GIFT OF MICHAEL REESE BIOLOSY LIBRARY 6 MANUAL OF BACTERIOLOGY BY THE SAME AUTHOR. Royal 8v0, izs. 6d. PHOTOGRAPHY OF BACTERIA. Illustrated by Eighty-six Photographs reproduced in Autotype. LONDON : H. K. LEWIS, 136, GOWER ST., W.C. MANUAL OF ACTERIOLOGY BY EDGAR M. CROOKSHANK M.B. (LoND.), F.R.M.S. DEMONSTRATOR OF PHYSIOLOGY, KING'S COLLEGE, LONDON SECOND EDITION, REVISED AND CONSIDERABLY ENLARGED ILLUSTRATED WITH COLOURED PLATES AND WOOD ENGRAVINGS LONDON H. K. LEWIS, 136 GOWER STREET, W.C. MDCCCLXXXVII. [All Rights Reserved.} 1U/ BIOLOGY LIBRARY* G 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, C&te OUorfc us JDeBicateD AS A TOKEN OF ADMIRATION AND RESPECT BY THE AUTHOR. 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 as much as possible in accordance Vlll PREFACE TO THE SECOND EDITION. with the chapters, and in chronological order, may be useful to those seeking further details. No doubt Professor Baumgarten's Jakresbericht, 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. PREFACE TO THE FIRST EDITION. A BRANCH of study, which has opened fresh paths in pathology and therapeutics, is so important that it may in time become a more essential part of the medical curriculum, and is perhaps worthy of an elementary handbook, which shall include a systematic sketch of the genera and species of micro-organisms, as well as the methods employed in the investigation of their life-histories. Having myself experienced the want of a practical aid to the study of Bacteriology by the methods introduced by Professor Koch, I thought that it might be useful to those wishing to commence this study by these recent methods, if I embodied my notes made in different laboratories in the form of a Manual for Students. The work is thus intended to help the student beginning the study of a subject, the literature of which, in English, is for the most part diffused in numerous periodicals. The methods of " pure cultivation " of Bacteria will, after all, be found to be remarkable for their extreme simplicity, and can be easily mastered by X PREFACE TO THE FIRST EDITION. the careful clinical worker, to whom it is hoped this little book may also prove useful. I take this opportunity of expressing my best thanks to Professor Virchow, who materially fur- thered my work in the Pathological Institute of Berlin by kind advice and generous assistance. I am most grateful to Dr. Babes, of Budapest, for his ever-ready co-operation ; and to Professor Johne, of Dresden, who also placed his laboratory at my disposal, and to whom I am particularly indebted for much of the material from which the microscopical preparations were made. I would also wish gratefully to acknowledge the great interest and courteous assistance shown me on the part of Dr. Hauser, of Erlangen ; Professor Pettenkofer, Professor Bellinger, and Dr. Biichner, of Munich ; and the officials of the new Hygienic Laboratory in Berlin. The original drawings from which the coloured plates of test-tube- and potato cultivations are reproduced were made by my wife from cultiva- tions prepared especially for the purpose, and selected as typical. The coloured plates of the microscopic appearances are reproductions of my own drawings, from specimens I had recently prepared. The drawings were made from parts selected as most characteristic from a great number PREFACE TO THE FIRST EDITION. XI of preparations, which have been since demon- strated at the meeting of the Royal Microscopical Society, on November 25th. All the plates have been most faithfully and skilfully reproduced by Messrs. Vincent Brooks, Day, & Son. In conclusion, I owe much to Professor Gerald F. Yeo, of King's College, London, for many valuable criticisms ; and to my colleague, Mr. Herroun, for his kindness in reading the proof- sheets. EDGAR M. CROOKSHANK. 24, MANCHESTER SQUARE, W., December, 1885. CONTENTS. PART I. GENERAL METHODS. CHAPTER I. PAGB INTRODUCTORY I CHAPTER II. APPARATUS, MATERIAL, AND REAGENTS EMPLOYED IN A BACTERIOLOGICAL LABORATORY . . . 5 a. Histological apparatus ...... 5 b. Reagents and materials employed in the processes of hardening, decalcifying, embedding, fixing, and cutting of tissues . . 7 c. Reagents for examining and staining microscopical preparations . 9 d. Reagents for mounting and preserving preparations . .16 c. Drawing and photographic apparatus . . . 17 f. Sterilisation apparatus . . . . . -19 g. Apparatus and material for preparing and storing gelatine- and agar-agar-peptone-broth . . . . .21 h. Apparatus for employment of nutrient jelly in test-tube and plate cultivations ....... 24 i. Apparatus for preparation of potato cultivations . . .28 j. Apparatus for preparation of solidified sterile blood serum . 29 k. Apparatus for storing, and for cultivations in, liquid media . 31 /. Apparatus for incubation . . . . . .32 ni. Inoculating and dissecting instruments and apparatus in common use 41 n. General laboratory requisites . . . . .42 XIV CONTENTS. CHAPTER III. PAGE MICROSCOPICAL EXAMINATION OF BACTERIA IN LIQUIDS, IN CULTIVATIONS ON SOLID MEDIA, AND IN TISSUES .45 a. Examination in the fresh state . . . . .46 b. Cover-glass preparations ; Methods of Ehrlich, Babes, and His . 48 c. Cover-glass impressions . . . . . S 2 CHAPTER IV. FREPARA TION AND STAINING OF TISSUE SECTIONS . 54 a. Methods of hardening and decalcifying preparations . . 54 b. Methods of embedding, fixing, and cutting . . -55 c. General principles of staining bacteria in tissue sections : methods of Weigert, Gram, and Weigert-Ehrlich . . -57 CHAPTER V. PREPARATION OF NUTRIENT MEDIA AND METHODS OF CULTIVATION 62 SOLID MEDIA : a. Preparation of sterile gelatine-, and agar-agar-peptone -broth . 64 b. Methods of employing nutrient jelly in test-tube- and plate-cultiva- tions ........ 69 c. Preparation and employment of sterilised potatoes, potato-paste, bread-paste, vegetables, fruit, and white of egg . . 82 d. Preparation and employment of sterile blood serum . . 86 LIQUID MEDIA : e. Preparation of sterilised bouillon, liquid blood serum, urine, milk, vegetable infusions, and artificial nourishing liquids . . 88 / 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 . . 91 CONTENTS. XV CHAPTER VI. PAGE EXPERIMENTS UPON THE LIVING ANIMAL . . 107 a. Inhalation of micro-organisms ..... 107 b. Administration with food .... . 107 c. Cutaneous and subcutaneous inoculation . . 108 d. Special operations . . . . . . . . 109 CHAPTER VII. EXAMINATION OF ANIMALS EXPERIMENTED UPON, AND THE METHODS OF ISOLATING MICRO-ORGAN- ISMS FROM THE LIVING AND DEAD SUBJECT a. Method of dissection and examination . . . . 1 1 1 b. Isolation of micro-organisms from the living subject . .114 PART II. GENERAL BIOLOGY OF BACTERIA. CHAPTER VIII. GENERAL MORPHOLOGY AND PHYSIOLOGY ' . .117 CHAPTER IX. ANTISEPTICS AND DISINFECTANTS . . . .150 CHAPTER X. IMMUNITY 162 PART III. SYSTEMATIC AND DESCRIPTIVE, WITH SPECIAL MICROSCOPICAL METHODS. CHAPTER XL CLASSIFICATION OF BACTERIA . . 175 XVI CONTENTS. CHAPTER XII. PAGE SYSTEMATIC AND DESCRIPTIVE . . . .194 GROUP I. COCCACE^; ....... 194 Genus I. Streptococcus . . . . . .195 ,, II. Merismopedia ...... 208 III. Sarcina . . . . . . .210 ,, IV. Micrococcus . . . . . .213 ,, V. ASCOCOCCUS ...... 222 GROUP II. BACTERIACE^E . . . . . . 224 Genus I. Bacterium ........ 225 ,, II. Spirillum . ..... 249 ,, III. Leuconostoc ...... 262 ,, IV. Bacillus . . x .... 265 V. Vibrio ....... 317 ,, VI. Clostridium . . . . . .318 GROUP III. LEPTOTRICHE^E . . . . . .321 Genus I. Crenothrix ...... 322 ,, II. Beggiatoa . 324 ,, III. Phragmidiothrix ...... 328 IV. Leptothrix . . . . . .329 GROUP IV. CLADOTRICHE^ ...... 330 Genus I. Cladothrix . . . . . -33 APPENDICES. A. Yeasts and moulds . . . 340 B. Flagellated protozoa in the blood . . -35 C. Examination of air . . 3 6r D*. Examination of soil ... 3^5 . Examination of water . , . 366 F. Chronological bibliography . . . 37 G. Table showing the magnifying power of Zeiss' objectives . . 430 LIST OF ILLUSTRATIONS. PLATES. PLATE I. Bacteria, Schizomycetes, or Fission-fungi . . Frontispiece II. Cultivations on nutrient agar-agar. Fig. I. Bacterium indicum. ,, 2. Bacillus cyanogenus. ,, 3. Bacterium prodigiosum. III. Cultivations in nutrient gelatine. Fig. I. Spirillum choleroe asiaticse. ,, 2. Bacterium cholerse gallinarum. ,, 3. Streptococcus cereus albus. IV. Cultivations in nutrient gelatine. Fig. i. Micrococcus tetragonus. ,, 2. Bacterium pneumonise crouposae. ,, 3. Saccharomyces niger. V. Cultivations in nutrient gelatine. Fig. I. Bacillus pyocyaneus. ,, 2. Sarcina lutea. ,, 3. Bacillus anthracis. VI. Plate-cultivation. First attenuation of the Spirillum Finkleri after twenty-four hours. VII. Plate-cultivation. Second attenuation of the Spirillum Finkleri after thirty-six hours. VIII. Cultivations on nutrient agar-agar. Fig. I. Sarcina lutea. ,, 2. Streptococcus pyogenes aureus. ,, 3. Bacillus pyocyaneus. IX. Potato-cultivations. Fig. I. Bacterium prodigiosum. ,, 2. Penicillium glaucum. b XV111 LIST OF ILLUSTRATIONS. PLATE X. Potato cultivations. Fig. i. Sarcina lutea. ,, 2. Saccharomyces rosaceus. XI. Bacillus tuberculosis. Fig. I. Pure cultivation on solid blood serum in a test-tube. ,, 2. Pure cultivation on solid blood serum in a glass-capsule. ,, 3. The same preparation as Fig. 2 ( x 80). ,, 4. Cover-glass impression-preparation of colonies ( x 7) Ehrlich's method. XII. Micrococcus tetragonus and Streptococcus pyogenes aureus. Fig. I . From a section of kidney of a mouse. Gram's method. ,, 2. From a section of liver of a rabbit. Gram's method. XIII. Cultivations on nutrient agar-agar. Fig. i. Sarcina lutea. ,, 2. Bacterium indicum. ,, 3. Saccharomyces rosaceus. XIV. Cultivations on nutrient agar-agar. Fig. I. Bacillus anthracis. 2. Bacillus subtilis. ,, 3. Streptococcus cereus albus. XV. Potato-cultivations. Fig. I. Bacillus anthracis. ,, 2. Bacterium indicum. XVI. Bacillus anthracis. Fig. I. From a section of mucous membrane of the stomach of a mouse. Gram's method and eosin. ,, 2. From a section of kidney of a mouse. Gram's method and eosin. XVII. Bacillus anthracis and Micrococcus telragonus. Fig. i. From a section of liver of a mouse. Weigert's and Orth's methods. ,, 2. From a section of lung of a mouse. Gram's method and eosin. XVIII. Bacillus tuberculosis. Fig. I. From a section of a lymphatic gland from a case of tuberculosis in a fcetal calf. Ehrlich-Koch method. ,, 2. From a section of lung from a case of artificial tuberculosis in a rabbit. Ehrlich-Koch method. XIX. Bacillus tuberculosis. Fig. I. From a section of liver of a hen. Ehrlich-Koch method. ,, 2. The same preparation. (Zeiss' ^ o.i. Oc. 4.) LIST OF ILLUSTRATIONS. XIX PLATE XX. Bacillus tuberculosis and Bacillus lepra. Fig. I. From a cover-glass-preparation of pus from a tubercular cavity of human lung. ,, 2. From a section of kidney from a case of leprosy. Ehrlich's method. XXI. Potato-cultivation of Bacillus cyanogenus. Fig. I. After three days' growth. ,, 2. After nine days' growth. XXII. Bacillus of septicamia of mice. Fig. i. From a section of kidney of a mouse. Gram's method and eosin. ,, 2. The same preparation. (Zeiss' r V o.i. Oc. 4.) XXIII. Bacillus lepra. Fig. i. From a section of skin from a case of leprosy. Ehrlich's method. ,, 2. The same preparation. (Zeiss' T V o.i. Oc. 4.) XXIV. Cultivations on nutrient agar-agar. Fig. I. Bacterium lineola. ,, 2. Micrococcus rosaceus. ,, 3. Streptococcus pyogenes citreus. XXV. Bacillus figurans. Fig. i. Cover-glass impression-preparation from a plate-culti- vation. ,, 2. The same preparation. (Zeiss' T V o.i. Oc. 4.) XXVI. Bacillus of swine- erysipelas. Fig. i. Pure cultivation in nutrient gelatine. ,, 2. Colonies on a plate-cultivation. 