuchsine, added to aniline water. A saturated alcoholic solution of the dye is added till precipitation commences (10 ccm. aniline water, and 10 20 drops of the colour solution). The cover-glasses are left in the solution for about half an hour ; then washed for a few seconds in strong nitric acid (one part commercial nitric 310 BACTERIOLOGY. acid to two of distilled water), and rinsed in distilled water. After-stain with vesuvin or methylene-blue, rinse in water, dry and preserve in Canada balsam (Plate III., Fig. i). Sections and cover-glass preparations may be stained by this method, as described by Koch.* Saturated alcoholic solution of methyl-violet or fuchsine . . >'.& .< \ ' > 3'JQ 11 Aniline water . . ,- 9 \ . ,.. . - ... -.-. . 100 Absolute alcohol. . . .^ .. , 10 Preparations are left for twelve hours in this solution (colouring of the cover-glass preparations can be expedited by warming the solution). Treat the preparations with (i 3) solution of nitric acid a few seconds. Wash in alcohol (60 per cent.) for a few minutes (cover-glass preparations need only be rinsed a few times). After-stain with diluted solution of vesuvin or methylene- blue for a few minutes. Wash again in 60 per cent, alcohol, dehydrate in absolute alcohol. Clear with cedar-oil, mount in Canada balsam. Rindfleisch' s method. Prepare a solution composed of Saturated alcoholic solution of fuchsine I o drops Aniline water . . . *.'.' 2 drams. Pour it into a watch-glass, and float the cover-glass ; warm the watch-glass over a spirit-lamp until steam rises. Remove it from the flame, and set it aside for five minutes. Take out the cover-glass, and transfer it for a few seconds to acidulated alcohol (two drops of nitric acid in a watch-glass full of alcohol). Wash in distilled water, dry, and preserve in balsam. * Mittheil. aus dem Gesundheitsamte, Zweiter Band, 1884, p. 10. SYSTEMATIC AND DESCRIPTIVE. 311 After - stain, if necessary, with bismarck - brown, or methylene-blue. Weigert-Ehrlich method (vide p. 78). Orth's modification of Ehrlich's method. Stain by the method of Ehrlich, but decolorise with acidulated alcohol (one of hydrochloric to one hundred parts of 70 per cent, alcohol). Gibbes' method.* Stain cover-glass preparations in magenta solution (No. 22) for 15 20 minutes. Wash in (i 3) solution of nitric acid, until the colour is removed. Rinse in distilled water. After-stain with methylene-blue, methyl-green, iodine-green, or watery solution of chrysoidin, five minutes. Wash in distilled water till no more colour comes away. Transfer to- absolute alcohol for five minutes ; dry, and preserve in Canada balsam. Leave sections in the stain for half an hour, then treat with nitric acid, and wash with distilled water. Transfer to methylene-blue till deeply stained, wash again in distilled water, and then in spirit. Pass through absolute alcohol and clove-oil, and preserve in Canada balsam. Gibbes' new method. Cover-glass preparations are placed in the double staining solution (No. 16), which has been warmed in a test-tube, and, as soon as steam rises, poured into a watch-glass. They are allowed to remain for five minutes, and then are washed in methylated spirit till no more colour comes away, dried in the air or over a spirit- lamp, and mounted in Canada balsam. If the solution is used without warming, the cover-glasses must be left in it for an hour. Sections are treated on the same prin- ciples, but must be left in the solution for several hours. The crumpling of the sections by the action of nitric acid is avoided. Baumgarteris method. Cover-glass preparations of sputum are made as already described (p. 65), and im- mersed in a very dilute solution of potash (i 2 drops * Gibbes, Practical Pathology. 1883. 312 BACTERIOLOGY. of a 33 per cent, solution of potash in a watch-glass of distilled water). The cover-glass is pressed down on a slide, and examined with a high power. The bacilli can be thus examined in the unstained condition, and to avoid any mistake from confusion with other species, the cover- glass can be removed, dried, passed through the flame, and stained with a drop of an aqueous solution of fuchsine, or gentian-violet The putrefactive bacteria are stained, but the tubercle bacilli remain absolutely colourless. Baumgartens new method. A solution is prepared as follows : Drop 4 5 drops of concentrated alcoholic methyl-violet solution into a small watch-glass full of water, (a) Stain the sections in this solution, wash them in water, and decolorise in absolute alcohol (five to ten minutes), or, before treating with alcohol, immerse the sections for five minutes in a half-saturated solution of carbonate of potash. Pass through clove-oil, and mount in a mixture of Canada balsam, free from chloroform, and clove-oil (equal parts). The object of this process is to differentiate the tubercle bacilli from chance bacteria, in- asmuch as the tubercle bacilli gradually are decolorised by the clove-oil. (&) Sections stained in the above solution are placed for five minutes in alcohol, and then in a concentrated solution of bismarck-brown in I per cent, solution of acetic acid. The after-treatment may be conducted as already described. Neelseris method. Cover-glass preparations may be quickly stained in Neelsen's solution (No. 25) warmed in a watch-glass till steam rises. Sections are left for from five to ten minutes in the solution, and then washed in a watery solution of sulphuric acid (25 per cent.) ; rinsed in distilled water, and immersed in methylene-blue solution. After two or three minutes they are passed through alcohol and oil of cloves, and mounted in Canada balsam. Balmer-Frantzel method. Dissolve two grammes of freshly powdered gentian-violet in 100 grammes of SYSTEMATIC AND DESCRIPTIVE. 313 aniline-water. Immerse the sections for twenty-four hours, and treat as in Ehrlich's method. Ziehl's method. Stain with Ehrlich's method, but omit the nitric acid ; after-stain with methylene-blue. The latter replaces the stain of all bacteria except the tubercle bacillus. Lichtheiiris method. Concentrated solution of fuchsine or gentian-violet is diluted with distilled water, and the sections stained for thirty-six hours. Peters' method. Sections are stained for half an hour in fresh aniline-gentian-violet solution. Transfer to 20 ccm. of absolute alcohol for eighteen hours, the alcohol being renewed two or three times. Rinse in distilled water for one minute, and immerse for three minutes in a watery solution of aniline-yellow (aniline-yellow *2 dissolved in distilled water 10, filter). Wash in absolute alcohol, clarify with clove-oil, and preserve in Canada balsam. FrankeUs method. Sputum preparations are rapidly double-stained by the following method : Prepare a solution by adding concentrated alcoholic methyl-violet or fuchsine solution, drop by drop till opalescence arises, to 5 ccm. of aniline-water heated to 100 C. Float the prepared cover glasses two minutes in the warmed solu- tion. The process of after-staining and decolorisation is effected by placing the preparation for one to two minutes in one of the following solutions : for fuchsine-stained preparations, a saturated solution of methylene-blue in a mixture of Alcohol . . . . . 50 Distilled water . . . . 30 Nitric acid . . . . 20 which is filtered before use ; for preparations stained in methyl-violet, a saturated solution of vesuvin may be used in Alcohol . . . . . 70 Nitric acid . . 3 314 BACTERIOLOGY. which must be filtered before use. The sections are washed in water (or weakly acidified 50 per cent, alcohol), dried and mounted in the usual way. Pfuhl-Petri's method. The colouring solution consists of 10 ccm. of a saturated alcoholic solution of fuchsine added to 100 ccm. of water. Float the cover-glasses for two minutes in the solution, heated till steam rises. Wash for one minute in glacial acetic acid, rinse in water, and after-stain in an alcoholic or watery solution of malachite- green for a half or one minute. Rinse again in water. Dry, and examine in glycerine, or preserve in Canada balsam. SenkewitscH s method. Stain cover-glass preparations in concentrated fuchsine solution. When strongly coloured, wash out the stain for one to two minutes in alcohol, to which one drop of nitric acid has been added for every 10 ccm. Rinse in water, dry, and mount in Canada balsam. Kaatzer's method. Place the cover-glass preparations for twenty-four hours in a solution of over-saturated alco- holic gentian-violet, or, if warmed to 80 C., for three minutes. Decolorise in a solution consisting of Alcohol 90 per cent. . . . 100 ccm. Water ... t "' f . 20 ccm. Strong hydrochloric acid . . 20 drops. Rinse in 90 per cent, alcohol, and after-stain with concen- trated watery solution of vesuvin for two minutes ; wash again in distilled water, dry, and mount in Canada balsam. Ehrlictis method and eosin. The author has found that after sections have been stained with methyl-violet and bismarck-brown by Ehrlich's method, as described by Koch (p. 3 I o), they may with advantage be immersed in a weak alcoholic solution of eosin, then rinsed in clean absolute alcohol, clarified with clove-oil, and mounted in Canada balsam. The giant cells are then stained pink, while SYSTEMATIC AND DESCRIPTIVE. 315 their nuclei are brown and the bacilli blue (Plate XXL, Fig. i). Bacillus anthracis (Bacteridie du charbon, Bacil- lus of splenic fever, wool-sorters disease ', or malignant pustule). Rods, 5 20 //, long and i 1*25 //, broad, and threads ; spore-formation present. As a thorough knowledge of the life-history of this bacillus is of the greatest importance, inasmuch as it is without any doubt the actual cause of wide- spread disease, the various steps to be followed in a practical study of it will be successively treated in detail. Its morphological and biological charac- FIG. 107. BACILLUS ANTHRACIS, X 1200. From a preparation of blood from the spleen of an inoculated mouse. teristics have been very completely worked out, and it serves as an excellent subject for gaining an acquaintance with the various methods that should be employed in studying micro-organisms. It is found that a mouse inoculated with the bacillus or its spores will die iji from twenty-four to forty- eight hours, or more rarely in from forty-eight to about sixty hours. Examination after death. The details to be ob- served in the autopsy have already been described (p. 141). The spleen is found to be consider- ably enlarged, and may be removed (p. 142), and 3 I 6 BACTERIOLOGY. examined by making cover-glass preparations, inoculations, and subsequently sections. Cover-glass preparations. In cover-glass prepara- tions of the blood of the spleen the bacilli are found in enormous numbers. Preparations should be made similarly with blood from the heart and exuda- tions from the lungs, etc. In the last-mentioned the bacilli are present in very small numbers, or alto- gether absent. They should be examined both un- stained and stained (p. 65). The rods are straight, or sometimes curved, rigid, and motionless, and vary in size in different animals. They stain intensely with aniline dyes, and are then seen to be composed of segments with their extremities truncated at right angles ; between the segments a clear linear space exists, which gives them a characteristic appearance (Fig. 107). By double staining (p. 67), the rods are seen to consist of a membrane or hyaline sheath with protoplasmic contents (Fig. 46). Drop-Cultures A little of the blood from the spleen or heart is employed to inoculate the liquid medium, bouillon or blood serum. Several of these cultures should be prepared, and some of them placed in the incubator. Examined from time to time it will then be observed that the rods grow into long homogeneous filaments, which are twisted up in strands, and then untwisted in long and graceful curves. In a few hours they begin to swell, become faintly granular, and finally, bright, oval spores develop (Plate I., Fig. 28). The ff V i I \ \y/\ ' >' / Fgl. PL ATE 23. fouangp.316. 2. i'dqar fr:c<+ J v*.k rrc etpvra. /lSe.Gower Street . Vincent BnoTet.Dty \Son.ZLth. PLATE 24. foJlplcuie 23. , ^SMlfe-V -i^f^^^&- v^Sfe- / > 'TTTS^^TS?! <^ X-'V)*- a- * SfNiV^ -= Fig 2,'atg LondoaPahlishedby H..K,Lewis,136.Gower Street . Vincent Bnahs.'Day Say.Son..liA.. SYSTEMATIC AND DESCRIPTIVE. 345 streak on the surface, forming a feather-like culti- vation * (Fig- 122). Cultivated from the air. Identical with Bacterium Zopfii, (p. 278). Bacillus mycoides. Rods with rounded ends, and varying in length. Spore-formation present. In plate-cultivations the colonies have a charac- teristic appearance like tufts of wool or masses of mycelium. In test-tube cultivations there is the same mycelial appearance. Nutrient gelatine is liquefied. The bacillus is found in garden earth. Bacillus tremulus. Rods shorter and thinner than those of Bacillus subtilis. They are provided with a flagellum at both ends, and exhibit characteristic trembling and rotatory movements. Spores thicker than the bacillus, and often placed laterally. They were observed on rotting plant infusions, forming a thick slimy skin. Bacillus of jequirity, Sattler. Rods 2 4-5 //, long and '58 /x, thick. They can be cultivated on nutrient gelatine and blood serum. Infusion of jequirity containing the bacilli, or an artificial cultivation of the bacilli, inocu- lated into the conjunctiva of healthy rabbits produces severe ophthalmia. The poisonous principle is, however, believed to be a chemical ferment, abrin, and not the bacillus. Boiling, which does not destroy the spores of the bacillus, destroys the ferment, and cultivations started with these spores, though teeming with jequirity bacilli, are quite harmless."]" The bacilli occur in infusions of the beans of Abrus precatorius, or jequirity. Bacillus caucasicus, Kern. Rods forming two spores, one at each end, otherwise similar to Bacillus sub- tilis. They occur in the form of whitish lumps in company Described by the author, "Notes from a Bacteriol. Laboratory," Lancet. 1885. t Klein, Micro-organisms and Disease. 1885. 34-6 BACTERIOLOGY. with Saccharomyces mycoderma in the production of a drink " kephir " from cow's milk. The fermentation is not due to the bacillus. Bacillus dysodes, Zopf. Cocci,longand short rods, and spores. They were observed in bread, making it greasy and unfit for food, and generating a penetrating odour resembling a mixture of peppermint and turpentine. A great loss may result to bakers if the fungus is intro- duced with the yeast. Bacillus Hansenii, Rasmussen. Rods 2-8 6 p. long, *6 *8 JJL wide. Cultivated on sterilised potato in four days they form a chrome-yellow layer with an agree- able fruitlike smell. Two or three days later the growth dries, and changes to orange-yellow in colour ; later it passes to yellowish or brown, and forms at the same time spores i "j p. long, i'i p, wide. The colouring matter is insoluble in most reagents. The bacilli occur on nourishing solutions, malt infusion, broth, wine, which have been kept at 3 i to 3 3 C, as a yellow or whitish skin. Bacillus erythrosporus, Cohn. Motile rods and threads ; rods exhibiting spore-formation. They grow well in nutrient gelatine, colouring the medium green by transmitted light. They were found to form a pellicle on meat-extract-solutions and on rotting albuminous liquids. Bacillus septicus, Klein.* Rods varying in size, non-motile. They form threads or leptothrix filaments, and are rounded at the ends. They are anaerobic, and form spores independently of access of air. In a nourish- ing fluid they are overcome by the presence of micrococci y Bacterium termo or Bacillus subtilis. They occur in the soil, in putrid blood, and many putrid albuminous fluids, and occasionally in the blood-vessels of man and animals after death. Bacillus saprogenes, Rosenbach. Three rod- * Klein, Micro-organisms and Disease. 1885. SYSTEMATIC AND DESCRIPTIVE. 347 formed organisms have been described by Rosenbach as intimately associated with putrefactive processes. No. I. Large rods (Fig. 123), which form an irregular sinuous streak with a mucilaginous appearance, when cultivated on nutrient agar-agar. Spore-formation present They grow also very readily on blood serum, and all cultivations yield the odour of rotting kitchen refuse. They are not pathogenic. No. 2. Rods shorter and thinner than No. i. They develop very rapidly on agar-agar, forming transparent drops, which become grey. They were isolated from a patient suffering from profusely-sweating feet. The cultivations yield a characteristic odour similar to the FIG. 123. BACILLUS SAPROCENES, No. I. [After Rosenbach.] last. They are pathogenic in rabbits. They appear to be identical with Bacillus f&tidus (Bacterium fcetidum, Thin). No. 3. See Bacterium saprogenes. Bacillus fcetidus (Bacterium f&tidum, Thin). Cocci, short rods, long rods, and leptothrix. The cocci, 1*25 I -4 in diam., occur singly or in pairs. Spore- formation present in the rods. They were isolated from the exudation in a case of profuse sweating of the feet, and the odour was noticeable in the cultivation (vide Bacillus saprogenes}. Bacillus putrificus Coli, Bienstock. Slender, motile rods, 3 p. in length, often less, sometimes forming long threads. Spore-formation present. Cultivations in gelatine are iridescent. They are constantly present in faeces. Bacillus coprogenes fcetidus, Schottelius. 348 BACTERIOLOGY. .Rods with rounded ends, shorter, but about same width as the hay-bacillus. Immotile ; spore-formation present. On nutrient gelatine the colonies are yellowish, and do not liquefy the medium. On potatoes they form a pale grey layer. They develop a strong rotting odour. They were isolated from the organs and intestinal contents of pigs reputed to be ill with swine-erysipelas. Bacillus aerophilus, Liborius. Slender rods, two- thirds the width of the hay-bacillus, and thread-forms. Spore-formation present. In nutrient gelatine they form dot-like colonies of greenish-yellow colour, which liquefy the gelatine. In test-tubes a somewhat funnel-shaped liquefaction results. On potatoes they develop a yellowish layer. Powerfully aerobic. Found as a contamination. Bacillus mesentericus fuscus, Fliigge. Small, short, actively-motile bacilli, often linked in twos and fours. Spore-formation present. They form white colonies on plate-cultivation, which later stream out in rays at the periphery, and liquefy the gelatine. In test-tube cultiva- tions a funnel-shaped turbidity is produced, and then a stratum of liquefied gelatine with subsiding flocculi. On potatoes they develop a smooth yellowish layer, which soon becomes folded and wrinkled, forming a delicate veil over the nutrient surface. They occur on unsterilised potatoes. Bacillus mesentericus vulgatus, Flugge (Potato bacillus). Rods, longer and thicker than the above, and sometimes thread-forms ; spore-formation present. The colonies, at first somewhat transparent, have later an opaque centre, and liquefy the gelatine. In test-tubes of nutrient gelatine a funnel-shaped turbidity results, and then an upper-stratum is completely liquefied, while a skin floats on the surface, and flocculent masses subside to the bottom of the liquid layer. They occur on potatoes. Bacillus phosphorescens, Fischer. Motile rods. Nutrient gelatine quickly liquefied. Cultivated from sea- water (West Indies). SYSTEMATIC AND DESCRIPTIVE. Genus V. Vibrio. SPECIES. 349 UNASSOCIATED WITH DISEASE : Vibrio rugula . . Zymogenic saprophyte. Vibrio rugula, Muller. Rods and threads, 6 1 6 n long, about -5 2-5 ^ thick. The rods are either simply bowed, or possessed of one shallow D E FIG. 124. VIBRIO RUGULA, x 1020. A. Bowed threads. B. Slightly-curved rods. C. Rods swollen preparatory to spore-formation. D. Rods swollen at the spore-forming end. E. Various stages of the developing spores. [After Prazmowski.] spiral (Fig. ). They bear a flagellum at each end. The rods form swarms when causing de- composition, and then, or after, grow out into threads, curved in a screw-like manner. In the next stage of development the rods cease to move, and become swollen with granular contents. One 350 BACTERIOLOGY. extremity develops an enlargement, giving the rod the appearance of a pin. The spore formed by the contraction of the plasma in the swollen end finally becomes globular. The vibrios appear in vegetable infusions, causing fermentation of cellulose. Genus VI. Clostridium. ASSOCIATED WITH DISKASE IN ANIMALS : Clostridium of symptomatic anthrax. Pathogenic. UNASSOCIATED WITH DISEASE : Clostridium butyricum . . . Zymogenic saprophytes. Clostridium polymyxa .... Clostridium of symptomatic anthrax (Rausch- brand, Charbon symptomatique*} Rods rounded at the ends, mostly with a shining spore at one end. They are especially distinguished from the bacilli of anthrax by being motile. Cultivated on blood- serum, threads develop, consisting of both rods and cocci. From blood-serum they can be cultivated on nutrient gelatine, and vegetable albumen. Cultivation does not deprive the micro-organism of its virulence, but heating the spores to 85 C. renders them harmless. Inoculation in the subcutaneous tissue of guinea- pigs, rabbits, calves, and sheep proves fatal. White rats, dogs, and fowls have an immunity. Injection into the veins in small quantity produces a febrile * Arloing, Cornevin et Thomas, Bull, de VAcad. de Med. 1881. SYSTEMATIC AND DESCRIPTIVE. 351 disorder, in larger quantities death. Animals in the former case suffer an abortive illness, which protects them against further inoculation. The micro- organism is the cause of a disease in cattle, " black- leg" "quarter-evil" or " Rauschbrand? At the autopsy the micro-organisms are found in the sub- cutaneous connective tissue, in the lymph glands, kidneys, spleen, and lungs. An irregular tumour is formed in the skin, which develops rapidly, and gives crepitus on palpation. The tumour, which is haemorrhagic effusion, occurring in the extremi- ties, impedes the animal's movements. The cattle infected die in thirty-six to forty-eight hours. Clostridium butyricum, Prazmowski (Bacillus amylobacter. Van Tieghem ; Bacillus butyricus. Bacillus of butyric acid fermentation). Rods 3 10 jju long, and under i //, wide, often indistinguish- able from Bacillus subtilis. They grow out into long, apparently unjointed threads. They are mostly actively motile, but also occur in zooglcea. The rods and threads are sometimes slightly bent, like vibrios. They are anaerobic. The shorter rods as a rule swell in the middle, becoming ellipsoidal, lemon, or spindle-shaped ; the long rods, and some- times the short ones, swell at one end ; in either case ellipsoidal spores are developed (Fig. 125). If they be cultivated in nutrient gelatine, the medium is liquefied, and a scum formed on the surface. They grow best between 35 and 40 C. The spores are widely distributed in nature, and 352 BACTERIOLOGY. grow readily on fleshy roots, old cheese, etc. They convert the lactic acid in milk into butyric acid, and produce the ripening of cheese. They occur FIG. 125. CLOSTRIDIUM BUTYRICUM. A. Active stage, (a, 6) Bent rods (vibrio-form) and threads, (c) Short rods. (d} Long rods. B. Spore-formation. C. Spore-germination. [After Prazmowski.] also in solutions of starch, dextrine, and sugar, and are the active agents in the fermentation of sauerkraut and sour gherkins. SYSTEMATIC AND DESCRIPTIVE. 353 METHOD OF STAINING THE BACILLUS OF BUTYRIC ACID FERMENTATION. Treat the bacilli with iodine-solution. At certain stages of the fermentation process the plasma takes a blue or violet-black coloration. The young rods give the former appearance, and the older ones the latter. It is most easily observed when the bacillus is cultivated in a sub- stance containing starch, or, if starch is wanting, in the presence of cellulose, calcium-lactate, or glycerine ; in bacilli cultivated in sugar solutions the reaction seldom appears. Clostridium polymyxa, Prazmowski. Threads consisting of rods which vary in length ; cocci, involution-forms, and spores are also present ; cul- tivated on nourishing solutions they develop a thick skin on the surface. On boiled beet and other roots they form a gelatinous scum, which often consists of crinkled, tough masses, several cm. in diam., somewhat similar to the Ascococcus Billrothii. They cause fermentation in solutions of dextrine, and more actively in potato or bean paste. Some cells give the iodine reaction weakly, as in Clostri- dium butyricum. GROUP III. LEPTOTRICHE^:. Genus I. Crenothrix. Threads articulated ; cells sulphurless ; habitat water. Genus II. Beggiatoa. Threads unarticulated ; cells with sulphur granules ; habitat water. 23 354 BACTERIOLOGY. Genus III. Phragmidiothtix. Threads jointless; suc- cessive subdivision of cells is continuous ; cells sulphurless ; habitat, water. Genus IV. Leptothrix. Threads articulated or un- articulated ; successive subdivisions of cells not continuous ; cells sulphurless. Genus I. Crenothrix. SPECIES. UNASSOCIATED WITH DISEASE : Crenothrix Kuhniana . . . Simple saprophyte. Crenothrix Kuhniana, Rabenhorst. Cocci, rods, and thread-forms. The cocci are globular, i 6 /A in diam. The threads are colourless, 1*5 5 //, thick, and club-shaped at the extremity, reach- ing a diam of 6 9 /A. The threads form colonies with a brick-red, olive-green, or dark-brown to brown-black coloration caused by impregnation with oxide of iron. The threads are distinctly articulated, and ensheathed. The segments are set free when the sheath bursts, and develop into new threads. In other cases the segments remain enclosed, and subdivide into discs, which, by vertical fission, break up into globular forms (cocci). These again develop into new threads, either within the sheath eventually penetrating it, or after they are set free (Fig. 126). The micro-organism appears in little whitish or brownish tufts in wells and drain-pipes, and it not only renders drinking-water foul, but may stop up the narrower pipes. SYSTEMATIC AND DESCRIPTIVE. 355 FIG. 126. CRENOTHRIX KUHNIANA. ASSOCIATED WITH DISEASE : Actinomyces . . ' ''.} . Pathogenic. Cladothrix dichotoma, Cohn. Threads re- sembling those of leptothrix ; slender, colourless, not articulated, straight or slightly undulated, and in places twisted in irregular spirals with pseudo- branchings. The development can be traced from the cocci to rods and threads. The latter are at the beginning simple threads, which were formerly described as Leptothrix parasitica, or if coloured by impregnation with iron, as Leptothrix ochracea. Later they form false branches by single rods turning aside, which by repeated division SYSTEMATIC AND DESCRIPTIVE. 