3, Cover-glass-preparation of blood from an inoculated pigeon. XXVIL A.ctinomyces. Fig. i. From a section of a maxillary tumour in a cow. Plaut's method. ,, 2. The same preparation. (Zeiss' v? o.i. Oc. 2.) XXVIII. Actinomyces. Fig. I. From a section of a maxillary tumour in a cow. Weigert's method. ,, 2. From a section of the lung of a cow. Weigert's method. XXIX. Yeast-fungi or Saccharomycetes, and Mould-fungi or Hyphomycetes. facing page 339 XX LIST OF ILLUSTRATIONS. WOOD ENGRAVINGS. F IG. PAGE 1. Koch's Steam-Steriliser . . . . 4 19 2. Hot-air Steriliser . . . . . . .20 3. Section of Hot-air Steriliser . . . . V . 20 4. Hot- water Filtering Apparatus, with Ring Burner . ..". . 22 5. Wire Cage for Test-tubes ...... 24 6. Platinum Needles . . . . . . 25 7. Damp-chamber for Plate-cultivations ... .26 8. Apparatus employed for Plate-cultivations . . . . . ,. .26 9. Box for Glass Plates . . . . . > -27 10 and ii. Glass Benches for Glass Plates or Slides . . -27 12. Israel's Case ........ 28 13. Damp-chamber, for Potato-cultivations . . . .28 14. Serum Steriliser ...... .29 15. Serum Inspissator . . . . . . -3 1 6. D'Arsonval's Incubator . . . . . -33 17. Schlosing's Membrane Regulator . . . -34- 1 8. Gas-burner protected with Mica-cylinder . . '. -35 19. Moitessier's Gas-pressure Regulator . . ... 36 20. Koch's Safety Burner . . . . . -36 21. Babes' Incubator . . . . ... -37 22. Reichert's Thermo-Regulator . . . . . . 39 23. Meyer's Thermo-Regulator . . . . . . 40 24. Siphon Bottle, with Flexible Tube, Glass Nozzle, and a Mohr's Pinchcock ...*.... 42 25. Dessicator ........ 43 26. Method of making a Folded Filter . . . . -67 27. Method of Inoculating a Test-tube, containing sterile nutrient jelly . 70 28. Method of Inoculating Test-tubes in the preparation of Plate-culti- vations ........ 75 29. Microscopical Examination of Colonies on Plate-cultivations, and Method of Isolating Colonies by Inoculation of Test-tubes . 79 30. Method of dividing Potatoes . . . . . .84 31. Method of Forming a Simple Moist Chamber . . -97 32. Simple Warm Stage . . . . . . .98 LIST OF ILLUSTRATIONS. XXI FIG. PAGE 33. Simple Warm Stage shown in Operation . . 99 34. Schafer's Warm Stage ....... 100 35. Strieker's Warm Stage . . . . . loo 36. Section of Israel's Warming Apparatus and Drop-culture Slide . 101 37. Israel's Warming Apparatus . . . . . . 101 38. Israel's Warming Apparatus in Operation .... 102 39. Simple Gas Chamber . . . . .103 40. Gas Chamber in use with Apparatus for generating Carbonic Acid . 104 41. Strieker's Combined Gas Chamber and Warm Stage . . -105 42. Simple Moist Chamber adapted for transmission of Electricity . 105 43. Apparatus arranged for transmitting Electricity . . .106 44. Slide with Gold-leaf Electrodes . . . . .106 45. From a Preparation of Bacillus anthracis . . . .119 46. Ascococcus Billrothii, x 65 (after Cohn) . . . .122 47. Streptococcus and Sarcinacoccus from a Drop-cultivation, x 1200 . 123 48. Streptococcus in the Blood of a Rabbit, x 1200 . . . 123 49. Streptococcus of Progressive Tissue Necrosis in Mice (after Koch) . 123 50. Spirochaeta from Sewage Water, x 1200 . . . .125 51. Bacteria showing Flagella . ...... 127 52. Bacillus megaterium . . . . . . .129 53. Clostridium biityricum, x 1020 . . . . .130 54. A Thread of Bacillus anthracis with Spores in a Drop- cultivation, x 1400 ........ 131 55. Leuconostoc Mesenteroides ; Cocci-chains with Arthrospores (after Van Tieghem and Cienkowski) . . . . . 131 56. Spores of Bacillus anthracis, stained with Gentian Violet, after passing the cover-glass twelve times through the flame, x 1200 . 133 57. Spore-bearing Threads of Bacillus anthracis, Double-stained with Fuchsine and Methylene Blue, x 1200 . . . . 133 58. Tubercle Bacilli in Sputum, x 2500 (from photographs) . . 135 59. Leprosy Bacilli from a Section of Skin, x 1200 . . . 135 60. Glanders Bacilli from a Section of a Glanders Nodule, x 1200 . 135 61. Bacterium of Chicken-cholera from Blood of Infected Hen, x 1200 . 136 62. Bacterium of Chicken-cholera from Muscle Juice of an Infected Hen, x 2500 (from a photograph) . . . .136 63. Comma Bacilli in Sewage Water stained with Gentian Violet, x 1200 136 64. Vibrios in Water contaminated with Sewage, x 1200. . . 137 65. Spirillum undula, x 1200 . . . . . . 137 66. Cladothrix dichotoma . . . . . . .184 67. Bacterium pneumonia crouposce, x 1500 (after Zopf) . . .189 68. Emmerich's Bacterium, x 700 (after Emmerich) . .. 190 XX11 LIST OF ILLUSTRATIONS. FIG. - PAGE 69. Colonies on Nutrient Gelatine, x 60 . . . . .190 70. Colonies on Nutrient Agar-agar, x 60 . . . .191 71. Colony of Bacillus anthracis, x 60 . . -. . .191 72. Bacterium of Rabbit Septicaemia . . . . .192 73. Streptococcus of Progressive Tissue Necrosis in Mice . . 206 74. Micrococcus of Pyaemia in Rabbits ; Vessel from the Cortex of the Kidney, x 700 . . . . . . .217 75. Ascococcus Billrothii (after Cohn) ..... 222 76. Bacterium Pneumoniae Crouposae. from Pleural Cavity of a Mouse, x 1500 (after Zopf) ...... 226 77. Bacterium Neapolitanum, x 700 (after Emmerich) . . . 229 78. Bacteria of Rhinoscleroma, x 1400 (after Cornil) . . . 229 79. Bacterium of Chicken Cholera ; Blood of Inoculated Hen, x 1200 . 233 80. Bacterium of Chicken Cholera, from Muscle Juice of Inoculated Hen, * 2500 . 233 81. Bacterium of Rabbit Septicaemia ; Blood of Sparrow, x 700 (after Koch) ........ 235 82. Bacterium Indicum ; Colonies on Nutrient Agar-agar, x 60 . . 242 83. Bacterium Zopfii ; Successive Changes in the same Thread, x 740 . 245 84. Cover-glass Preparation of the Edge of a Drop of Meat Infusion, x 600 (after Koch) . . . . . .251 85. Colonies of Comma Bacilli on Nutrient Gelatine, natural size (after Koch) . . . . . . . . 251 86. Colonies of Koch's Comma Bacilli, x 60 . . . 252 87. Cover-glass Preparation from the Contents of a Cholera Intestine, x 600 (after Koch) ...... 252 88. Cover-glass Preparation of Cholera Dejecta on Damp Linen (two days old), x 600 (after Koch) . . . . .252 89. Section of the Mucous Membrane of a Cholera Intestine, x 600 (after Koch) ....... 253 90. Pure Cultivation of Finkler's Bacillus, twenty-four hours old . . 254 91. ,, ,, two days old . . . 254 92. ,, Koch's Cholera Bacillus, twenty-four hours old . 254 93. ,, ,, ,, two days old . . 254 94. Comma-shaped Organisms with other Bacteria in Sewage-con- taminated water, x 1200 . . . . . .256 95. Pure Cultivation of Spirillum Finkleri in twenty-four hours . . 258 96. ,, ,, ,, thirty-six hours . . 258 97. Spirillum sputigenum, x I2OO ...... 259 98. Spirillum tyrogenum, x 1200 ...... 260 99. Spirillum pliccOik (Marsh Spirochaete), x 1200 . . .261 LIST OF ILLUSTRATIONS. XXlli FIG. PACK 100. Spirillum itndula, x 1500 ...... 262 101. Leuconostoc mesenteroides . ... 263 1 02. Leprosy Bacilli from a Section of Skin, x 1200 . . . 266 103. Bacillus typhosus from a Potato-cultivation, x 1500 . . . 269 104. Bacillus tuberculosis from Tubercular Sputum, stained by Ehrlich's method, x 2500 . ...... 272 105 and 106. Bacillus anthracis, x 1200 ..... 282 107. Pure Cultivation of the Bacillus anthracis in Nutrient Gelatine . 284 108. Colonies in a Plate-cultivation, x 70 . . . . . 285 109. Cover-glass impression-preparation, x 70 . . . 286 HO. Spores of Bacillus anthracis unstained, x 1500 . . . 290 111. Spores of Bacillus anthracis, x 1200 . . . . .291 112. From a double-stained preparation of Bacillus anthracis, x 1200 . 291 113. Bacillus Mallei, x 1200 ...... 292 1 14 and 1 15. Pure Cultivations of the Bacillus of Septicaemia of Mice . 298 1 16. From a preparation of Bronchial Mucus of a Pig (after Klein) . 299 117. Blood of Fresh Spleen of a Mouse, after inoculation with Swine Fever (after Klein) ....... 300 118. Bacilli from an Artificial Culture with Spores (after Klein) . . 300 119. Bacillus cyanogenus, x 650 (after Neelsen) .... 305 120. Bacillus megaterium (after De Bary) ..... 310 121. Pure Cultivation of Bacillus figurans in Nutrient Agar-agar . 311 122. Bacillus saprogcnes, No. I (after Rosenbach) . . . 314 123. Vibrio rugula, x 1020 (after Prazmowski) . . . . 317 124. Clostridium butyricum (after Prazmowski) . . . 319 125. Crenothrix Kiihniana (after Zopf) . . . . . 323 126. Beggiatoa alba (after Zopf) ...... 325 127. Several Phase-forms of Beggiatoa Roseopersicina (after Warming) . 326 128. Cladothrix dichotoma (after Zopf) . . . . . 331 1 29. Parasites in the Blood of Rats (after Lewis) . . . . 35 1 130. Hcematomonas cobitis (after Mitrophanow) . . . -353 131. Organisms in the Blood of the Carp (after Mitrophanow) . . 354 132. " Surra " Parasites occurring singly and fused, x 1200 . . 356 133. A Monad in Rat's Blood, x 3000 . . . . . 357 134 and 135. Monads in Rat's Blood, x 1200 .... 358 136. Hesse's Apparatus ....... 363 137. Apparatus for estimating the number of Colonies on a Plate-culti- vation . . . . . . . 368 DESCRIPTION OF ELATE I. 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 pneumonic? crouposce], 6. Cocci in groups of four (merismopedia). 7. Cocci in packets (sarcina). 8. Bacterium termo. 9. Bacterium termo, x 4000 (after Dallinger and Drysdale). 10. Bacterium lineola. 11. Bacillus subtilis. 12. Bacillus tuberculosis. 13. Bacillus lepra. 14. Bacillus malaria (after Klebs). 15. Bacillus typhosus (after Eberth). 1 6. Spirillum undula (after Cohn). 17. Spirillum volutans (after Cohn). 1 8. Spirillum cholera Asiatics: from an artificial cultivation. 19. Spirillum Obermeieri (after Koch). 20. Spiroch&te plicatilis (after Fliigge). 21. Vibrio rugula (after Prazmowski). 22. Cladothrix Fcerstcri (after Cohn). 23. Cladothrix dichotoma (after Cohn). 24. Monas Okenii (after Cohn). 25. Monas Warmingii (after Cohn). 26. Rhabdomonas rosea (after Cohn). 27. From a cover-glass-preparation of blood from the spleen of a mouse which had died of anthrax ; stained with fuchsine (Zeiss' T V o.i. Oc. 4). 28. From a drop-cultivation of 'Bacillus anthracis (Zeiss' ^ o.i. Oc. 4). 29. From a cover-glass impression-preparation of a potato- cultivation of Bacillus anthracis (Zeiss' % o.i. Oc. 4). 30. From a preparation of Bacillus anthracis^ cultivated in nutrient gelatine (torula-form). 31. Involution-form of Crenothrix (after Zopf). 32. Involution-forms of Vibrio serpens (after Warming). 33. Involution-forms of Vibrio rugula (after Warming). 