36, lengthen into threads. A thread appears to be first composed of long rods, then of short rods, FIG. 129. CLADOTHRIX DICHOTOMA. A. Branching schizomycete : (a) Vibrio-form ; (6) Spirillum-form [slightly magnified]. B. A screw-form with (a) Spirillum-form ; (6) Vibrio-form. C. Long spirochaeta-form. D. Fragment with spirillum-form at one end, vibrio-form at the other. E. Screw-forms: (a) continuous; (b) composed of rods; (c) composed of cocci. F. Spirochaeta-form : (a) continuous ; (6) composed of long rods ; (c) short rods ; (ct) cocci. [After Zopf.] and lastly of cocci. The iodine reaction must be applied to distinguish these forms, especially when 364 BACTERIOLOGY. the sheath of the threads has a yellow, rust-red, olive-green, or dark-brown coloration. The cocci may grow into rods while still in the sheath, and finally become leptothrix threads, surrounded by a delicate gelatinous sheath, from which the false branching proceeds. Fragments may break off, which are actively motile, and appear as vibrios, spirilla, and spirochaeta-forms (Fig. 129). They may also occur in zooglcea. They are the commonest of all bacteria in both still and running water, in which organic substances are present. They are observed also in the waste water of certain manufactures, such as sugar. Ar- tificially they can be cultivated on infusions of rotting algae and animal substances, forming on these media small tufts, about i 3 p and floating masses. Cladothrix Fcersteri (Streptothrix Forsteri, Cohn). Cocci, rod-forms, and leptothrix-threads. The threads are twisted in irregular spirals, and branch sparingly and irregularly. Screw-forms are produced by the threads breaking up into small pieces. They occur in the lachrymal canals of the human eye, in the form of closely felted masses. There are some little-known species, which possibly belong to this group. Sphaerotilus natans. Cells 4 9/x long, 3^ thick, united in a gelatinous sheath to form threads. The cells comprise rods and cocci-forms ; the cocci are set free, and SYSTEMATIC AND DESCRIPTIVE. 365 develop into rods, which again form threads. In the last a false branching has been observed. The plasma of the cells breaks up into minute, strongly refractive portions, which develop into round spores, at first of a red, and afterwards a brown colour. They occur in stagnant and flowing water contaminated with organic matter, and form floating flakes of a white, yellow, rust-red, or a yellow- brown colour. Myconostoc gregarium, Cohn. The threads are very thin, colourless, unarticulated, but fall apart into short cylindrical links when dried. They form gelatinous masses, 10 17 jut in diameter, singly or heaped into slimy drops on water in which algae are decomposing. Spiromonas volubilis, Perty. Colourless, trans- parent cells, 15 1 8 //, long. Rapidly motile, and revolv- ing round a longitudinal axis. They occur in marsh water and putrefying infusions. Spiromonas Cohnii. Colourless cells, consisting of ij spirals, with both ends acutely pointed and provided with a flagellum. Breadth of the cells, i'2 4^1. They occur in water containing decomposing matter. APPENDIX. Plate 28. facing p. 366 Fig.l || Tig. 10. ^^ ^-/A( \Fig.il. YEAST-FUflGI OK SACCHAR.OMYCETES AND MOULD-HIKGI OR HYPHOHCETES. - Groofchouik del. Vvu&ntErooks, HcyJiSon., . APPENDIX A YEASTS AND MOULDS. Yeast-fungi and mould-fungi, like bacteria or fission-fungi, are achlorophyllous Thallophytes. They belong to two separate orders, the Saccharomycetes and Hyphomycetes, which are intimately related to each other, but quite distinct from bacteria. Their germs occur widely dis- tributed in air, soil, and water, and are constantly en- countered in bacteriological investigations. In addition many species are of hygienic and pathological interest or importance in being either accidentally associated with, or actually the cause of various morbid processes. For a complete account of all the described species and full details of the various forms of development,* reference must be made to botanical treatises. A description of certain species is appended here, and may afford some useful information to the worker in a bacteriological laboratory. YEAST-FUNGI OR SACCHAROMYCETES. Saccharomyces cerevisiae (Torula Cells round or oval, 8 9 p, long, singly or united in small chains. Spores occur three or four together in a mother-cell, 4 5 p in diameter (Plate XXVIII., Fig. 2). Sacch. ellipsoideus. Elliptical cells, mostly 6 /z, long, singly or united in little branching chains. Two to * Sachs, Text-book of Botany. 1882. 24 37O APPENDIX, four spores found in a mother-cell, 3 3*5 ft in diam. It is widely distributed, and is the principal agent in ac- cidental fermentation. Sacch. conglomeratus. Cells round, united in clusters, consisting of numerous cells produced by budding from one or a few mother-cells. There are 2 to 4 spores in each mother-cell. They occur on rotting grapes and in wine at the commencement of fermentation. Sacch. exiguus. Conical or top-shaped cells, 5 ft long, and reaching 2*5 ft in thickness, in slightly branching colonies. Spore-forming cells are isolated, each contain- ing 2 or 3 spores in a row. Present in the after-fermenta- tion of beer. Sacch. pastorianus. Cells oval or club-shaped. Colonies consist of primary club-shaped links, 18 22 fi long, which build lateral, secondary round or oval daughter-cells, 5 6 ft long. Spores 2 to 4. In the after-fermentation of wine, fruit-wines, or fermenting beer. Sacch. apiculatus. Cells lemon-shaped, both ends bluntly pointed, 6 8 ft long, 2 3 ft wide. Bud- ding occurs only at the pointed ends. Rarely united in colonies. Spores unknown. They occur with other yeasts in various accidental fermentations. Sacch. sphaericus. Cells varying in form ; the basal ones of a colony oblong or cylindrical, 10 15 fi long, 5 ft thick ; the others round, 5 6 ft in diam. United in ramified families. Spores unknown. Sacch. mycoderma (Mycoderma cerevisice et vim). Cells oval, elliptical, or cylindrical, 6 7 ft long, 2 3 ft thick, united in richly branching chains. Spore-forming cells reaching 20 ft long. Spores I to 4 in each mother- cell. Forms the so-called " mould " on fermented liquids, and develops on the surface without exciting fermenta- tion. When forced to grow submerged, a little alcohol is produced, but the fungus soon dies. They occur on wine, beer, fruit-juices, and sauerkraut. Sacch. albicans (Oidium albicans}. Cells partly YEASTS AND MOULDS. 371 round, partly oval or cylindrical, 3-5 5 p, thick, the cylindrical cells 10 20 times as long as they are thick, The bud-colonies mostly consist of rows of cylindrical cells, from the ends of which oval or round cells shoot out. Spores form singly in roundish cells. They occur on the mucous membrane of the mouth, especially of infants, in greyish-white patches which consist of epithe- lium, bacteria, yeasts, and the mycelia of various moulds. They can be easily cultivated in a nutrient solution con- taining sugar and ammonic tartrate.^ The cells germinate according to the richness of the fluid in sugar ; they either grow into long threads, or, in a very strongly saccharine solution, many daughter-cells are formed, budding out in various directions (Plate XXVIIL, Fig. 3). Sacch. glutinis. Cells round, oval, or short cylinders, 5 11 JJL long, 4 ^ wide, isolated, or united in twos. Cell-membrane and contents are colourless in the fresh state, but when dried and remoistened possess a pale-reddish nucleus in the middle. Spore-formation unknown. Forms rose-coloured, slimy spots on starch, paste, or on sterilised potatoes. The colouring matter is not changed by acids or alkalies. Sacch. rosaceus (Pink Torula). Cells 9 IO ft in diam. Forms a coral-pink growth in nutrient gelatine, nutrient agar-agar (Plate VII., Fig. 3) or on sterilised potatoes (Plate XV., Fig. 2). They are present in the air. Sacch. niger (Black Torula). Cells also present in the air. Cultivated in nutrient gelatine they form a black crust (Plate V., Fig. 3). '"'" ' f Vf '''- ; -' '->-"* !;J ''. ,' MOULD-FUNGI OR HYPHOMYCETE$. The mould-fungi have been divided into five orders : "j* Hypodermii, Phy corny cetes^ Ascomycetes, Basidiomycetes, and * Grawitz, Virch. Archiv, vol. 70. t Fliigge, Fermente u. Mikrofiarasiten. ' 1883. 37 2 APPENDIX. Myxomycetes. The following species, with the orders to which they belong, are of especial interest : HYPODERMII. UstilagO carbo (mildew, smut). Spores, brown, circular ; episporium smooth ; sporidia, ovoid cells. The spores or conidia occur as a black powder in the ears and panicles of wheat, barley, and oats. Tilletia caries. Spores round, pale, brown ; epi- sporium with reticulated thickenings. In germinating sporidia grow out radially from the end of the promy- celium ; these, at their lower part, conjugate by a cross branch, and separate from the promycelium, and at some point of the pair a hypha grows out, on which abundant secondary sporidia develop. The latter are long, oval cells, which can in turn germinate. The fungus occurs in the form of a stinking powder in grains of wheat, which renders the meal impure, and gives it a disagree- able smell. Urocystis OCCulta. The spores consist of several cells united together ; partly large, dark-brown cells in the interior, and outside, several flat, semicircular, colourless cells. Spores, "024 mm. Promycelium germinates as in Tilletia, but the cylindrical cells produce a hypha, without, as a rule, previous conjugation. Occurs as a black powder in rye straw, in long disintegrated stripes, which are at first greyish. The affected plant produces abortive ears. Empusa muscae Spores, 'Oil mm. in diam. A spore or conidium alighting upon the white area of the under surface of the body of the house-fly, germinates into a hypha. The latter, penetrating the skin, forms toruloid cells, which multiply by germination, and are disseminated in the blood throughout the body of the fly. These cells again grow into hyphae, which penetrate the skin, each forming a conidium, which is cast off with con- siderable force. The parasite is fatal to flies, especially YEASTS AND MOULDS. 373 in the autumn. They are often observed attached to the walls or window-panes, surrounded by a powdery sub- stance, consisting of the extruded conidia. Empusa radicans. The spores form long hyphae, which pierce the transparent skin of the caterpillar of the cabbage-white butterfly. The terminal cells ramify, and fill the body of the caterpillar with a network of mycelial filaments. The caterpillars attacked become restless, then motionless, and death ensues. Tarichium megaspermum. The spores are -05 mm. in diam., black in colour, and provided with a thickened episporium. They occur at the sides and ends of mycelial threads, attacking caterpillars (Agrotis segetuni). PHYCOMYCETES. Saprolegnia. Colourless threads, formingdense radi- ating tufts, occur on living and dead animal and vegetable matter in fresh water. The filaments penetrate into the substratum, and branch more or less in the surrounding water. The cylindrical ends of threads are shut off by a septum-forming zoosporangia, or mother-cells, in the interior of which a number of spherical spores, zoospores, develop. These are set free through an apical opening in the thread, and, after a time coming to rest, give rise to new plants (Plate XXVIIL, Fig. 4). In the sexual mode of reproduction, a spherical bud, the oogonium, develops at the end of a mycelial thread ; from the thread, small processes or antheridia sprout out laterally towards the oogonium, and blend with its protoplasm (Plate XXVIIL, Fig. 4). The latter breaks up into a number of oospores, which clothe themselves with a membrane, while still within the mother-cell, and, eventually being set free, grow into fresh mycelial filaments. The parasite attacks fish and tritons, and produces a diseased condition of the skin, which may be ultimately fatal In salmon it pro- duces the common disease of salmon. Peronospora infestans. Mycelium, -005 mm. in 374 APPENDIX. thickness. Twigs with as many as five branches, each bearing an egg-shaped conidium. The contents of the conidia falling off and reaching a drop of moisture, break up into a number of swarming zoogonidia, which in turn develop upon plants. Fixing themselves to the cuticle of the host, they throw a germinating filament into an epidermal cell ; piercing first its outer wall, and then its inner wall, the filament reaches an intercellular space, where the mycelium develops. This continues to grow and spread throughout the plant. In tubers it can hiber- nate and develop in the young shoots in the following spring. The parasites appear in the form of brown patches on the green parts of the plants, especially the leaves. The attacked parts wither and turn yellow or brown in colour. If the under surface of a diseased leaf be examined, a corresponding dark spot may be observed, accompanied with a faint greyish-white bloom which covers it. The latter consists of the conidia-bearing branches of the fungus. Pilobolus. Hyphae, I 2 mm. high. Fruit-hyphae, possessing spherical receptacles containing conidia. When ripe the receptacles with their conidia are detached at their bases, and spring by their elasticity to some distance. The mould occurs as glassy tufts on the excrement of cows, horses, etc. A cultivation can generally be obtained by keeping fresh horse-dung under a bell-glass. Mucor mucedo.- Hyphse colourless, simple or branched, I 15 cm. long; sporangia are yellowish-brown or black. Spores ovoid, -008 mm. long and '0037 wide. Occurring as the familiar white mould on fruits, bread, potatoes, excreta, and penetrating into the interior of nuts and apples. A network of fibrils develops in the substance of nutrient gelatine, with formation of sporangia on the free surface. The germination of the spores and development into hyphae can be observed in a few hours, if the fungus be cultivated in a decoction of horse-dung. Mucor racemosus. Hyphae, at most 1-5 cm. long; YEASTS AND MOULDS. 375 sporangia, yellowish to pale-brown ; spores round. By continued cultivation in liquids saturated with carbonic acid, the hypha becomes shorter, and exhibits a yeast-like sprouting. These yeast-like or toruloid cells can, when the carbonic acid is withdrawn, germinate into normal mycelium. Mucor Stolonifer, Lichtheim. Mycelium grows in the air, and then bends down and re-enters the nutrient substratum ; sporangia black and spores globular. The mycelium can penetrate through the shell of eggs, and form conidiophores within them. Mucor aspergillus, Lichtheim. Fruit-hyphae, thinned at the base, and with many fork-like divisions; dark-brown spores. ..'. Mucor phycomyCCS, Lichtheim. Mycelium thick- walled ; olive-green fruit-hyphae, black sporangia, and oblong spores. Mucor macrocarpus, Lichtheim. Spindle-formed, pointed spores. Mucor fusiger, Lichtheim. Ovoid spores. Mucor mellittophorus, Lichtheim. Spores ellip- tical. Found in the stomach of bees. Mucor COrymbifer, Lichtheim. Forms branched fruit-hyphae ; sporangium has a smooth membrane. Found in the external auditory meatus; occurring also upon bread. Pathogenic in rabbits. Mucor rhizopodiformis, Lichtheim. Occurs on bread. The spores of Mucor rhizopodiformis and Mucor corymbifer y when introduced into the vascular system of rabbits, can germinate in the tissues, especially in the kidneys, where they set up hsemorrhagic inflammation. Dogs are immune, and only artificial mycosis is known.* * Lichtheim, Zeistchr. f. Klin. Med., vii. ; Hiickel, Beitr. z. Anat. u. Phys., herausgeg. v. Ziegler u. Nauwerck. 1885. 376 APPENDIX. ASCOMYCETES. Oidium Tuckeri. Fruit-hyphse,bearing single ovoid conidia. Observed in the form of brown patches, covered with a white mildew-like layer on the leaves, branches, and young fruit of the vine, producing a " grape-disease." Oidium lactis. Fruit-hyphse, simple, erect, and colourless, bearing at their ends a series or chain of conidia (Plate XXVIIL, Fig. 5). In some cases the fruit- hypha branches beneath the chain of spores. Spores are short cylinders, '0077 '0108 mm. long. The fungus is sometimes found as a whitish mould on milk, bread, paste, potato, and excrement, and is believed to be iden- tical* with the fungus of certain human skin diseases, Favus (Achorion Schoenleinii\ Herpes tonsurans (Tri- cophyton tonsurans), and Pityriasis versicolor (Microsporon furfur) (Plate XXVIIL, Pig. 6). Cultivated artificially in nutrient gelatine, the conidia germinate into filaments of varying length, which by subdivision form septate mycelial hyphae ; these and their branches give rise in turn to spores or conidia. The differences observed in various diseases are attributed to differences in the nutrient substratum. Others f maintain that, in artificial cultiva- tions of the spores of Tricophyton tonsurans, the fructifica- tion is identical with Penicillium. Oidium albicans. Vide Saccharomyces albicans. Aspergillus glaucus (Eurotium aspergillus glau- cus). Mycelium, at first whitish, becoming grey-green or yellow-green. Spores grey-green, thick-walled, -009 '015 mm. in diam. Sometimes found on various substances, chiefly cooked fruit (Plate XXVIIL, Fig. 10). Aspergillus repens (Eurotium repens, De Bary). Fruit-heads fewer than in the above, which are at first pale and then blue-green to dark-green in colour ; conidia mostly oval, smooth, -005 -008 mm. long, colourless or pale to grey-green. * Grawitz, Vir chow's Archiv, vol. 70. t Morris and Henderson, Journ. Royal Microsc. Society. 1883. YEASTS AND MOULDS. 377 Aspergillus flavus. Gold-yellow, greenish, and brown tufts ; fruit-heads round ; yellow, olive-green, or brown. Conidia round, seldom oval ; sulphur-yellow to brown in colour, "005 "007 mm. in diam. Saprophytic in man, pathogenic in rabbits. Aspergillus fumigatus. Greenish, bluish, or grey tufts. Fruit-heads long and conical. Conidia round, seldom oval, smooth, mostly pale and colourless. Diam. 0025 to "003 mm. Observed saprophytically in human lungs, external auditory meatus, and middle ear. The spores introduced into the vascular system of rabbits, or into the peritoneal cavity, establish metastatic foci in the kidneys, liver, intestines, lungs, muscles, and sometimes in the spleen, bones, lymphatic glands, nervous system, and skin. Aspergillus niger (Eurotium aspergillus niger, De Bary). Dark chocolate-brown tufts. Conidia round, black-brown, or grey-brown, when ripe ; '0035 to '005 mm. This mould can be cultivated readily on bread moistened with vinegar, on slices of lemon, and on acid fruits and liquids. It flourishes best of all, according to Raulin,* in a liquid of the following composition : Grammes. Water . ' . _. j' . ' . 1500* Sugar-candy. . . . "V 70* Tartaric acid '%' . Jtj . 4* Nitrate of ammonia p4 . li . f V-< : 4* Phosphate . . y . ->H, V f .5 Carbonate of potassium i . ' . *6 magnesium . 8 ; - ; -4 Sulphate of ammonia '$* *?' . "25 zinc ... -07 iron : . ; i i;'- "07 Silicate of potassium . ' '. ." '07 It was also found that the fungus grew best when the quid was spread out in a layer 2 or 3 cm. in depth in a * Duclaux, Health Exhibition Handbook. London : 1884. 3.78 APPENDIX. shallow dish, and a temperature of 35 C. proved to be the most favourable. The abstraction of zinc from the nutritive liquid reduced the weight of a crop from 2 5 (the average) to 2 grammes, and the presence of To-trJinro- part of nitrate of silver, or -guthnr P art f corrosive sublimate, stopped the growth altogether. It is sapro- phytic in the living body. METHOD OF EXAMINING ASPERGILLUS NIGER. Species of aspergillus stain intensely with carmine, fuchsine, or methyl-violet ; but to examine Aspergillus niger with a high-power, a little special technique is employed, as follows : A drop of glycerine is placed on a clean slide, and a drop of alcohol on a cover-glass. With a fine pair of forceps a few of the fruit-hyphae with their black heads are immersed in the alcohol. The cover- glass is then turned over on to the drop of glycerine, and the slide held in the flame of a Bunsen burner till the spores or conidia are dispersed. To make a permanent preparation, remove the cover-glass, and transfer the fruit- hyphae so treated to a mixture of glycerine and water (i to 5) ; a drop may be conveniently placed ready on a slide provided with a ring of Canada balsam. The speci- men is then permanently mounted by employing a circular cover-glass, and surrounding it with a ring of cement in the usual way (Plate XXVIIL, Figs. 8 and 9). Aspergillus OChraceus. At first flesh-coloured, and then ochre-yellow heads. Aspergillus albus. Pure white fruit-heads. Aspergillus clavatus. Club-shaped fruit-heads on long stems. Penicillium glaucum. Occurs as a white, and later a blue-green mould, on which dew-like drops of liquid may appear (Plate XIV., Fig. 2). Its spores are present in large numbers in the air, and are liable to contaminate cultivations. Diam. of the spores '0035 mm. ; threads vary in diameter between '004 and '0007 1 mm., ACTINOMYCES. 379 according to the nourishing material ; the fruit-hypha bears terminally a number of branched cylindrical cells, from which chains of greenish conidia are developed (Plate XXVIL, Fig. 7). It is the commonest of all moulds. Botrytis Bassiana. Hyphae and spores colour- less. Hyphse usually simple, but sometimes united in arborescent stems (Plate XXVIIL, Fig. 1 1). It is the cause of muscardine, a fatal disease of silkworms, and occurs also in various other caterpillars and insects. -.,; . ;i *.--. "i ' _. '' t v! :'. r UNCLASSED. Chionyphe Carted. Mycelium, penetrating the skin and subcutaneous tissue, sets up suppuration and ulceration. Described as the cause of a disease known in India as " madura-foot." APPENDIX B. ACTINOMYCES. Actinomyces is a fungus associated with a disease occur ring occasionally in man and very commonly in cattle. The micro-organism appears in the form of a rosette of pyriform or club-shaped elements, and hence the name " ray-fungus." The fungus is believed to commonly effect an entrance by the mouth, being taken in with the food, possibly through the medium of a wound of the gum or a carious tooth. In whatever manner it has gained access to the living organism, it sets up inflammation in its neighbour- hood, resulting in the formation of a neoplasm, composed chiefly of round cells. The nodules may break down and suppurate, or go on increasing in size. Fibrous tissue develops between the nodules, and large tumours often result, containing purulent cavities and excavations. In 380 APPENDIX. the slimy detritus the little pale-yellow grains of fungus can be detected. In cattle the lower jaw is usually affected, and then the upper jaw and neighbouring parts. The organism may also occur in nodular tumours in the lung, subcutaneous and intermuscular tissues. It is the cause of " wens " and " wooden tongue,". and also of other diseases of cattle which have been variously described before their true nature was understood as bone-canker, bone-tubercle, osteo-sarcoma, etc. In man the pulmonary formations tend to break down early, forming fistulae and sinuses, with the clinical cha- racter of empyema. In some cases there are symptoms of chronic bronchitis with fcetid expectoration. In other cases the disease, originating in the lung, spreads to the praevertebral tissues. If the actinomyces invade bones, as has been especially observed in the bodies of the vertebrae, caries results. The organism has been known to produce disease of the intestinal canal. The fungus has also been detected in the crypts of the tonsils of healthy pigs, and a similar, if not identical, one in the spermatic duct of the horse.* The disease has been transmitted from cattle to cattle by inoculation, j" and rabbits have been infected by means of pieces of actinomycotic tumours from human subjects, introduced into the peritoneal cavity. THE FUNGUS IN CATTLE. Examination in the Fresh State. The fungus may be detected with the naked eye in the muco-purulent discharge, or in a scraping from the cut surface of a growth. The tufts of the fungus vary in' size under different circumstances, from that of a grain of fine sand, to that of a pin's head. If the pus or scraping * Johne, Bericht uber das Veterinarwesen imKonigreich Sachs en fiirdasjahr. 1884. t Johne, Deutsche Zeitschr, f. Thiermedicin. 1881. ACTINOMYCES. 381 be spread out on a slide and examined against a dark background, the grains appear to be white or yellowish- white in colour ; but if examined by transmitted light, they appear distinctly brownish. On pressing the cover-glass on the slide the grains readily flatten out, being of a soft tallowy consistency ; or in the process of gently pressing the cover-glass on the slide with slight lateral movement, a distinct gritty sensation is transmitted to the finger, owing to the presence of calcareous matter. On examina- tion with a low power the fungus will be recognised in the form of irregular patches scattered over the field, which might readily be regarded as collections of granular debris of a brownish or yellowish-brown colour, but on careful examination this apparent granular debris is ob- served to have a more or less characteristic appearance. On examining with a higher power, spherical, ovoid, or reniform bodies are to be seen which are either typical rosettes of clubs or granular masses, with here and there a club-shaped body at the periphery. Pus cells, round cells, fat granules, and minute spherical bodies may also be distinguished. If the grains consist of typical rosettes, and be merely covered with the cover-glass, and examined without being flattened out between the cover-glass and the slide, they will recall to mind, on focussing in turn the centre and the periphery, the appearance of the capitulum of a composite flower. The central portion appears to consist of spherical forms ; these are the ex- tremities of the component elements, and as we focus the edge of the rosette these elements are seen laterally, and their characteristic club-form is readily distinguished. The central portion may be flattened against the cover-glass, and as the individual clubs vary considerably in size, the appearance of an irregular mosaic is produced (Plate XXVIIL, Fig. i). By pressing upon the cover we break up the rosette, and then the clubs are recognised either singly or in twos, or attached together in the form of wedge or fan-shaped 382 APPENDIX. segments. Calcareous material, if present, may readily be demonstrated by the action of acids. It will be found that on the addition of hydrochloric acid, nitric acid, or acetic acid, the calcareous deposit is dissolved while the clubs are not affected, and even with the addition of the strongest acids the only result will be to dissolve out the calcareous matter and clarify the tufts of the fungus, the form of the clubs being still recognisable. They are not affected by ether or potash, and thus the effects of chemical reagents clearly distinguish them from fat crystals or calcareous particles. By breaking up the growth into small fragments, we may readily study the shape of the individual club-like elements. By using high power ob- jectives, and properly arranging the illumination, various forms will be clearly delineated. In some cases the club will be found to be bifid at the extremity; in other cases there are lateral offshoots or daughter-clubs. Here and there will be found clubs closely pressed together like a bunch of bananas, and in other cases the broken-off pieces have a palmate form. By teasing out the grains in water, and pressing them apart between the slide and cover-glass, we find that the central portion is composed, as a rule, of a structureless core. More rarely there are the delicate filaments which are found in the disease in man. If the grains be mounted in glycerine, the appearance of the organism in the fresh state may be preserved. The granules may be stained by picking them out with needles and transferring to a watch-glass containing alcohol, to which a few drops of concentrated alcoholic solution of eosin have been added. They remain in the solution until distinctly stained, and they are then placed on a glass slide in a drop of glycerine. The muco-pus may be spread out into as thin a film as possible on a cover-glass, allowed to dry, fixed by warming slightly over the flame, and stained by the methods of Plaut or Gram. The characters of the fungus can be so readily recognised in the perfectly fresh state ACTINOMYCES. 