34. Involution-forms of Clostridium Poly my xa (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). BACTERIOLOGY. CHAPTER I. IN1R OD UCTOR Y. 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 importance. The further researches of the prac- tical 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 infectious diseases, and more espe- cially the discovery by Koch of the tubercle and cholera bacilli, have claimed the attention of the whole thinking world. The scientific importance of these latter dis- i 2 BACTERIOLOGY. coveries is evidenced by the fact that in Germany medical men were summoned from all parts of the country to Berlin to attend a course of instruction in the latest methods employed in the investigation of bacteria. In this way the methods of pure cultivation devised by Koch, and the means of recognising the cholera bacillus, are being widely disseminated. To a medical man, those bacteria which are con- nected with disease, and more especially those which have been proved to be the causa, if not the actual materies morbi, are of predominant interest and im- portance. 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. It is not 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. To ascertain beyond all doubt whether a micro-organism is actually the causa causans of a disease, Koch has laid down the following postulates : a. The micro-organism must be found in the blood, lymph, or diseased tissues of man, or animal, suffering from, or dead of, the disease. INTRODUCTORY. 3 b. The micro-organisms must be isolated from the blood, lymph, or tissues, and cultivated in suit- able media, i.e., outside the animal body. These pure cultivations must be carried on through succes- sive generations of the organism. c. A pure-cultivation thus obtained must, when introduced into the body of a healthy animal, pro- duce the disease in question. d. Lastly, in the inoculated animal the same micro-organism must again be found. These points would 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 isolating 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 4 BACTERIOLOGY. chapter upon their classification, followed by a de- scription 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 special methods for examining bacteria in air, soil, and water, and several are of interest in being, like many bacteria, micro-organisms associated with disease. A short account is also given of the Flagellated Protozoa, which have been found to occur in the blood of animals. To the pathologist these are of interest owing, more especially, to the discovery of closely allied micro-parasites in the blood in cases of malaria. The special methods just referred to, with description of the apparatus employed, and a Chronological Bibliography, are also comprised in the Appendix. CHAPTER II. 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 fnstruments are supplied by Leitz, Zeiss, and Hartnack in Germany, and Powell and Lealand in England. Zeiss' micro- scope, with -fj and T *g- oil-immersion lenses, or Powell and Lealand's with -^ and -fj, is recom- mended for investigators; while Leitz', with ^ 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 1$ ; Zeiss', with the same, and with iV, 20 more. Refer to foot-note on p. 44. O 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, used in preparing slides with rings, for mounting preparations of Aspergillus, etc. 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. UN7VKRSTTY APPARATUS, MATERIAL, AND REAGENTS. somewhat cumbrous instrument, is much to be preferred. 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, such as Williams' or Roy's, and a Valentin's Knife, are useful for the examination of tissues in the fresh state, but other- wise are supplanted by the above. (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 . . . 5- Alcohol . . . . . roo Distilled water .... 20 Chloride of sodium . . . 5- 8 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 . . . . .10 Lastly a few drops of phenol for preservation Gum. Kleinenberg's solution. Saturated watery solution of picric acid . . . . . .100 Strong sulphuric acid . . 2 Filter and add Distilled water .... 300 Muller's fluid. Bichromate of potash . . 2 Sulphate of sodium i Distilled water . . . .100 Osmic acid. Distilled water . . . .100 Osmic acid ..... '5 APPARATUS, MATERIAL, AND REAGENTS. 9 Paper trays. Paraffin. 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 TOO Hydrochloric acid i IO BACTERIOLOGY. 5. Alum carmine (Grenacher). Carmine . ..... i Five per cent, solution of alum . 100 Boil twenty minutes, filter when cold. 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. (') Aqueous solution. Bismarck brown . . 2 Alcohol 15 Distilled water . 85 10. Borax carmine (Grenacher). Borax ...... 2 Carmine ..... '5 Distilled water . . . .100 To the dark purple solution add a 5 p. c. sol. of acetic acid until a red colour is produced ; set aside 24 hours, filter, and add a drop of carbolic acid. APPARATUS, MATERIAL, AND REAGENTS. I I 11. Cedar oil. 12. Eosin. (a) Saturated alcoholic solution. (6) Aqueous solution. Distilled water .... 100 Eosin . . v . . 5 13. Ether. 14. Fuchsine. (a) Saturated alcoholic solution. (6) Aqueous solution. Fuchsine ..... 2 Alcohol . , . . . 15 Water 85 15. Gentian violet. (a) Saturated alcoholic solution. (6) 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, T 2 BACTERIOLOGY. Dissolve aniline oil . , . > 3 In rectified spirit . . . 1 5 and add slowly to the above. Lastly, slowly add distilled water . 15 Keep in stoppered bottle. 17. Glycerine, pure. 1 8. Haematoxylin 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 . . . . 2*5 APPARATUS, MATERIAL, AND REAGENTS. 13 22. Magenta solution (Gibbes). Magenta ..... 2 Aniline oil ..... 3 Alcohol (Sp. Gr. -830) .; . .20 Distilled water 20 23. Methylene blue. (a) Concentrated alcoholic solution. (<$) Aqueous solution. Methylene blue .... 2 Alcohol . . . . . -15 Water 85 (c) Koch's solution. Concentrated alcoholic solution of methylene blue i Ten per cent, potash solution . . 2 Distilled water .... 200 (a} Loffler's solution. Concentrated alcoholic solution of methylene blue . . . .30 Solution of potash i 10,000 . . 100 24. Methyl violet. (a) Concentrated alcoholic solution. 14 BACTERIOLOGY. (b) Aqueous solution. Methyl violet . . . . 2.25 Distilled water . . . .100 (c) Koch's solution. Aniline water . . . . .100 Alcoholic solution of methyl violet 1 1 Absolute alcohol . . . .10 25. Neelsen's solution. Dissolve fuchsine i In a 5 per cent, watery solution of carbolic acid . . . .100 Add alcohol . . . , .10 26. Nitric acid, pure. 27. Orseille (Wedl). Dissolve pure ammonia-free orseille in Absolute alcohol . . .20 Acetic acid .... 5 Distilled water .... 40 until a dark red liquid results : filter. 28. Picric acid. (a) Concentrated alcoholic solution. (b) Saturated aqueous solution. APPARATUS, MATERIAL, AND REAGENTS. 15 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. () 10 M 33 32. Safranine. (a) Concentrated alcoholic solution. (6) 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. 1 6 BACTERIOLOGY. Water, distilled. Water, sterilised. Distilled water can be kept for use in a wash bottle, or far better in a siphon apparatus. Steri- lised 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 bottom. The numbered reagents can be conveni- ently 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 crystal of thymol, should be added as a preservative. 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. APPARATUS, MATERIAL, AND REAGENTS. I? 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 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 microscopical 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, 2 1 8 BACTERIOLOGY. 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 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,f who employed the electric light, obtained some excellent pictures of preparations mounted in balsam. Van Ermengem* 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- * Koch, Verfahren zur Untersuchung zum Conserviren und Photographiren der Bacterien . 1877. t Hauser, Uber Faulniss Bacterien und deren Bcziehungen zur Septicdmie. 1885. \ Van Ermengem, Bull, de la Soc. Beige de Microscopic, No. X., pp. 170-2. 1884. APPARATUS, MATERIAL, AND REAGENTS. 1 9 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 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 interior into two chambers the upper or "steam chamber," and the lower or " water-chamber.' ' A gauge outside marks the level of the water . ~F IG . i. KOCH'S ,11 i i ,1 1 u STEAM-STERILISER. 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 * Photography of Bacteria. 1887. 20 BACTERIOLOGY. burner. It is employed for sterilising nutrient media in tubes or flasks, for cooking potatoes, or hastening the filtration of agar-agar. When the thermometer indi- cates 1 00 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. FIG. 2.-HOT-AIR STERILISER. p ota toes Or Small flasks are lowered into the cylinder in a tin receiver with a perforated bottom, which rests fc-ii L*_i_A_A_ A upon the grating and admits of its contents being exposed to the steam. Hot-air Steriliser. A cubi- cal chest of sheet iron with double walls, supported on four leors ; it may also be suspended on the wall of the laboratory, with a sheet of asbestos inter- vening (Figs. 2 and 3). It is heated with a rose gas-burner from below, and the temperature of the interior indicated by a thermometer inserted through i i ^i e FIG. 3. SECTION a hole in the roof ; in a second O p HOT-AIR STERILISER. APPARATUS, MATERIAL, AND REAGENTS. 21 opening a gas regulator can be fixed. Test-tubes, flasks, funnels, cotton wool, etc., may be sterilised by exposure to a temperature of 150 C. for an hour or more. (G) APPARATUS AND MATERIAL FOR PREPARING AND STORING GELATINE-, AND AGAR-AGAR- PEPTONE-BROTH. 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 con- centric rings, so that the mouth of the vessel may be graduated to any size required. Test-tube Water-bath. This consists ot 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 22 BACTERIOLOGY. burner to a tubular prolongation of the water chamber. In a more recent model, as repre- sented in Fig. 4, this prolongation is dispensed with, and the tempera- ture is maintained 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 require- ments. Bohemian hard glass flasks are employed in several sizes, for boil- ing" nutrient media. The FIG. 4. HOT-WATER FILTERING APPARATUS Conical forms are CS- WITH RING BURNER. . ,, , , pecially 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. APPARATUS, MATERIAL, AND REAGENTS. 23 Cylindrical glasses graduated in cubic cen- timetres, 10 ccm., 100 ccm., 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 con- stantly 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). Agar-Agar. 7'his is also called Japanese Isin- glass ; it consists of the shrivelled filaments of certain Algae (Gracilaria lichenoides and Gigartina speciosa).