383 that methods of staining are in diagnosis of secondary importance, though there are certain minute points which can only be satisfactorily determined by means of suitable dyes. Cultivation Experiments. . Repeated attempts by the author to cultivate the fungus from bovine specimens have failed. A section of a tongue, for example, may be made with a sterilised knife, a small portion of the growth transplanted on such medig. as blood serum, nutrient gelatine, and nutrient agar-agar. The little pieces of tissue remain without showing any visible growth ; and though on microscopical examination the presence of the clubs can still be demonstrated, there is no distinct evidence of any increase of the fungus. PREPARATION AND EXAMINATION OF TISSUES, In order to examine the microscopical appearances, the tissues should be hardened in absolute alcohol and em- bedded in celloidin. The sections, when stained, are to be dehydrated as a rule in strong spirit instead of absolute alcohol, as the latter dissolves the celloidin. If the sections are very friable, they can be cleared with clove-oil on the slide. By these means the little fungus tufts, which have a great tendency to fall out of the sections, may be pre- served in situ after passing through the various staining processes. To cut the sections, we can use either Jung's microtome cutting in alcohol, or the freezing microtome. In the latter case, after the celloidin has hardened it is necessary to shave off all that surrounds the piece of tissue. It is placed in water until it sinks, and then transferred to gum and frozen and cut in the ordinary way. Staining Methods. ... ,\ , There are several methods by which the organism can be stained in the tissues, but it is best to employ for APPENDIX. this purpose Gram's method and modifications of Plaut's method. Gram's Method. By Gram's method the clubs in the bovine disease are distinctly stained, especially if the sections contain the fungus at a suitable stage. Use freshly prepared staining solution. A few drops of aniline-oil are placed in a test-tube, which is filled up with distilled water, the mouth of the tube closed with the thumb, and the mixture shaken up thoroughly. An emulsion forms, which is then filtered until a perfectly clear solution of aniline-water is obtained. To this is added, drop by drop, an alcoholic solution of gentian- violet until precipitation commences. About fifteen to twenty drops in a small capsule of aniline-water will be sufficient. Sections are floated in this dye for about ten minutes, then transferred to iodine-potassic-iodide solution until they turn brown like a tea-leaf. They are then decolorised in alcohol ; then stained in a weak alcoholic solution of eosin, dehydrated in strong commercial alcohol, cleared in clove-oil, and mounted in balsam. It will be found that the clubs are stained blue, and that there is a central area, which is, as a rule, tinged by the eosin. There are various modifications of the method, and some of them are extremely successful in affording not only a picture of the fungus, but also the structure of the sur- rounding tissue. Very instructive results may be obtained by combining the method of Gram with Ehrlich's his- tological stain. In this case, after the section has been decolorised in alcohol, it is ready to be transferred to logwood and treated as described below (p. 385). Weigerfs Method. This also gives very beautiful results. The sections are placed for an hour in Wedl's solution of orseille, which is prepared as follows : Add liquid extract of orseille to a mixture of absolute alcohol 20 parts, strong acetic acid 5 parts, distilled water 40 parts, until a dark- red liquid results. This must be filtered before use. The sections are left in this solution PLATE 29 ^ ** f , * * * '* /:,%""**/' ,*;>* i v% /;;:;. .*" :tvA.\ /. y. % ij- ..;--.' b^'xV: Vst;.-^ fek.MftV.j;$i t. ;Iii.\*P.?.Vfc7 Cv ^ ^4V. - > \/* ?%** > * % %V^ ?i i ** * *->* ^*A S ^ >%ti* * % Fig 2. ay Fort . litii PLATE 30 lab :'-}. F&l. q a*- CheokshanJt fcc-. &t j> i-nxt . KXewic. 136. Gowsr Street ri, !cen tfi,wi.. ACTINOMYCES. 385 for an hour, then just rinsed in alcohol, and transferred to a solution of gentian-violet. Such sections show the nuclei of a violet-blue colour, and the peripheral part of the central core in the larger masses of the fungus also takes a blue colour, while the club-shaped structures are stained a striking wine-red colour (Plate XXIX.). Plant's Method and Modifications. This is one of the most valuable methods for staining the clubs. The original method was to float sections for ten minutes in magenta solution warmed to 45 C. This solution consisted of magenta 2 parts, aniline-oil 3 parts, alcohol of specific gravity 0*830, 20 parts, distilled water 20 parts (Gibbes). The sections were then rinsed in water, stained in con- centrated alcoholic solution of picric acid for from five to ten minutes, immersed in water five minutes, 50 per cent, alcohol fifteen minutes, passed through absolute alcohol and clove-oil, and preserved in Canada balsam. The clubs are stained a brilliant red and the tissue yellow. Instead of employing the magenta solution, we now use Ziehl-Neelsen solution (Plate XXX.). By removing the picric acid in Plaut's method by prolonged immersion in alcohol, and then staining with gentian-violet or methylene-blue, a very successful con- trast can be obtained. The most instructive histological picture can be obtained by first staining with Neelsen's solution, removing the stain from the tissue in the way that has been already described, and then transferring the sections to distilled water, and subsequently staining with Ehrlich's histological stain. EhrlicJis New Histological Stain. This is a combina- tion of Ehrlich's logwood with orange-rubin. It is of especial value for sections of actinomycosis, and par- ticularly in combination with carbolised fuchsine. It is employed in the following way : The sections must be placed in alcohol or distilled water, and then in Ehrlich's logwood for about half a minute. From this solution they are transferred to distilled water, washed to remove 25 386 APPENDIX. the excess of stain, and then placed in a large dish of tap-water, where they are left for half an hour or more, until the sections turn blue ; if preferred, they may be left overnight. They are then stained for one or two minutes in a solution of rubin. S, and orange, and washed again in distilled water to remove the excess. They must then be dehydrated in alcohol, cleared in clove-oil, and mounted in balsam. Preparation of Large Sections. The method of cutting large sections of organs, which has somewhat recently been introduced, is one which may be very advantageously employed in studying actinomy- cosis. The value of these sections depends, not only upon their affording an instructive picture of the naked- eye appearances, but they can also be studied with a pocket lens, or under the microscope with a J or -J-in. objective. By staining the sections the relation of the morbid to the healthy structures is brought out in greater contrast, and thus the topography of the disease can be studied more minutely than by simply observing the cut surfaces of organs or growths. And, further, it affords a means of rendering permanent many of the instructive appearances observed at the autopsy, without preserving the whole structure in the form of a museum specimen. Very satisfactory results can be obtained with material hardened either in spirit or Muller's fluid. The fresh material is cut with a large keen-edged knife into slices about a quarter of an inch, or less, in thickness. These slices are placed between filter-paper in large porcelain dishes, such as are employed for photographic purposes, and well covered with the hardening solution, which should be frequently changed. By covering the slice with a small pane of glass, which is lightly weighted, any curling or turning up of the edges is prevented, and the slice not only kept flat, but hardened with smooth sur- faces. Several weeks are required for hardening in ACTINOMYCES. 387 M tiller's fluid. The slices, after a short time in water, are placed in gum, and then frozen and cut ; the slices which are hardened in alcohol are soaked in water until all trace of the spirit has been removed. A large micro- tome on the Bruce model is used to freeze and cut the sections. But in some cases it will be found better to embed the slices in celloidin, and cut under alcohol with a large microtome of Jung's pattern. The sections are carefully removed from the blade of the knife with a large camel's-hair brush, and in the case of frozen sections floated in water. The next process is to float a section out in spirit, and with the camel's-hair brush to unfold it and spread it out on a sheet of glass. The glass with the section is lifted out and examined, and if the section is sufficiently thin, transferred to the staining solution. In the same way the section is passed through the various stains, as it should be prevented from rolling up or folding in the dye, or it may not be evenly stained throughout. Modifications of this process will suggest themselves, such as pouring off the dye and leaving the section spread out at the bottom of the dish, and then using the same dish for the next process. The sections are so easily injured, that it is better, as much as possible, to avoid handling them. If the sections are only a few inches in diameter, such as transverse sections of the anterior portion of the tongue of an ox, they can readily be transferred from dish to dish by means of a large spatula made by soldering a piece of sheet German silver to thick copper wire. To stain them employ carbolised fuchsine and picric acid, or alum cochineal, or logwood and orange-rubin. The processes of staining are precisely the same as with ordinary sections ; but, from their unusual size, experience and practice are required in their manipulation. When the section is dehydrated, it is ready to be cleared in clove-oil. The glass on which it is to be permanently mounted should be selected without scratches or flaws, APPENDIX. and thoroughly cleaned and polished. It is slipped under the section, which is evenly spread out upon it, and then lifted out of the dish. The excess of spirit is drained off, the glass placed on a level surface, and clove-oil poured on the section. It is left until completely clarified ; the clove-oil, as much as possible, drained off, and the rest entirely removed by gentle pressure with several thick- nesses of best filter-paper. In this way several large sections can be cleared at the same time. But when only one or two sections are dealt with, they are cleared in clove- oil in a dish, and the mounting glass at this stage passed underneath them as already described. Another plan which will be found of advantage is as follows : A piece of clean thick filter-paper rather larger than the section is slipped underneath it, and then raised with the section upon it. After allowing the excess of clove-oil to drain back into the dish, it is carefully laid on the glass with the section downwards, and gently pressed down. By taking up a corner, the filter-paper is peeled off, and the section left behind on the glass. Any creases or folds are adjusted with needles. After removal of the clove-oil, balsam is run over the section, and a cover-glass gently and dexte- rously lowered, so as to avoid the presence of air-bubbles. The preparations are set aside to harden in a warm place and on a level surface, and are then ready for fixing in suitable frames. Naked -eye Appearances of Large Sections. In the sections of an actinomycotic tongue it is at once apparent that the new growth is more or less limited to the periphery of the section. In parts there are dense clusters of little nodular neoplasms, the fungus systems, each having a rounded form, and averaging in size that of a small pea. In other parts small nodules, varying in size from a millet-seed to a hemp-seed, have a linear arrangement between bundles of muscular fibres. The ACTINOMYCES. 389 appearance is suggestive of an invasion of the tongue along the lymphatics. In many of the nodules the largest tufts of the fungus can be seen, with the naked eye, to occupy a more or less central position. In parts the muscular fibres are replaced by fibrous tissue. If now these sections be placed under the microscope, the minute structure may be examined; but as it is obvious that still better results may be obtained by small sections, any part which it is necessary to examine with high powers can be selected from a corresponding part of the growth and prepared in the ordinary way. In the case of a "wen" the whole growth can be excised with the surrounding tissues, sliced and treated in the way already described, and sections stained by different methods. The nature of the growth is at once recognisable as actinomycosis from the characteristic honeycombed appear- ance produced by the trabeculae of fibrous tissue which form a spongy structure, from the loculi of which the fungus tufts and caseous matter have for the most part dropped out. In other parts this structure is intact, and the tufts of the fungus can be detected with the naked eye, and readily recognised with a pocket lens. A fungus system may be studied more minutely in ordinary sections of the tongue. Each nodule is composed of the actinomyces surrounded by round cells and epithe- loid cells, and beyond this fibrous tissue often forming a distinct capsule. In some specimens the fungus is sur- rounded by a single row of large multinucleated cells, and in other specimens the fungus is found in the interior of large oval giant cells. THE FUNGUS IN MAN. Examination in the Fresh State. A close examination of pus from a case of actinomy- cosis reveals the yellowish grains which a casual observer 39 APPENDIX. might mistake for grains of iodoform. On collecting some of the discharge in a test-tube, and holding it between the light and the eye, the tufts of fungi appear as brownish or greenish-brown grains, embedded in a mucopurulent matrix. On spreading some of the discharge on a glass slip, and examining in the same way described for the bovine disease, the largest tufts of the fungus are found to be about the size of the head of a small pin. They have a distinctly sulphur-yellow colour by reflected light, but appear of a yellowish or greenish-brown tint by trans- mitted light. With a needle or a platinum wire flattened at the end into a miniature spatula, the grains can be readily picked out of the discharge, or taken off the dressing, transferred to a clean slide, and gently covered with a cover- glass. Examined with an inch objective, they have the appearance of more or less spheroidal masses of a pale greenish-yellow colour. On removing the preparation from the microscope, and gently pressing down the cover-glass with the finger, the grains flatten out like specks of tallow, and on again examining with the same power they are found to have fallen apart into a number of irregular and sometimes wedge-shaped frag- ments of a faintly brown colour, affording a characteristic appearance. By preparing another specimen, and cover- ing it with a cover-glass without completely flattening out the grains, the spherical, oblong, and reniform masses of which the tufts are composed can be recognised with a <|th in. objective, like the rosettes of clubs which are observed in cattle. By examining the peripheral part of a rosette with a rath in., and especially after pressing the grains into a thin layer, with or without the addition of a drop of glycerine, the characteristic clubs are most readily demonstrated, and the most varied shapes observed by carefully examining the form of the individual elements. As in the bovine fungus, every variation in form is found from single clubs to clubs with lateral offshoots, clubs 1'LVH- M nj p. \ -' t (W Fig. 2. 'r ACTINOMYCES. 391 bifid at the extremity, palmate or fan-shaped groups, and banana-like bunches. In many cases the clubs are divided by transverse fission into two, three, or more segments. As a rule, the clubs are irregular in shape, and of about equal size, while a few are conspicuous by their length. In other parts of a preparation the clubs are replaced by long slender forms, which are sometimes transversely divided into a number of short links. With suitable illumination many clubs are seen to taper off into slender filaments. In addition there are free fila- ments, which are twisted, branched, and sometimes distinctly spirilliform. Many of the clubs are composed of layers differing in their refractive power, and many have the appearance of a central channel. There are also in the preparation small highly refractive bodies, fat granules, granular detritus, round cells, pus cells, and sometimes blood corpuscles (Plate XXXI.). The grains differ, as a rule, from those from a bovine source, in the absence of that sensation of grittiness so often transmitted to the finger when pressing the cover- glass upon them, and in the slightly greater tendency of the tufts to retain their compact form. By teasing the grains in a drop of water on a slide, and examining the preparation with a sixth or a twelfth objective, the explanation of the latter is forthcoming, for by this process the clubs are gradually washed away, and a central core remains which is composed entirely of a dense network of filaments. This can readily be observed by using a small diaphragm, and it will be found that the rosettes of clubs are now replaced by tangled masses having some resem- blance to miniature tufts of cotton-wool. These filaments constitute the mycelial network which is seen in sections stained by the method of Gram ; this can be readily verified by making a cover-glass preparation of the grains and staining by that method. The characters of the fungus can readily be studied by proper illumination with- out staining. The clubs have a faintly greenish tint, and 392 APPENDIX. in form and arrangement are quite characteristic and easily recognisable. Permanent preparations may be made by mounting the fungus in glycerine. THE FUNGUS IN STAINED SPECIMENS. The fungus may be stained in alcoholic solution of eosin, in the manner already described for the bovine organism, or in orange-rubin, and in either case mounted in glycerine. But although the fungus can be detected without any staining process, there may sometimes be doubtful appear- ances, and then cover-glass preparations should be made and stained by the method of Gram and eosin. The filaments can be readily recognised, and this is of great value, as it forms an additional means for the diagnosis of the disease. In combination with orange-rubin we have a test that is as characteristic and useful as staining for tubercle bacilli. The discharge, scraping from a growth, sputum, or the isolated fungus is squeezed between two cover-glasses, which are then slid apart ; they are allowed to dry, passed through the flame in the ordinary manner, and then stained. The cover-glasses can be cleared in clove-oil, the excess of clove-oil being removed by gentle pressure between pieces of blotting-paper, and then the preparation can be mounted in balsam and rendered per- manent. On examination of these specimens the masses of filaments will be found to be stained blue, and the tissue elements pink. These filaments vary very much in extent and character in different preparations. In some cases there are masses of short threads which are straight, or sinuous, or twisted, and sometimes branched. In other parts the field is occupied by very short, straight, or curved and sometimes spiral fragments ; in others, again, there are comparatively long strands. On examination with a high power, and with careful illumination, some filaments will be observed to be moniliform, while others .are provided with a terminal oval body. There are also free spherical PLATE 32. cg p. 392 ACTINOMYCES. 393 round bodies stained blue, which possibly represent the spores of the organism. If orange-rubin be used instead of eosin, the clubs will be stained and easily recognised. And this method enables one to determine the exact relation of the threads to the club-shaped bodies. This is an interesting point, as it has been suggested that the threads are not connected with the clubs, but are merely an adventitious micro-organism growing in the track of the ray-fungus. The threads are stained blue and the clubs crimson (Plate XXXIL). In the younger clubs the protoplasm of the thread can be traced into the interior of the club. In some of the older clubs the central portion takes a yellowish stain, and in others the protoplasm is not continued as a thread, but is collected into a spherical, ovoid, or pear-shaped mass. In others, again, irregular grains, stained blue, are scattered throughout the central portion. The sheath of the thread is stained pink ; and the protoplasm, stained blue, fills the sheath, or consists of small spherical and irregular grains, giving a distinctly beaded appearance. The effect of various reagents should be tried upon the isolated grains. The grains are picked out of the pus and transferred to watch-glasses containing strong potash, xylol, and benzole. If returned to a slide and covered with a cover-glass, the clubs are found unaltered. Water or weak potash washes away the clubs, and the filaments become easily distinguished ; ether and strong acids have no effect upon them. Corallin soda, Hanstein's violet, and iodine zinc-chloride fail in giving any particular re- action. Hoffman's blue stains the clubs, but without bringing out any structural details which could not be observed in the unstained specimens. CULTIVATION EXPERIMENTS. Bostrom succeeded in cultivating the fungus outside the animal body ; and finding that his cultures consisted of what appeared to be cocci forms, short rods, and threads, 394 APPENDIX. but no clubs, he advanced the theory that the fungus belonged to the bacteria, forming one of the Cladothrix group, and possibly closely allied to the Streptothriv Fcersteri of Cohn. In accordance with this view, Bostrom regarded the clubs as comparable to the degenerate or involution forms, which are often found in old cultivations of bacteria. They constituted, in his opinion, the lifeless organism. According to the old theory the actinomyces was a hyphomycetous fungus, and the clubs were the flask-shaped structures or gonidia. In the hope of throwing some light on this point, the author has made attempts at cultivation upon a number of different media. Glycerine agar-agar, a favourable medium for the growth of the tubercle bacillus, suggested itself as a suitable soil for actinomyces. An actinomycotic abscess was opened, the discharge collected in sterilised tubes, and cultivations prepared with as little delay as possible. Some of the discharge was spread out on a sterilised glass slide, and the grains isolated with sterilised needles and quickly transplanted on the surface of the nutrient medium. The tubes were placed in the incubator at 37 C, and the result watched from day to day. For several days there was no promise of success to the naked eye, but gradually the grains began to change, and by the end of a fortnight there was an appreciable increase in size. Numerous cover-glass preparations were made from what was originally a single grain, and on examination by the method of Gram the appearance was very striking. There could be no doubt as to the increase of the mycelial structure. The dense masses of filaments covered almost the w r hole area of the preparation. In parts less thickly covered there were a vast number of oval bodies and rod-like segments with terminal enlarge- ments. These " crocus " forms corresponded with the appearances previously described as met with in the interior of certain clubs. From this it would appear that ACTINOMYCES. 395 some other condition is necessary for the development of the fully formed club, this being the result of the sheath undergoing some change, possibly mucilaginous, resulting in the formation of a thick investment of the clubbed mass of protoplasm at the end of the thread. These club-shaped bodies represent organs of fructifi- cation, rather than the results of degeneration or death. The difficulty in accepting the view of their being entirely lifeless forms lies in the fact that one can observe daughter clubs growing from the mature clubs ; and, further, in the bovine fungus the author has been able to trace by this process the stages in the development of a single club to a completely formed rosette. In the unstained condition, the clubs are found, on the whole, to be very regular in their form and arrangement, while by certain staining methods they can be shown to have a somewhat complex structure. If we take all the characters into account, and particularly the minute structure and the relation to each other of the threads and clubs, we are justified in the opinion that the club in the early stages is an integral part of the living fungus, and that these characters bring the fungus into relation with a higher group of micro-fungi, the Basidiomycetes, although the filaments, regarded by themselves, correspond with the characters of streptothrix. LIFE-HISTORY. It has not been possible to trace every step in the life- history of many of the Basidiomycetes ; but if we regard the ray-fungus in the light of what is known to occur in many species, we may explain its life-history as follows : The spores sprout into hyphae, which form ex- cessively fine, straight, or sinuous, and sometimes distinctly spirilliform threads, which branch irregularly and sometimes dichotomously. The extremities of the branches develop the club-shaped bodies. The clubs are closely packed together, so that a more or less globular body is formed, 396 APPENDIX. with a central core composed of a dense mycelium. The threads can be differentiated by the method of Gram into an external sheath, and protoplasmic contents. The club-shaped body externally appears to be mucilaginous, while internally it is continuous with the protoplasm of the thread. It is difficult to say what further changes occur in the club-shaped bodies ; in all probability they represent the organs of fructification. If so, the proto- plasm in the interior of the club may possibly undergo changes leading to the development of spores, which are ultimately set free ; in some cases the terminal segment of a club is separated by transverse fission in the form of a globular body, a process resembling the formation of spores by abj unction. In others, the forms sprout- ing from the club are suggestive of teleutospores. In whatever way they may be formed, there can be little doubt that spores are set free in the vicinity of a rosette, and give rise to fresh individuals ; the ultimate result recalling, as has previously been suggested, the appear- ance of "fairy rings." There can be little doubt that spores and young fungi are taken up by wandering cells, and conveyed to a distance from the parent fungus, and thus fresh centres of growth are established. According to the view here propounded, the filaments or threads may be regarded as extremely delicate hyphse forming a dense mycelium, and the more familiar clubs as the basidia of the hymenial layer. There are occasionally long slender forms, very different from the ordinary clubs ; they possibly represent fiaraphyses or abortive elements. FLAGELLATED PROTOZOA. 397 APPENDIX C. FLAGELLATED PROTOZOA IN THE BLOOD.