^ Peptonum Siccum (Savory and Moore). 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. * Ilueppe, Die Methoden der Bakterien Forschung. 1885. 24 BACTERIOLOGY. 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 re- commended. 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 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 FIG. 5.- WIRE CAGE called into use for holding cul- FOR TEST.TUBES. ^ , , tivations. Pegged racks are also recommended for draining test-tubes after washing. APPARATUS, MATERIAL, AND REAGENTS. 25 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, 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 FIG. 6. PLATINUM NEEDLES. 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 nutrient jelly. For other purposes the needles may also be bent at the extremity into a small hook or a loop* (Fig. 6) Tripod Levelling-stand A triangular wooden frame supported upon three screw-feet, which * A looped platinum needle is called in Germany an " ose," a term which, on account of its brevity, may be conveniently adopted 26 BACTERIOLOGY. 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 FIG. 7. DAMP-CHAMBER FOR PLATE CULTIVATIONS. and dishes, for making a dozen or more damp chambers (Fig. 7), and for completing the ap- 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. paratus for pouring out liquefied nutrient jelly on glass plates or slides (Fig. 8). Iron Box. A box of sheet-iron (Fig. 9) for APPARATUS, MATERIAL, AND REAGENTS. 27 ' containing glass plates during their sterilisation in the hot-air steriliser, and for ff^V- storing them until required for use. Glass Plates. Small panes of glass, about six inches by four. Not less than three dozen are re- quired for a dozen damp chambers. Glass Benches. These are necessary for arranging the glass plates or slides in tiers in the damp chambers (Fig. 10). Metal shelves may be substituted for them, but the former are to be preferred. They can be easily made, in any number required, by Box FOR GLASS PLATES. FIGS. 10, ii. GLASS BENCHES FOR GLASS PLATES OR SLIDES. cementing a little piece ot plate glass at either end of a glass slip (Fig. 1 1). 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 thermometers. 28 BACTERIOLOGY. 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 150 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. 13. DAMP-CHAMBER FOR POTATO- CULTIVATIONS. APPARATUS, MATERIAL, AND REAGENTS. 29 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 3O BACTERIOLOGY. 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 FIG. IS.-SERUM INSPISSATOR. * WO front Ones move 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. APPARATUS, MATERIAL, AND REAGENTS. 31 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. Professor 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. Professor Sternberg, of America, 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 * Magnin and Sternberg, Bacteria. 1884. 32 BACTERIOLOGY. atmospheric germs on removal from the ordinary 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 rec- tangular 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'Arsonval'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 " luve UAr~ sonval" (Fig. 16) is a very efficient apparatus, and is provided with a heat regulator, which APPARATUS, MATERIAL, AND REAGENTS. 33 enables the temperature to be maintained with a minimum variation. It consists of a cylindrical copper vessel, with double walls, enclosing a wide interspace for containing 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 caout- chouc stopper, through which a glass tube passes. The mouth of the cylinder itself is horizontal, and is ,. , FIG. 16. D'ARSONVAL'S INCUBATOR. 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 cylin- der. The cylinder is continued below by a cone, also double-walled, and there is a perforated grating at 3 34 BACTERIOLOGY. the line of junction of the cylinder and cone. The cone 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 heating apparatus. Situated above this leg is the heat regulating apparatus (Fig. 17), attached to a circular, lipped aperture in the outer wall of the in- cubator. To the lip is fixed with six screws the corresponding lip of a brass box, with a tightly stretched diaphragm of india-rubber intervening. Thus the diaphragm FIG. 17. SCHLOSING'S MEMBRANE separates the cavity of REGULATOR. . . r . the box from the 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 APPARATUS, MATERIAL, AND REAGENTS. 35 removed, and the water-chamber filled completely with distilled or rain water at the temperature required. The 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 GAS Bu * NER PROTECTED * WITH MICA CYLINDER. to the burner represented in Fig. 18. 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 FIG. 18. BACTERIOLOGY. passes through a pressure regulator (Fig. 19). To provide against the danger h (f\\ resulting from acci- dental extinction of 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 FIG. 19. MOITESSIER'S GAS-PRESSURE REGULATOR. very simple, and is recommended by the author in preference to all others (Fig. 21). PIG. 20. KOCH'S SAFETY BURNER. It consists of a double-walled chest with sides APPARATUS, MATERIAL, AND REAGENTS. 37 and roof jacketed with felt. Water fills the inter- space 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 appa- ratus can be suspended on the wall or supported on legs, and is heated from below by means of protected burners. The gas should pass FIG. 2i. BABES' INCUUATOR. 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 (K) 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 cc. 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 BACTERIOLOGY. t, g only touches i by three projecting ridges. 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 n n 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 h, which is replaced on g, by its downward pressure counteracts this upward pressure of the gas and opens the valve c c. 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 APPARATUS, MATERIAL, AND REAGENTS. 39 contain mercury. The regulator is fixed in the 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 at the required temperature. If the p IG . 22 . temperature of the incubator falls the ^SaSS^ mercury contracts, and gas passing REGULATOR - 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 envelopes 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 BACTERIOLOGY. opening of the inner tube, which is carried down with it, is obstructed by the mercury. Meyer's Thermo-Regulator is represented in Fig. 23. No. I. shows the construction of the FIG. 23. MEYER'S THERMO-REGULATOR. regulator ; its inner tube terminates in an oblique opening, and is also provided with a minute lateral aperture, which prevents the complete shutting off of the gas supply. No. II. illustrates APPARATUS, MATERIAL, AND REAGENTS. 41 the method of introducing the mercury by suction through a filling tube, which is substituted for the inner tube of the regulator. No. III. represents Frankel's modification of the same instrument. 1 (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 with 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 Szirgery. 1882. BACTERIOLOGY. (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 APPARATUS, MATERIAL, AND REAGENTS. 43 surplus stain from cover glasses, etc., and the latter 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 the sulphuric acid in confined air. A cultivation of Bacterium prodigiosum^ for example, 44 BACTERIOLOGY. 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 \ oil. imm. for five guineas, and are prepared to supply a microscope completely equipped for bacteriological work at a very low price. CHAPTER III. MICROSCOPICAL EXAMINATION OF BACTERIA IN LIQUIDS, IN CULTIVATIONS ON SOLID MEDIA, AND IN TISSUES. Preliminary Remarks. In conducting bac- teriological researches, the importance of absolute cleanliness 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 atten- 46 BACTERIOLOGY. tion to minute technical details, will surely be followed with disappointing results in the contami- nation of one's cultures, resulting in the loss of much time. When using platinum needles, either for inoculating fresh tubes in carrying 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 \vhich 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 sterilised ose 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 MICROSCOPICAL EXAMINATION OF BACTERIA. 47 sterilised needle to a drop of sterilised water on a slide, thinning it out, and covering with a cover-glass as already described. A more satis- factory 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 re-agents. This micro-chemical reaction is made the basis of Baumgarten's method (p. 278). 48 . BACTERIOLOGY. (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 other fluid to be examined, is conveyed to it with a large ose. 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 wash UNIVERSITY MICROSCOPICAL EXAMIN^IO^ OF BACTERIA. 49 bottle. Turn the cover-glass on to a slide, remove excess of water with filter paper, and wipe the exposed surface ; examine with Zeiss' DD (about 230 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 culture from a solid medium may be stained and examined in the same way after spreading it out with a 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. Double coloration of cover-glass prepara- 50 BACTERIOLOGY. 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 pre- parations 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 XX., 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 ose, is spread out on a cover glass into as thin a film as possible : when almost dry, a drop or two of a weak aqueous solution of methyl violet is MICROSCOPICAL EXAMINATION OF BACTERIA. 51 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. 48 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 2 10 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 minutes on a fuchsine aniline- water solution, as 5 2 BACTERIOLOGY. 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 Miiller'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 intense staining of the whole preparation results. (C) COVER-GLASS IMPRESSIONS. One of the most instructive methods for examin- ing micro-organisms is to make what is called in German a " Klatsch Prdparat" It enables us in many cases to study the relative position of in- dividual micro-organisms one to another in their growth on solid cultivating media, and in some cases produces the most exquisite preparations for MICROSCOPICAL EXAMINATION OF BACTERIA. 53 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 de- scribed. In the case of plate-cultures, especially where no liquefaction has taken place, the growth is bodily transferred to the cover-glass and a vacant area left on the gelatine or agar-agar, correspond- ing exactly with the form and size of the cover-glass employed (Plate XXV., Figs, i and 2). CHAPTER IV. 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. Muller'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 Muller'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 PREPARATION AND STAINING OF TISSUE SECTIONS. 55 in water, to wash out the picric acid, and then transferred through weak spirit to absolute alcohol. Ebner's solution also gives excellent results, es- pecially 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 Muller'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 paraffin, or celloidin, and mounted on cork, or, if firm enough, they may be fixed upon cork without any embedding material at all. Paraffin, 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 5 6 BACTERIOLOGY. 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 paraffin must be cut per- fectly dry, and the sections prevented from rolling up by gentle manipulation with a camePs-hair brush. They must then be picked off the blade of PREPARATION AND STAINING OF TISSUE SECTIONS. 57 the knife with a clean needle, and dropped into a watch-glass containing xylol. This dissolves out the paraffin ; 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, one employs gelatine, or glycerine-gelatine, liquefied over a Bunsen burner in a porcelain cap- sule. 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. (c) GENERAL PRINCIPLES OF STAINING BACTERIA IN TISSUE SECTIONS: 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- 5 BACTERIOLOGY. 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 how- ever prepared, one should always examine with a low power (Zeiss' AA) first; this enables one to recognise the tissue under examination in most cases, and even to examine in many cases the topo- graphical distribution of masses of bacteria. With Zeiss' DD., Oc. 2, a power of about 250 diams., 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 Zeiss' microscopes are, like most good modern instruments, provided with a triple nosepiece, there 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 follows : Place the sections for from six to eighteen hours in a one 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 PREPARATION AND STAINING OF TISSUE SECTIONS, 59 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 de- colorised 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 parts of water, and filtering the emulsion. Half a gramme of the best finely powdered gentian violet is dissolved in the clear filtrate, and the solution filtered before use. The sections are then trans- ferred to a solution of iodine in iodide of potassium till they become dark brown in colour, and then decolorised in absolute alcohol. The time required for complete decolorisation in alcohol varies from a few minutes to twenty-four hours. They are then treated with clove-oil and mounted in Canada balsam. It is much better, however, to employ 6O BACTERIOLOGY. the aniline-gentian-violet solution quite freshly prepared, and the following- useful method is invariably used by the author : Place four or five drops of pure aniline in a test-tube, fill it three- quarters full with distilled water, close the mouth of the tube with the thumb and shake it up thoroughly. Filter the emulsion twice, and pour the filtrate into a watch-glass or glass capsule. To the perfectly clear aniline water thus obtained add drop by drop a concentrated alcoholic solution of gentian-violet till precipitr Jon commences. Stain sections in this solution from ten minutes to half an hour, then transfer to iodine-potassic-iodide solution, and decolorise in alcohol. The process of decolorisation may be hastened by placing the section in clove-oil and returning it to alcohol, and again to clove-oil. If examined, after it has been finally treated with clove-oil and mounted in Canada balsam, the tissue appears colourless or tinged faintly yellow, while micro-organisms, e.g., bacilli and micrococci, are stained blue or blue- black. Double staining is obtained by transferring the sections after decolorisation to a solution of eosin, bismarck brown, or vesuvin, again rinsing in alcohol, clearing in clove-oil, and mounting in balsam. Another instructive method is to place the decolorised sections in picro-carminate of ammonia for three or four minutes, and then treat with alcohol, oil of cloves, and balsam. In this way the nuclei are well stained. A somewhat similar PREPARATION AND STAINING OF TIS result is obtained by placing the sections for a few minutes in Orth's solution (picro-lithium-carmine) ; transferring to acidulated alcohol, then to an alco- holic solution of picric acid, and treating with clove- oil and balsam (Plate XVII., Fig. i). Weigert-Ehrlich Method. This is a method in which nitric acid is employed as a decolorising agent. It is as follows : Filtered, saturated, watery solution of aniline one hundred parts ; saturated alcoholic solution of a basic aniline dye (methyl- violet, gentian-violet, fuchsine), eleven parts; are mixed and filtered. Rapid staining is obtained by warming the solution. Decolorise with nitric acid (i in 2), and stain with a contrast colour. As delicate sections are apt to be injured by immersion in the nitric acid, they may be transferred from the fuchsine solution to distilled water, then rinsed a few minutes with alcohol, and finally placed in the following contrast stain for one to two hours : Distilled water . . . 100 cc. Saturated alcoholic solution of methylene blue. , . 20 cc. Formic acid 10 mm.* The method of Weigert-Ehrlich is employed for staining both leprosy and tubercle bacilli. The ani- line-fuchsine solution may also be prepared by the simple plan described for Gram's method (page 60). The more special methods for staining will be given with the description of those species of micro- organisms to which they apply. * Watson Cheyne, Practitioner. 1883. CHAPTER V. PREPARATION OF NUTRIENT MEDIA AND METHODS OF CULTIVATION. To cultivate micro-organisms artificially, and, in the case of the pathogenic bacteria, to fulfil the second of Koch's postulates, they must be supplied with nutrient material free from pre-existing micro- organisms. Hitherto various kinds of nutrient liquids have been employed, and in many cases they still continue to be used with advantage, but as a general rule they have been in a great measure supplanted by the methods of cultivation on sterile solid media about to be described. The advantages of the latter method are obvious. In the first place, in the case of liquid media, in spite of elaborate precautions and the expenditure of much labour and time, it was almost impossible or extremely difficult to obtain a pure culture. If a drop of liquid con- taining several kinds of bacteria be introduced into a liquid medium, we have a mixed cultivation from the very first. If in the struggle for existence some bacteria were unable to develop in the presence ol others, or a change of temperature and soil allowed PREPARATION OF NUTRIENT MEDIA. 63 one form to predominate over another, then one might be led to the conclusion that many bacteria were but developmental forms of one and the same micro-organisrn ; while possibly the contamination of such cultures might lead to the belief in the trans- formation of a harmless into a pathogenic bacterium. In the case of solid cultivating media, on the other hand, the chance of contamination by gravitation of germs from the air is avoided by the fact that test- tubes, flasks, etc., can be inverted and inoculated from below. The secret of the success of Koch's method, however, depends upon the possibility, in the case of starting with a mixture of micro-organisms, of being able to isolate them completely one from another, and to obtain an absolutely pure growth of each cultivable species. When sterile nutrient gelatine has been liquefied in a tube and inoculated with a mixture of bacteria in such a way that the in- dividual micro-organisms are distributed throughout it, and the liquid is poured out on a plate of glass and allowed to solidify, the individual bacteria, instead of moving about freely as in a liquid medium, are fixed in one spot, where they develop individuals of their own species. In this way colonies are formed each possessing its own characteristic biological and morphological appearances. If an adventi- tious germ from the air falls upon the culture, it also grows exactly upon the spot upon which it fell, and can be easily recognised as a stranger. To maintain the individuals isolated from one another 64 BACTERIOLOGY. during their growth, and free from contamination, it is only necessary to thin out the cultivation, and to protect the plates from the air. The slower growth of the micro-organisms in solid media, and the greater facility afforded thereby for examining them at various intervals and stages of development, is an additional point in favour of these methods ; and the characteristic macroscopical appearances so frequently assumed are, more especially in the case of morphological resemblance or identity, of the greatest importance. The colonies on nutrient gelatine (examined with a low power) of Bacillus antfiracis and Proteus mirabilis ; the naked eye ap- pearances in test-tubes of nutrient gelatine of the bacillus of mouse-septicaemia (Figs. 114, 115), and of anthrax (Fig. 107), and the brilliant and curious growth of Bacterium indicum upon nutrient agar- agar (Plate II., Fig. i), may be quoted as examples in which the appearances in solid cultivations are pathognomonic. SOLID MEDIA (A) PREPARATION OF STERILE GELATINE-, AND AGAR-AGAR-PEPTONE-BROTH. Sterile Gelatine-Peptone-broth, or Nutrient Gelatine, is prepared as follows : Take half a kilogramme of beef (one pound), as free as possible from fat. Chop it up finely, transfer it to a flask or cylindrical vessel, and shake it up well with a PREPARATION OF NUTRIENT MEDIA. 65 litre of distilled water. Place the vessel in an ice- pail, ice-cupboard, or in winter in a cold cellar, and leave for the night. Next morning commence with the preparation of all requisite apparatus. Thoroughly wash, rinse with alcohol, and allow to dry, about 100 test-tubes. Plug the mouth of the test-tubes with cotton wool, taking care that the plugs fit firmly, but not too tightly. Place them in their wire cages in the hot-air steriliser to be heated for an hour at a temperature of 150 C. In the same manner cleanse and sterilise several flasks and a small glass funnel. In the meantime the meat infusion must be again well shaken, and the liquid portion separated by filtering and squeezing through a linen cloth. The red juice thus obtained must be brought up to a litre by again transferring it to a large measuring glass and adding distilled water. It is then poured into a sufficiently large and strong beaker; and set aside after the addition of 10 grammes of peptonum siccum. 