* WHEN examining blood the bacteriologist must be pre- pared to meet with minute organisms which at the first glance under moderate amplification may be mistaken for vibrionic or spiral forms of bacteria. The organisms referred to belong not to the vegetable but to the animal kingdom. They may occur associated with disease, but FIG. 130. PARASITES IN THE BLOOD OF RATS [after Lewis]. they appear to be more commonly found in the blood of apparently perfectly healthy animals. Flagellated organisms in the blood of rats and hamsters. Lewisf described peculiar organisms in the blood of healthy rats in India. When first noticed they were thought to be vibrios or spirilla. A drop of blood under examination appeared to quiver with life, and on diluting the blood motile filaments could be seen * Abstract of paper by the Author, Journ. Roy. Micros. Soc., read November loth, 1886. j* Lewis, Microscopic Organisms in the Blood of Man and Animals. Calcutta, 1879 (with photographs) ; and Quart. Journ. Micr. Sci., Ixxiii. (1879), pp. 109-14, and xxiv. (1884), pp. 357-69. APPENDIX. rushing through the serum, and tossing the blood - corpuscles about in all directions. Under careful exami- nation the filaments were found to consist of a thicker portion or body, with at one end a flagellum (Fig. i 30). After fixing with osmic acid, they measured 0*8 I /x,in width, and 20 30 //, in length ; the flagellum was about as long as the body, so that the total length of the organism was about 50 p. Lewis detected these parasites in 29 per cent, of the species Mus decumanus and Mus rufescens. Though they had many features in common with motile organisms of vegetable origin, they appeared to approach much more closely to the Protozoa, more par- ticularly several of the species of Dujardin's Cercomonas. Wittich* discovered, in the blood of hamsters, whip- like bodies with lively movements. They resembled frog's spermatozoa, possessing a thick portion continued into a long lash-like thread. Wittich considered them identical with the organisms described by Lewis, and they also were observed in apparently healthy animals. Koch j~ later met with the same organisms. Flagellated organisms in the blood of horses, mules, and camels. In India a fatal disease, known by the natives as Surra, occurs in horses, mules, and camels. The malady is described as a blood disease, characterised by fever, accompanied by jaundice, petechiae of mucous membranes, great prostration, and rapid wasting, terminating in death. Evans J observed the presence of a parasite in the blood, and by means of subcutaneous inoculation, and by the introduction into the stomach of blood con- taining the parasites, the disease was transmitted to healthy animals. * "Spirillen im Blut von Hamstern," CentralbL.f. Med. Wiss. 1881, No. 4. t Mittheilungen aus dem Kaiser lich. Gestmdh. Ami. 1881. % Evans, Report published by the Punjab Government Military Department, No. 439. 1880. FLAGELLATED PROTOZOA. 399 Steel,* who was deputed to investigate this disease in British Burma, also found the parasite in all cases, and further observed that it appeared as the temperature rose and disappeared during the apyrexial periods. This observer concluded that the organism was a spiral bac- terium, and named it, after its discoverer, Spirochceta Evansi (see p. 402). FlG. 131. H^EMATOMONAS COBITIS. a, First variety ; b, second variety ; c, third variety. d, First variety in a state of diminished activity. corpuscle, as if to obtain a point d'appui, while lashing its flagellum in all directions (Fig. 135, b\ At other times, when the parasite has impinged with its posterior extremity against a corpuscle, or the stiff filament is apparently en- tangled in debris, the movements of the organism give one the idea of its endeavouring to set itself free. In the active state the thicker portion, or body, appears to twist and bend from side to side with great activity. The organism can turn completely round with lightning rapidity, so that the flagellum, at one moment lashing FIG. 135. MONADS IN RAT'S BLOOD, x 1200. a, A monad threading its way among the blood-corpuscles ; b, another with pendulum movement attached to a corpuscle ; c, angular forms ; d, encysted forms ; e and f, the same seen edgewise. in one direction, is suddenly observed working in the opposite direction. Then suddenly the organism makes progression, and it can be distinctly seem to move in the direction of the flagellum, the flagellum threading its way between the corpuscles and drawing the rest of the organism after it. By treating cover-glass preparations with osmic acid, the appearances corresponded exactly with photo- graphs of the organisms observed by Lewis in India, so that the author has no doubt of their identity, in spite of the descriptions not completely according. A great like- ness to the organisms described by Mitrophanow, and to the Surra parasite, as just described, was obvious ; and 406 APPENDIX. after staining the rat parasites, the closest examination confirmed the belief that they were morphologically identical with the stained parasites of Surra. The cover-glasses with a thin layer of blood should be passed through the flame of a Bunsen burner in the way commonly employed for examining micro-organisms, and stained with an aqueous solution of fuchsine, methyl-violet, or bismarck-brown. The following method will, how- ever, be found most instructive : Use freshly prepared saturated solution of fuchsine or methyl-violet in absolute alcohol, and put a drop with a pipette on the centre of FIG. 136. MONADS IN RAT'S BLOOD, showing membrane under different aspects, blood-corpuscles some crenated, and stained discs, x 1200. the preparation ; do not disturb the drop-form for a few moments ; then, before the alcohol has evaporated, wash off the excess of stain. It will be found that where the drop rested the organisms will be very deeply stained, while in the surrounding area the colour will vary in intensity. By the effect of the different degrees of staining much maybe learnt (Fig. 136). In one organism the body and entire membrane will be equally stained ; in another the margin of the membrane only. In some the posterior stiff filament is stained, and at its base a darkly stained speck is very striking ; and in other cases, again, the posterior filament is only faintly tinged, or an unstained spot occurs near its base. FLAGELLATED PROTOZOA. 407 The morphological identity of the rat and Surra parasites is thus established, and both seem morpho- logically identical with the organism of Mitrophanow. If we follow Mitrophanow, we must, therefore, enlarge his genus of Hcematomonas. The author ventures, how- ever, to disagree with Mitrophanow in the advisability of adopting this entirely new generic name. Mitrophanow suggested this new term because of the special habitat normal fish-blood of the species he discovered. But the characteristic features of these organisms are the characteristic marks of the genus Trichomonas* They are, therefore, embraced by the genus Trickomonas, and there is no need to create a new one. If it were not for the different description given by Mitrophanow of the organism in the blood of Cobitis fossilis, the author would be inclined to say that all these organisms belonged to one and the same species, which might well be named Trichomonas sanguinis. The monads in the rat and the Surra parasite have been shown to be morphologically identical with each other, and both, as far as one can judge from the description, are morphologically identical with the monad in the blood of the carp. We have, how- ever, seen that the organism in Surra is believed to be pathogenic, and too much stress must not be laid on morphological identity. There is strong evidence in favour of believing in its pathogenic properties ; but, at the same time, it must be borne in mind that the organism has never been isolated apart from tJte blood, and the disease then produced by its introduction into healthy animals. It is quite possible that the parasites in Surra are only associated with the disease, the impoverished blood affording a suitable nidus for their development, while the contaminated water may be the common source of the organism and of the disease. On the other hand, the organism in the rat is found in apparently perfectly healthy, well-nourished animals. * Vide Leuckart, The Parasites of Man, translated by Hoyle, 1886. 408 APPENDIX. APPENDIX D. H^MATOZOA OF MALARIA (Laveran). IN 1880, Laveran in Algiers noticed the existence of peculiar structures in the blood of a patient suffering from malaria. It occurred to him that he had discovered the " Microbe du paludism" Laveran followed up his ob- servation by researches which were communicated to the Academy of Medicine in Paris, in 1881 and 1882, and they were subsequently published in extenso in a treatise on the subject in 1884.* Laveran described various bodies which he was led to regard as different stages in the life-history of the same micro-parasite. The most striking forms were cylindrical elements with pointed extremities. They were crescent- shaped and pigmented in the middle. There were other forms, more frequently found, which were either free in the serum or in contact with the red blood-corpuscles. They were more or less spherical, pigmented, and endowed with amceboid movement. Other forms, again, were pro- vided with motile filaments three or four times as long as the diameter of a red blood-corpuscle. And, lastly, there were little masses of hyaline material, which Laveran regarded as dead forms. These observations at first attracted little attention ; but they have been confirmed and extended by Richard,t Councilman and Abbot,J Marchiafava and Celli, Golgi,|| * Laveran, Traite des Fievres Palustres. 1884. t Richard, Comptes Rendus: 1882. \ Councilman and Abbot, American Journal of Medical Sciences. 1885. Marchiafava and Celli, Archivio per le Scienze Mediche. 1885, 1886. Atti della R. Academia Medica. 18861887. Archiv. Ital. de Biolog. 18871 888. || Golgi, La Riforma Medica. 1888. Archimo per le Scienze Mediche. 1886. Fortschritte der Medicin. 1889. H^MATOZOA OF MALARIA. 409 Sternberg,* Osier, f Crookshank, and Vandyke Carter,^ and their importance fully recognised. The different forms assumed by the haematozoon of malaria may be described in two groups : those within the red blood-corpuscles and those free in the serum. Intra-corpuscular bodies. These are of three kinds : First, structureless protoplasmic bodies, much smaller than, and within or attached to, the red blood- corpuscles (Fig. 137). These rapidly change their shape, FIG. 137. NON-PIGMENTED AncEBOiD FORMS [after Marchiafava and Celli]. exhibiting amoeboid movement. They were first described by Marchiafava and Celli, and possibly represent the first stage in the life-history of the haematozoon. Marchia- fava and Celli suggested the name Plasmodium malaria. Second, minute masses of finely granular or of hyaline FIG. 138. PIGMENTED AMCEBoiD FORMS [after Golgi]. protoplasm enclosing granules of pigment (Fig. 138). These forms are sometimes present in large numbers, and at other times can be found only with difficulty. They are more or less spherical, but exhibit amoeboid move- ment, and rapidly change their form. The pigment * Steinberg, Medical Record. 1886. t Osier, British Medical Journal. 1887. \ Carter, Scientific Memoirs Med. Officers Army of India. 1888. 4IO APPENDIX. granules are also in active movement. There may be one or more of these amceboid bodies to a blood- corpuscle, and they vary in size ; one may occupy the whole of the corpuscle. In cases of pernicious malaria similar bodies may be seen in tissue sections, in the corpuscles filling the capillaries. Third, forms which appear like isolated grains, and larger homogeneous bodies surrounded by clear spaces which change in outline. Extra-corpuscular bodies. These are the most striking and perhaps the most interesting forms. First, the semi lunar bodies of Laveran. These are crescent- shaped bodies, sometimes pointed at the extremities, but more usually rounded off (Fig. 139). They are not FIG. 139. SEMI LUNAR BODIES OF LAVERAN [after Golgi] always curved ; some, indeed, are almost spherical, and others sausage-shaped. They are motionless. In many specimens a delicate line is visible on the concave side of the crescent, connecting the extremities. On careful examination this is found to be the edge of a very delicate membrane. The body is composed of homo- geneous protoplasm. Centrally placed is a collection of pigment granules, which on careful examination can be distinctly seen to be in movement. The semi lunar bodies vary in number in different cases. Sometimes several can be seen in the field at the same time, and in other cases they are only observed after a long and patient search. They are, as a rule, free in the serum ; but they have also been seen within the red blood- cells. Second, finely granular masses of protoplasm, H^MATOZOA OF MALARIA. 411 which arise, according to Golgi, from the intra-cor- puscular pigmented bodies. The pigment is collected in a rosette, and the protoplasm by segmentation gives rise to a number of small spherical forms, which are FIG. 140. ROSETTE FORMS WITH SEGMENTATION [after Golgi]. ultimately set free (Fig. 140). Golgi believes that these changes occur in definite relation to the development of the paroxysm. Third, spherical, pear-shaped, or ovoid bodies, rather smaller than the red blood-corpuscles, and provided with one or more actively motile flagella FIG. 141. FLAGELLATED FORMS [after Vandyke Carter]. I. A flagellated spherule; (a) the same in the interior of a phagocyte; ~(b] free motile filaments. (Fig. 141). These flagella are long lash-like filaments, which by their activity set the neighbouring blood- corpuscles in motion. Free filaments in active move- ment have also been observed. Fourth, small spherical pigmented bodies about one quarter the size of a red blood-corpuscle, which exhibit amoeboid movement. 4*2 APPENDIX. Inoculation experiments. Marchiafava and Celli assert that inoculation of a healthy subject with blood containing the parasites will produce a paroxysm of ague with development of the haematozoa. The pathogenic power of these parasites, however, has not been established. As in Surra disease, there has been no cultivation of the parasite outside the animal body, and reproduction of the disease with a pure cultivation. In favour of its being a pathogenic organism, Laveran points out its invariable presence in some form or other in cases of malaria ; the marked changes it effects in the red blood-cells ; the increase in the number of the parasites in proportion to the severity of the attack ; and, lastly, their disappearance after the administration of quinine. Others, again, have doubted the parasitic nature of these be/dies, and have looked upon them as repre- senting pathological changes in the blood-cells.* Laveran first of all suggested the name Oscillaria malaria, but subsequently he recognised that these bodies belonged to the animal, not to the vegetable king- dom. Osier has suggested that, temporarily at any rate, the organism should be placed in the genus Hcematomonas of Mitrophanow, thus : " Genus, Hcematomonas ; species, Htcmatomonas malaricz. Definition Body plastic ; ovoid or globose ; no differentiation of protoplasm, which con- tains pigment grains ; flagella variable from one to four, highly polymorphic, occurring in (i) amoeboid form, (2) crescents, encysted form, (3) sporocysts, (4) cellular free pigmented bodies." EXAMINATION OF THE HAEMATOZOA OF LAVERAN. In the living condition. Select a patient by pre- ference who has had several attacks of malaria, and is markedly anaemic. Examine before the invasion of the febrile paroxysm. Take two perfectly clean cover-glasses * Cattaneo and Monti, Archivio fier le Scienze Mediche. 1888. H^MATOZOA OF MALARIA. 413 and two clean slides. Wash one of the fingers of the patient with soap and water, and then cleanse with alcohol. Apply a ligature, and with a clean needle puncture the thin skin near the root of the nail. Touch the drop of blood which collects with a clean slide. Cover quickly with a cover-glass, and gently press it if the layer of blood be too thick. Examine with a T ^ o.i. In stained preparations. Puncture the finger again if necessary, touch the droplet of blood with a clean cover-glass, apply another cover-glass, press them gently together, and then slide them apart. Stain with two or three drops of alcoholic solution of methylene-blue, wash off excess and examine in water, or allow the preparation to dry, and mount in balsam. APPENDIX. APPENDIX E. CHRONOLOGICAL BIBLIOGRAPHY. A. METHODS. B. MORPHOLOGY AND CLASSIFICATION. C. GENERAL BIOLOGY. D. ZYMOGENIC SAPROPHYTES AND FERMENTATION. E. CHROMOGENIC SAPROPHYTES. F. SIMPLE SAPROPHYTES. G. PTOMAINES AND PUTREFACTION. H. ANTISEPTICS AND DISINFECTANTS. I. IMMUNITY. J. BACTERIA ASSOCIATED WITH DISEASES IN MAN AND ANIMALS XIX. OPHTHALMIC DISEASES. XX. OSTEOMYELITIS. XXI. PLEURO-PNEUMONIA. XXII. PNEUMONIA. XXIII. PUERPERAL FEVER. XXIV. PYAEMIA AND SEPTICAEMIA. XXV. RELAPSING FEVER. XXVI. RHINOSCLEROMA. XXVII. SCARLATINA. XXVIII. SWINE-ERYSIPELAS. I. ACTINOMYCOSIS. II. ACUTE YELLOW ATROPHY. III. ANTHRAX. IV. CATTLE PLAGUE. V. CEREBRO-SPINAL MENINGITIS. VI. CHICKEN-CHOLERA. VII. CHOLERA. VIII. DENTAL CARIES. IX. DIPHTHERIA. X. ERYSIPELAS. XL ENDOCARDITIS. XII. GLANDERS. XIII. GONORRHOEA. XIV. HYDROPHOBIA. XV. LEPROSY. XVI. MALARIA. XVII. MALIGNANT CEDEMA. XVIII. MEASLES. XXIX. SWINE-TYPHOID. XXX. SYMPTOMATIC ANTHRAX. XXXI. SYPHILIS. XXXII. TETANUS. XXXIII. TUBERCULOSIS. XXXIV. TYPHOID FEVER. XXXV. VARIOLA AND VACCINIA. XXXVI. 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Zeitschr. f. Hygiene. Beumer. Deut. Med. Woch. Frankland. Proc. Roy. Soc. Bischof. Journ. Soc. Chem. Industry. Pfeiffer. Zeitschr. f. Hygiene. 452 APPENDIX. APPENDIX F. TABLE SHOWING THE MAGNIFYING POWER OF ZEISS' OBJECTIVES. Ocular: 12345 a, a 2 a 3 a* aa A, AA B, BB C, CG D, DO E F G H J K L A 1 7 11 15 22 12 17 24 34 20 27 38 52 412 717 1024 22 30 41 56 75 38 52 71 97 130 70 95 130 175 235 120 145 195 270 360 175 230 320 435 580 270 355 490 670 890 405 540 745 1010 1350 260 340 470 640 855 320 430 590 805 1075 430 570 785 1070 1430 570 760 1045 1425 1900 770 1030 1415 1930 2570 260 340 470 640 855 380 505 695 950 1265 605 810 1110 1515 2020 a, a, a 3 a* aa A, AA B, BB C, CC D, DO E F G H J K L 1 TS 1 18 INDEX. Abbe condenser, 23 Abrin, 345 Abrus precatorius, 345 Abscesses, 229 Acetate of potash, 34 Acetic acid, 27 Achorion Schoenleinii, 376 Actinomyces, 377. methods of staining, 383 Actinomycosis, 379 Acute infectious osteomyelitis, coccus of, 246 Acute yellow atrophy, 250 ./Eroniscopes, 129 /Eroscopes, 129 Agar-agar, 41 glycerine, 87 nutrient, preparation of, 86 Air, examination of, 126 Aitken's test-tube, 48, no Alcohol, 25 acidulated, 27 Alopecia areata, 266 Alum carmine, 27 Ammonia, 28 Aniline, 28 water, 28 Animals, examination of, 141 Anthrax, 315 Antiseptics, 180 Ascococcus, 257 Billrothii, 257 Ascomycetes, 376 Asiatic cholera, 284 Aspergillus albus, 378 clavatus, 378 flavus, 377 fumigatus, 377 glaucus, 376 niger, 377 ochraceus, 378 repens, 376 Asphalte lac, 34 Attenuation of virus, 193 B Babes' incubator, 53 method, 67 Babes' of staining comma-bacilli, 291 of staining bacillus of leprosy 301 Bacillus, 299 acidi lactici, 339 aerophilus, 348 alvei, 335 amy! obacter, 351. anthracis, 315 butyricus, 351 caucasicus, 345 cavicida, 273 columbarum, 267 cuniculicida, 269 cyanogenus, 337 dysodes, 346 erythrosporus, 346 figurans, 344 Fitzianus, 339 fluorescens, 336 foetidus. 347 Hansenii, 346 ianthinus, 337 indicus, 274 in gangrenous septicaemia, 305 in septicaemia of man, 305 in swine-erysipelas, 333 in swine-typhoid, 333 in syphilis, 301 in tetanus, 334 leprae, 299 liordermos, 278 luteus, 276 malariae, 304 mallei, 325 megaterium, 343 mesentericus fuscus, 348 vulgatus, 348 multipediculus, 278 mycoides, 345 Neapolitanus, 263 cedematis maligni, 327 of blue milk, 337 of choleraic diarrhoea from meat- poisoning, 304 of diphtheria, 265 of glanders, 325 of jequirity, 345 of pneumo-enteritis of the pig, 333 454 INDEX. Bacillus of rhinoscleroma, 264 of septicaemia of mice, 330 of splenic fever, 315 of ulcerative stomatitis in the calf, 332 oxytocus perniciosus, 273 parvus ovatus, 252 phosphorescens. 348 pneumonicus agilis. 272 prodigiosus, 275 pseudo-pneumonicus, 263 putrificus coli, 347 pyocyaneus, 336 pyogenes foetidus, 305 ramosus liquefaciens, 278 saprogenes, No. I, 347 No. 2, 347 No. 3, 266 foetidus, 347 septicus, 346 agrigenus, 269 sputigenus, 271 subtilis, 340 tremulus, 345 tuberculosis, 305 tumescens, 342 typhosus, 302 ulna. 342 urese, 276 violaceus, 337 virens, 147 vitulorum, 266 Bacteria, chromogenic, 172 classification of, 205 distribution of, 178 general morphology of, 152 pathogenic, 174 saprogenic, 173 zymogenic, 172 Bacteriaceae, 215 Bacteridie du charbon, 315 Bacteridium cyaneum, 253 Bacterium, 260 aceti, 277 geruginosum, 336 brunneum, 276 cavicida, 273 chlorinum, 147 choleras gallinarum, 267 coli commune, 273 crassum sputigenum, 272 decalvans, 266 fluorescens liquefaciens. 276 putidum, 276 fcetidum, 347 fusiforme, 280 hyacinthi, 274 ianthinum, 337 Bacterium indicum, 274 in diphtheria of calves, 266 of man, 265 lactis aerogenes, 273 lineola, 282 liodermos, 278 litoreum, 280 luteum, 276 merismopedioides, 279 multipediculum, 278 navicula, 280 Neapolitanum, 263 of Davaine's septicaemia, 270 of diphtheria of pigeons, 267 - of fowl-cholera, 267 of rhinoscleroma, 264 of septicaemia in rabbits, 269 of yellow milk, 274 oxytocum perniciosum, 273 Pasteuri, 271 Pasteurianum, 277 Pflugeri, 280 photometricum, 280 pneumoniae cruposse, 261 pneumonicum agile, 272 prodigiosum, 275 pseudo-pneumonicum, 263 ramosum liquefaciens, 278 rubescens, 357 rubrum, 275 saprogenes, 266 septicum agrigenum, 269 sputigenum, 271 syncyanum, 337 synxanthum, 274 termo, 281 ureae, 276 violaceum, 276 viride, 147 xanthinum, 274 Zopfii, 278 Balance and weights, 40 Balmer-Frantzel method, 312 Baumgarten's method, 311 new method, 312 Bees, bacillus in disease of, 335 Beggiatoa, 356 methods of examining species of, 356 alba, 356 mirabilis, 356 roseo-persicina, 357 Bergamot oil, 25 Biere malade, 256 Bismarck-brown, 28 Blackleg, 350 Black torula, 371 Bleeding host, 275 INDEX. 455 Blood rain, 275 serum, liquid, 107 sterile, 104 Blue milk, bacillus of, 337 Borax carmine, 28 Botrytis Bassiana, 379 Bouillon, 1 06 Bread-paste, 103 Brush, 46 Bulbed tubes, 49 Butyric acid fermentation, bacillus of, 351 Camera-lucida, 34 Canada-balsam, 34 Caoutchouc caps, 42 Carbonate of soda, 41 Carious teeth, 361 Cattle plague, 236 Cedar oil, 28 Cell-contents, 149 Cell-wall, 148 Celloidin, 25, 74 Cerebro-spinal meningitis, 238 Charbon symptomatique, 350 Chemical composition, 147 disinfectants, 183 Cheshire's trough, 89 Chionyphe Garten, 379 Cholera, comma-bacillus of, 284 fowl, 267 nostras, comma-bacillus in, 291 Choleraic diarrhoea from meat- poison- ing, bacillus of, 304 Cladothrix, 362 dichotoma, 362 Fcersteri, 364 Cladotrichese, 216 Classification, 205 Fliigge's, 210 Hueppe's, 217 Zopf's, 215 Clostridium, 350 butyricum, 351 of symptomatic anthrax, 350 polymyxa, 353 Coccaceae, 215 Cocci, methods of staining, 258 Cohn-Mayer fluid, 108 Cohnia roseo-persicina, 357 Collection of water samples, 133 Comma-bacillus of Finkler, 291 of Koch, 284 in cholera nostras, 291 Cork, 25 Cornil and Alvarez, method of, 265 Cotton-wool, 40 Cover-glass impressions, 69 preparations, 65 double coloration, 67 Crenothrix, 354 Kiihniana, 354 Cutaneous inoculation, 138 Cutting tissues, 72 D Damp chambers, 26, 45, 92 D'Arsonval's incubator, 49 Decalcifying preparations, 71 Dental caries, 361 Desiccator, 60 Diphtheria, 265, 266 Diplococcus albicans tardissimus, 242 Disinfectants, 180 Dissecting boards, 58 case, 58 Dissection, 141 Distribution of bacteria, 178 Double coloration, 67 Double-stain spore-bearing bacilli, 68 Double-staining, 67 Dressing-case, 58 Drinking water, 131 Drop-cultures, 112 Drop-culture slides, 49 Ebner's solution, 25 Ehrlich's method, 67 and eosin, 314 Electricity, application of, 121 Embedding tissues, 72 Empusa muscse, 372 radicans. 373 Endocarditis ulcerosa, 236 Eosin, 28 Erysipelas, 230 malignum, bacillus des, 333 Ether, 29 Eurotium aspergillus glaucus, 376 aspergillus niger, 377 repens, 376 Examination of plate-cultivations, 96 of test-tube cultivations, 89 Experiments on living animals, 137 Farrant's solution, 34 Favus, 376 Filter, making, 85 paper, 41 Fire blight, 253 456 INDEX. Flagella, to stain, 69 Flagellated protozoa in blood, 397 Flagellum, 157 Flannel, 41 Folded filter, 85 Foot-and-mouth disease, 235 Form, 152 Foulbrood, 335 Fowl-cholera, bacterium of, 267 Frankel's method, 313 Friedlander, method of, 262 Frogspawn fungus, 296 Fuchsine, 29 Gangrene, 250 Gas burners, 52 chambers, 120 pressure regulator, Moitessier's, 54 Gases, effect of, 170 Gelatine. 26. 40 nutrient, preparation of, 82 plates, 94 Gelatinous envelope, 150 Gentian-violet, 29 Gibbes' first method, 311 magenta solution, 29 new method, 311 solution for double-staining, 29 Glanders, bacillus of, 325 Glass bells, 43 benches, 44 capsules, 48 - dishes, 43, 45 jar, 46 plates, 44 rods, 44 vessels, 39 Glycerine agar-agar, 87 gelatine, 26 gum, 34 pure, 29 Gomme de sucrerie, 296 Gonorrhoea, 241 Gram's method, 76 solution, 30 Growth, circumstances affecting, 169 products of, 171 H Hsematomonas carassii, 400 cobitis, 399 Evansi, 404 Hsematozoa of malaria, 408 Haematoxylin solution, 30 Haemophilia neonatorum, 249 Hardening pieparations, 71 Hay-bacillus, 340 methods of staining, 342 Heat regulator (Meyer's), 57 (Reichert's), 55 (Schlosing's), 51 Herpes tonsurans, 376 Hesse's apparatus, 127 His' method, 68 Hollis' glue, 34 Hot air and steam, 189 Hot-air steriliser, 37 Hot-water filter, 39 Hydrophobia, 250 Hyphomycetes, 371 Hypodermii, 372 I Immunity, 192 Impression-preparations, 69 Incubators, 49 Babes', 53 D'Arsonval's, 49 Inoculation of test-tubes, 88 cutaneous and subcutaneous, 138 of potatoes, 101 protective, 193 Intermittent fever, bacillus of, 304 Iodine solution, 30 (Gram), 30 Iron box, 44 Isolation of micro-organisms. 90 Israel's case, 44 warming apparatus, 118 J Japanese isinglass, 41 Jequirity, bacillus of, 345 K Kaatzer's method, 314 Kephir, 345 Kleinenberg's solution, 26 Koch's apparatus for examination of air, 127 method for staining comma- bacilli, 290 original method for staining the tubercle-bacillus, 309 postulates, 17 solution (methylene-blue), 31 solution (methyl- violet), 31 Laboratory requisites, 23 Lactic acid, 41 INDEX. 457 Leprosy, 299 Leptothrix, 361 buccalis, 361 gigantea, 362 ochracea, 362 parasitica, 362 Leptotrichese, 215 Leuconostoc, 296 mesenteroides, 296 Leukaemia, 239 Levelling apparatus, 91 Lichtheim's method, 313 Light, 171 Liquid media, 106 Lister's flasks, 48, 109 Lithium-carmine solution, 30 Litmus papers, 41 Lochial discharges, 242 Loffler's solution, 31 Lustgarten, method of, 301 M Madura-foot, 379 Magenta solution (Gibbes'), 30 Malaria, 304 Malignant oedema, bacillus of, 327 pustule, 315 Marsh-spirochaste, 294 Measles, 249 Merismopedia, 241 gonorrhoea, 241 Methylene-blue, 30 Methyl- violet, 31 Metschnikoft's theory, 201 Meyer's thermo-regulator, 57 Micrococcus, 245 albicans amplus, 242 amylivorus, 253 aurantiacus, 253 bombycis, 253 candicans, 254 candidus, 254 cereus albus, 248 flavus, 248 chlorinus, 254 cholerae gallinarum, 267 cinnabareus, 255 citreus conglomerate, 242 coronatus, 256 crepusculum, 255 cyaneus, 253 flavus desidens, 256 liquefaciens, 255 tardigradus, 255 foetidus, 255 fulvus, 256 Micrococcus haematodes, 254 indicus, 274 in acute yellow atrophy, 249 in gangrene, 249 in haemophilia neonatorum, 249 in measles, 249 in rabies, 249 in scarlatina, 249 insectorum, 253 in typhus, 249 in whooping-cough, 249 lacteus faviformis, 256 luteus, 254 of progressive suppuration in rabbits, 251