5 grammes of common salt. 100 grammes of best gelatine. In about half-an-hour the gelatine is sufficiently softened, and subsequent gently heating in a water- bath causes it to be completely dissolved. The danger of breaking the beaker may be avoided by placing a cloth several times folded at the bottom of the water-bath. 66 BACTERIOLOGY. The next process requires the greatest care and attention. Some micro-organisms grow best in a slightly acid, others in a neutral or slightly alkaline medium. For example, for the growth and cha- racteristic appearances of the comma bacillus of Asiatic cholera a faintly alkaline soil is absolutely essential. This slightly alkaline medium will be found to answer best for most micro-organisms, and may be obtained as follows : With a clean glass rod dipped into the mixture, the re-action upon litmus paper may be ascer- tained, and a concentrated solution of carbonate of soda must be added drop by drop, until red litmus paper becomes faintly blue. If it has been made too alkaline it can be neutralised by the addition of lactic acid. Finally, the mixture is heated for an hour in the water-bath and filtered while hot. For the filtration the hot-water apparatus can be used with advantage, furnished with a filter of Swedish paper made in the following way. About eighteen inches square of the best and stoutest filtering paper is first folded in the middle, and then, as in Fig. 26, creased into sixteen folds. The filter is made to fit the glass funnel by gathering up the folds like a fan, and cutting off the superfluous part. The creasing of each fold should be made firmly to within half an inch of the apex of the filter, which part is to be gently inserted into the tube of the funnel. To avoid PREPARATION OF NUTRIENT MEDIA. 67 bursting the filter at the point, the broth when poured out from the flask should be directed against the side of the filter with a glass rod. During filtration the funnel should be covered over with a circular plate of glass, and the process of filtration must be repeated, if necessary, until a pale, straw-coloured, perfectly transparent filtrate results. The sterilised test-tubes are filled for about a third of their depth by pouring in the gelatine Fig. 26. METHOD OF MAKING A FOLDED FILTER. carefully and steadily, or by employing a small sterilised glass funnel. The object of this care is to prevent the mixture touching the part of the tube with which the plug comes into contact ; otherwise, when the gelatine sets, the cotton wool adheres to the tube, and becomes a source of embarrassment in subsequent procedures. As the tubes are filled they are placed in the test-tube basket, and must then be sterilised. They are either lowered into the steam steriliser, when the 68 BACTERIOLOGY. thermometer indicates ico C., for twelve minutes for four or five successive days ; or they may be transferred to the test-tube water- bath and heated for an hour a day for three successive days. If the gelatine shows any turbidity after these processes, it must be poured back from the test- tubes into a flask and boiled up for ten minutes, after the addition of the white and shell of an egg beaten up together. It is once more filtered, and the processes of sterilisation just described must be repeated. Sterile Agar - Agar - Peptone - broth, or Nutrient Agar-Agar. Agar-Agar has the ad- vantage of remaining solid up to a temperature of about 45. The preparation of a sterile nutrient jelly is conducted on much the same principles as those already described. Instead, however, of 100 grammes of gelatine, only about 20 grammes of agar-agar are employed (1*5 2 per cent.), and to facilitate its solution it must be allowed to soak in salt-water overnight. For the filtration, flannel is substituted for filter paper, or may be used in combination with the latter. The hot- water apparatus is invariably employed, unless, to accelerate the process, the glass funnel and receiver are bodily transferred to the steam steriliser. If the conical cap cannot be replaced, cloths laid over the mouth of the steriliser must be employed instead. It may be necessary to repeat the process of filtration, but it must not be TEST-TUBE CULTIVATIONS. 69 expected that such a brilliant transparency can be obtained as with gelatine. The final result, when solid, should be colourless and clear, but if only slightly milky it may still be employed. After the final treatment in the steam steriliser some of the tubes may be placed in the blood- serum apparatus, and left to gelatinise with an oblique surface. A little liquid gradually collects at the bottom of the surface, being expressed by the contraction of the nutrient jelly. (B) METHODS OF EMPLOYING NUTRIENT JELLY IN TEST-TUBE- AND PLATE-CULTIVATIONS. Test-Tube-Cultivations.- To inoculate test- tubes containing nutrient jelly, the cotton wool plug must be twisted out, by which means any adhesions that may exist are broken down. A sterilised needle charged with the blood, pus, etc., containing the micro-organisms, or with a colony from a plate-culture, is steadily thrust once, and once only, into the nutrient jelly. The tube should be held with its mouth downwards, to avoid, as far as possible, accidental contamination from the gravitation of germs in the air, and the plug, which has been removed with the thumb and index finger of the right hand and held between the fourth and fifth fingers of the left, is replaced as rapidly as possible (Fig. 27). The chances of error arising from contamination BACTERIOLOGY. of the cultivations are further reduced by avoiding draughts at the time of inoculation, and it is best that these manipulations should be carried on in a quiet room in which the tables and floor are wiped "with damp cloths, rather than in a laboratory in which the air becomes charged with germs through constant sweeping and dust- ing, and the entrance and exit of classes of students. In conducting any investi- gation a dozen or more tubes should be inoculated, and if by chance an ad- ventitious germ, in spite of these precautions, gain an entrance, the contaminated tube can be rejected and the experiments continued with the remaining pure cultivations. Where, however, one tube is inoculated trom another containing a liquid medium, as in the process of preparing plate-cultures, or where a culture is made from a tube in which the growth has liquefied the gelatine, it is obvious that the tubes cannot be inverted, and they must then be held and inoculated as in Fig. 28. To inoculate those tubes of nutrient agar-agar which have been gelatinised obliquely, the sterilised needle with the material to be cultivated is streaked over the surface from below upwards. FIG, 27. METHOD OF INOCULATING A TEST-TUBE CONTAINING STERILE NUTRIENT JELLY. TEST-TUBE CULTIVATIONS. 7 I Examination of test-tube cultivations. The appearances produced by the growths in test- tubes can be in most cases sufficiently examined with the naked eye. The illustrations in the ac- companying plates (Plates II., III., IV., V., VIII., XIII., XIV., XXIV.) are given as examples of the various changes produced in the nutrient media. In some cases the jelly is partially or completely liquefied, while in others it remains solid. The growths may be abundant or scanty, coloured or colourless. The nutrient jelly may itself be tinged or stained with products resulting from the growth of the organisms. When liquefaction slowly takes place in the needle track, or the organism grows without producing this change, the appearances which result are often very delicate, and in some cases very characteristic. The appearance of a simple white thread, of a central thread with branching transverse filaments, of a cloudiness, or of a string of beads in the track of the needle, may be given as examples. In such cases much may be learnt by examining the growth with a magnifying glass. Here, however, a difficulty is encountered, for the cylindrical form of the tube so distorts the appearance of its contents, that the examination is rendered somewhat difficult. To obviate this, the following very simple contrivance may be employed with advantage. Cheshire's Trough. This consists of a rect- angular vessel, four inches in height, two inches in 7 2 BACTERIOLOGY. width, and one inch in depth. It may be easily constructed by cementing together two slips of glass to form the back and front, with three slips of stout glass with ground edges forming the sides and base. The front may be constructed of thin glass, and the base of the vessel made to slope so that the test-tube when placed in the trough has a tendency to be near the front. The trough is filled with a mixture of the same refractive index as the nutrient gelatine. The latter has a refractive index rather higher than water, which is about 1*333 ; alcohol has a refractive index of 1*374. The trough is filled with water, and alcohol is then added until the proper density is reached. The test-tube is placed in the trough, and held in position by means of a clip. The trough can be fixed on the inclined stage of the microscope, and the contents of the tube conveniently examined with low power objectives. PL ATE- CULTIVATIONS. The key to the success of Koch's method of cultivation on solid media consists in the employ- ment of plate-cultivations. By this means, as has already been mentioned (p. 63), a mixture of bacteria, whether it be in fluids, excreta, or in artificial cultivations, can be so treated that the different species are isolated one from the other, and perfectly pure cultivations of each of the cultivable bacteria in the original mixture esta- blished in various nutrient media. We are enabled PLATE-CULTIVATIONS. 73 also to examine under a low power of the microscope the individual colonies of bacteria, and to distinguish, by their characteristic appear- ances, micro-organisms which, in their individual form, closely resemble one another, or are even identical. The same process, with slight modifica- tion, is also employed in the examination of air, soil, and water, as will be referred to later. The preparation of plate-cultivations, therefore, must be described in every detail, and to take an example, we will suppose that a series of plates are to be prepared from a test-tube-cultivation. Arrangement of Levelling Apparatus. In order to spread out the liquid jelly evenly on the surface of a glass plate, and hasten its solidifica- tion, it is necessary to place the glass plate upon a level and cool surface. This is obtained in the following manner : Place a large shallow glass dish upon a tripod stand, and fill it to the brim with cold water; carefully cover the dish with a slab of plate glass, or a pane of window glass, and level it by placing the spirit-level in the centre and adjusting the screws of the tripod. Replace the level by a piece of filter paper, the size of the glass plates to be employed, and cover it with a shallow bell glass (Fig. 8). Sterilisation of Glass Plates. The glass plates are sterilised by filling the iron box (p. 27), and placing it in the hot-air steriliser, at 150 C., from one to two hours. As these plates are used 74 BACTERIOLOGY. also for other purposes, a quantity ready sterilised should always be kept in the box. Preparation of Damp Chambers. The damp chambers for the reception of the inoculated plates are prepared thus : Thoroughly cleanse and wash out with (i-iooo) sublimate solution a shallow glass dish and bell (Fig. 7). Cut a piece of filter paper to line the bottom of the glass dish, and moisten it with the same solution. In a glass beaker or an ordinary glass tumbler, with a pad of cotton wool at the bottom, place the tube containing the cultivation, the three tubes to be inoculated, and three glass rods which have been sterilised by heating in the flame of a Bunsen burner. Provide yourself with a strip of paper 01 a large label, a pencil, a pair of forceps, and inoculating needles. All is now ready at hand to commence the inoculation of the tubes. Method of Inoculating the Test-tubes. Liquefy the gelatine in the three tubes by placing them in a beaker containing water at 30 C., or by gently warming them in the flame of the Bunsen burner. Keep the tubes, both before and after the inoculation, in the warm water, to maintain the gelatine in a state of liquefaction. Hold the tube containing the cultivation and a tube of the liquefied gelatine (to be called the " original ") as nearly horizontal as possible between the thumb and index finger of the left hand. With the finger and thumb of the right hand loosen the plugs ot PLATE-CULTIVATIONS. j - the tubes. Take the sterilised ose in the right hand and hold it like a pen. Remove the plug from the culture-tube by using the fourth and fifth Angers erf the right hand as forceps, and place it between the fourth and fifth fingers of the left. Remove the plug of the " original " in the same way, placing it between the third and fourth fingers of the left hand (Fig. 28). With the ose take up a droplet FIG. 28. METHOD OF INOCULATING TEST-TUBES IN THE PREPARATION OF PLATE-CULTIVATIONS. of the cultivation and inoculate the '' original," twisting the ose several times in the liquid gelatine. Replace the plugs and set aside the cultivation. Hold the freshly inoculated tube between the fore- finger and thumb of either hand, almost horizontally, then raise it to the vertical, so that the liquid gelatine gently flows back. By repeating this motion and rolling the tube between the fingers and thumbs the micro-organisms which have been introduced are 7 6 BACTERIOLOGY. distributed throughout the gelatine. Any violent shaking, and consequent formation of bubbles, must be carefully avoided. From the so-called " original" inoculate in the same manner afresh "tube of liquefied gelatine, introducing into it three droplets with a sterilised ose. This tube is then called the " first attenuation," or No. i. After treating No. i as has been already described in the case of the original, the same process is repeated with a third tube, which is inoculated in the same way from No. i. This is the "second attenua- tion" or No. 2, and in some cases a " third atten- uation " is carried out from No. 2. The last tube must be inoculated in different ways, according to experience for different micro-organisms. Some- times a sufficient separation of the micro-organisms is attained by inoculating the last tube with a fine straight needle dipped from one into the other from three to five times. The next process consists in pouring out the gelatine on a glass plate and allowing it to solidify. Preparation of the Gelatine-plates. The directions to be observed in pouring out the gelatine are as follows : Place the box containing sterilised plates horizon- tally, and so that the cover projects beyond the edge of the table ; remove the cover, and withdraw a plate with sterilised forceps ; hold it between the finger and thumb by opposite margins, rapidly transfer it to the filter paper under the bell-glass, and PLATE-CULTIVATIONS. 7 7 quickly replace the cover of the box. On removing the plug from "the original," an assistant raises the bell-glass, and the contents of the tube are poured on to the plate ; with a glass rod the gelatine must be then rapidly spread out in an even layer within about half an inch of the margin of the plate. The assistant replaces the bell-glass, and the gelatine is left to set. Meanwhile a glass bench or metallic shelf is placed in the damp chamber, ready for the reception of the plate-cultivation, and when the gelatine is quite solid the plate is quickly trans- ferred from under the bell-glass to the damp chamber; precisely the same process is repeated with tubes i and 2. and the damp chamber, labelled with the details of the experiment, is set aside for the colonies to develop. Not only plate- cultures should be care- fully labelled with date and description, but the same remark applies equally to all preparations, tube-cultures, potato-cultures, drop-cultures, etc. In plate-cultivations write the source of the mate- rial, the date, and the number of inoculations; for example, thus : Finkler* s comma-bacilli. From tube-cultivation on " agar-agar," 5th Feb- ruary, 1885 Lower plate (Orig.) , I ose from cultivation. Middle plate, No. i . 3 osen from Orig. Upper plate, No. 2 . .3 osen from No. i. 73 BACTERIOLOGY. Corresponding with the fractional cultivation of the micro-organisms obtained in this manner, the colonies will be found to develop in the course of a day or two, varying with the temperature of the room. The lower plate will contain a countless number of colonies which, if the micro-organism liquefies gelatine, speedily commingle, and produce, in a very short time, a complete liquefaction of the whole of the gelatine. On the middle plate, with the first attenuation, the colonies will also be very numerous, but retain their isolated position for a longer time ; while on the uppermost plate, the second attenuation, the colonies are completely isolated from one another, with an appreciable surface of gelatine intervening [Plates VI. and VII.]. Examination of Plate - cultivations. The macroscopical appearances of the colonies are best studied by placing the plate on the slab of blackened glass, or on the porcelain slab if the colonies are coloured. To examine the microscopical appearances a selected plate is placed upon the stage of the microscope ; it is better to have a larger stage than usual for this purpose. The smallest diaphragm is employed, and the appearances studied principally with a low power. These appearances should be carefully noted, and a rapid sketch of the co'ony made. The morphological characteristics of the micro-organisms of which the colony is formed can then be examined in the following way. A small PLATE-CULTIVATIONS. 7 9 ose, or a platinum needle bent at the extremity into a miniature hook, is held like a pen, and the hand steadied by resting- the little finger on the stage of the microscope. The extremity of the needle is steadily directed to the space between the lens and the gelatine without touching the latter, until, FIG. 29. MICROSCOPICAL EXAMINATION OF COLONIES ON PLATE- CULTIVATIONS, AND METHOD OF ISOLATING COLONIES BY INOCULATION OF TEST-TUBES. on looking through the microscope, it can be seen in the field, above or by the side of the colony under examination (Fig. 29). The needle is then dipped into the colony, steadily raised, and withdrawn. Without removing the eye from the microscope this manipulation can be seen to be successful by the colony being disorganised or completely re- O . : i( .. ''BACTERIOLOGY. moved from 'the gelatine. It is, however, not easy to be successful at first, but with practice this can be accomplished with rapidity and precision. A cover-glass-preparation is then made in the manner already described, viz., by rubbing the extremity of the needle on a perfectly clean cover-glass and examining by Babes' rapid method, or by thinning out the micro-organisms in a droplet of sterilised water previously placed on the cover glass, drying, passing three times through the flame, and staining with a drop of fuchsine. Inoculations should be made in test-tubes of nutrient gelatine and agar-agar, from the micro- organisms transferred to the cover-glass before it is dried and stained, from any remnants of the colony which was examined, or from other colonies bearing exactly similar appearances. In this way pure cultivations are established, and the macro- scopical appearances of the growth in test-tubes can be studied. The plates should be replaced in the damp, chambers as soon as possible ; drying of the gelatine, or contamination with micro- organisms gravitating from the air during their exposure, may spoil them for subsequent examina- tion. Nutrient agar-agar can also be employed for the preparation of plate- cultivations, but it is much more difficult to obtain satisfactory results. The test-tubes of nutrient agar-agar must be placed in a beaker with water and heated until the agar-agar is completely liquefied. The gas is then PLATE-CULTIVAT turned down and the temperature^ allowed to fall until the thermometer stands just above 50 C. The water must be maintained at this temperature, and the test-tubes must be in turn rapidly inoculated and poured out upon the glass plates, as already described. Glass plates may also be employed in a much simpler method. The nutrient jelly is liquefied, poured out, and allowed to set. A needle charged with the material to be inoculated is then streaked in lines over the surface of the jelly. This method is of especial value in inoculating different organisms side by side, and watching the effect of one upon the other, or a micro-organism in this way may be sown upon gelatine which has been already altered by the growth of another micro-organism ; the change produced in the gelatine, as in the case of the fluorescing bacillus, extending far beyond the limits of the growth itself (Plate VIII., Fig. 3). Nutrient jelly may also be spread out on steri- lised microscopic slides and inoculated as just described, or cultivations may be made in shallow glass dishes, glass capsules, etc., which must be sterilised on the principles already laid down, and after inoculation placed in damp-chambers for the growths to develop. 82 BACTERIOLOGY. (C) PREPARATION AND EMPLOYMENT OF STERILISED POTATOES, POTATO -PASTE, BREAD-PASTE, VEGE- TABLES, FRUIT, AND WHITE OF EGG. Potato Cultivations. Sterilised potatoes form an excellent medium for the cultivation of many micro-organisms, more especially the chromogenous species. Potato-cultivations also give in some cases very characteristic appearances, which are of value in distinguishing bacteria which possess morphologi- cal resemblances [Plates IX. and X.] Preparation of Sterilised Potatoes. Potatoes, preferably smooth- skinned, which are free from " eyes " and rotten spots, should be selected. If they cannot be obtained without eyes and spots, these must be carefully picked out with the point of a knife with as little destruction of the surface as possible. The potatoes are well scrubbed with a stiff brush and allowed to soak in sublimate solution for half an hour. They are then transferred to the potato-receiver and steamed in the steam-steriliser for twenty minutes to half an hour, varying accord- ing to the size of the potatoes. When cooked, the potato-receiver is withdrawn and left to cool, the potatoes being retained in it until required for use. Damp chambers are prepared ready for their reception, the vessels being cleansed and washed with sublimate as described for plate-cultivations. Small glass dishes of the same pattern as the PREPARATION OF NUTRIENT MEDIA. Sj large ones (Fig. 13) may be employed for single halves of potatoes. Potato knives and several scalpels which have been sterilised in an Israel's case by heating them in the hot-air steriliser to 150 for one hour, must be ready to hand. The potato knives may also be sterilised by heating them in the flame of a Bunsen burner and placing them on their backs with their blades projecting over the edge of the table. Scalpels may be sterilised in the same way and laid upon a sterilised glass plate and covered with a bell-glass. It must not be forgotten, however, that heating the blades in the flame destroys the temper of the steel, and therefore knives and other instruments should preferably be sterilised in the hot-air steriliser^ en- closed in an Israel's case, or simply enveloped in cotton wool. Inoculation of Potatoes. The coat sleeves should be turned back, and the hands, after a thorough washing with good lathering soap, be dipped in sublimate solution. An assistant opens the potato receiver, and a potato is selected, and held between the thumb and index finger of the left hand (Fig. 30). With the knife held in the right hand, the potato is almost completely divided in the direction which will give the largest surface. The assistant raises the cover of the damp chamber, and the potato is introduced, and while withdrawing the knife, allowed to fall apart. The cover is quickly replaced, and another 84 BACTERIOLOGY. potato treated in the same way is placed in the same damp chamber. The four halves are then quite ready for inoculation. As an extra pre- caution the left hand is again dipped in sublimate, and one half of a potato is taken up between the tips of the thumb and index finger, care being taken to avoid touching the cut surface. Holding FIG. 30. METHOD OF DIVIDING POTATOES. it with its cut surface vertical, a small portion of the substance to be inoculated is placed on the centre with a sterilised needle or ose. With a sterilised scalpel the inoculated substance is rapidly spread over the surface of the potato with the flat of the blade to within a quarter of an inch of the margin, and the potato is then as PREPARATION OF NUTRIENT MEDIA. 85 quickly as possible replaced in the moist chamber. With another sterilised scalpel a small portion of the potato from the inoculated surface of the first half is in the same way spread over the surface of the second half, this forming as in plate-cultiva- tions a " first attenuation." Exactly the same is repeated with a third potato, and even a fourth, so that a still further attenuation or fractional culti- vation of the micro-organisms may be obtained. In some cases it is necessary to place the cultures in the incubator, others grow very well at the tempera- ture of the room. As in plate-cultivations, the potato may also be inoculated by simply streaking it in lines with a needle charged with the material to be inoculated. POTATO-PASTE. Potato-paste is sometimes employed where it is desirable to obtain an extensive growth of certain bacteria. The potatoes are boiled for an hour, and the floury centre squeezed out of their skins. This is then mashed up with sufficient sterilised water to produce a thick paste, and is heated in the steam steriliser for half an hour for three successive days. BREAD-PASTE. Some micro-organisms, more especially mould fungi, grow very well on bread-paste. This is prepared by removing the crust from a stale loaf 86 BACTERIOLOGY. of bread and allowing it to dry in the oven. It is then broken up, and reduced to a fine powder with a pestle and mortar. Small, carefully cleansed, conical, or globe-shaped flasks are plugged with cotton wool and sterilised in the oven. When cool a small quantity of the powder is placed in them, and sterilised water added in the proportion of one part for every four of the powder. The paste is sterilised by steaming in the steriliser at 1 00 C. for half an hour for three successive days. The flasks can be reversed, and are inocu- lated in the usual way with a platinum needle. CULTIVATIONS ON VEGETABLES, FRUIT, WHITE OF EGG, ETC. Boiled carrots and other vegetables, and various kinds of stewed fruit, are also occasionally em- ployed for the cultivation of bacteria. The sterili- sation of these media must be carried out on the principles already explained. White of egg may be poured out on sterilised glass plates, or in shallow glass dishes, boiled in the steam-steriliser and after inoculation, placed in a damp chamber. (D) PREPARATION AND EMPLOYMENT OF STERILE BLOOD SERUM. The tubercle-bacillus, the bacillus of glanders, and a few other micro-organisms, thrive best when culti- PREPARATION OF NUTRIENT MEDIA. 8? vated on solid blood serum. This medium has the additional advantage of remaining solid at all tem- peratures. The technique required for its preparation and sterilisation is as follows : Several cylindrical vessels, about 20 cm. high, are thoroughly washed with sublimate solution (i-iooo), and then with alcohol, and finally rinsed out with ether. The ether is allowed to evaporate, and the vessels are then ready for use. The skin of the animal selected calf, sheep, or horse is washed with sublimate at the seat of operation, and the bleeding is performed with a sterilised knife. The first jet of blood from the vein is rejected, and that which follows is allowed to flow into the vessels until they are almost full. The ground-glass stoppers, greased with vaseline, are replaced, and the vessels set aside in ice, as quickly as possible, for from twenty-four to thirty hours. By that time the separation of the clot is completed, and the clear serum can then be transferred 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 tem- perature 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 88 BACTERIOLOGY. 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 some- times 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 propor- tion of 5 per cent. Inoculation of the Tubes. A small portion of the material to be inoculated is taken up with a sterilised needle or ose, 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 in isolating the material to be inoculated will be referred to later (p. 112). 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 LIQUID MEDIA. 89 media advocated by Koch. It must not be supposed, however, that the methods of cultivation in liquids are discarded entirely by the German 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 or bouillon of beef, pork, or chicken may be made in the same manner as described for the preparation of gelatine-peptone- broth, 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 gO BACTERIOLOGY. 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 and hands, it may be kept without change. If procured 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 pur- poses, and more rarely decoctions of plums, raisins, malt, and horse-dung. They are mostly prepared LIQUID MEDIA. QI 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 (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 9 2 BACTERIOLOGY. 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. 31) were especially introduced by Sir Joseph Lister as a means of storing liquid nutrient media. They are so constructed that after removal of a portion of the contents, or restoring the vessel to the vertical position, a drop of liquid always remains in the extremity of the nozzle, which prevents the regur- gitation of unfiltered air. Sternberg's Bulbs. The method of introducing liquid into the bulbs employed by Professor Stern- berg, 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 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 t remain transparent 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 LIQUID MEDIA. 93 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 contain- ing 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 in- oculating 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 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, 94 BACTERIOLOGY. 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 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 inocu- lated by aspiration, and the point of the S tube sealed in the flame of a Bunsen burner. Drop-Cultures. This method of cultivation * Duclaux, Ferments et Maladies. 1882. t Miquel, Les Organismes Vivants de T Atmosphere. 1883. LIQUID MEDIA. 95 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. Mean- while 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 ose a drop of sterile bouillon is transferred to the cover glass, and this is inoculated by touching it with a sterilised needle charged with the material without disturbing the form of the drop. It is quite sufficient just to touch the drop instead of transferring a visible quantity of blood, juice, or growth, as the case may be. The slide is then in- verted and placed over the cover-glass, so that the drop will come exactly in the centre of the excava- tion, 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 96 BACTERIOLOGY. 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 em- ployed 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 adhering 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 camel's-hair brush outside the groove, and the cover-glass with the drop-cultivation is inverted and placed over the 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* : * Schafer's Course of Practical Histology. 1877. LIQUID MEDIA. 97 A small piece of putty or modelling wax is rolled into a cord about two inches long and inch thick. By uniting the ends a ring is formed, which is placed on the middle of a clean glass slide (Fig. 31). 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 FIG. 31. METHOD OF FORMING A SIMPLE MOIST-CHAMBER. be constructed by cementing a glass ring with flat surfaces to an ordinary slide. Vaseline is applied with a cameF s-hair brush to the upper surface of 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 7 9 8 BACTERIOLOGY. either place the microscope bodily within an in- cubator, with the eyepiece protruding* through an opening, so that we may observe what is going on without moving the preparation, or we must employ 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 projects. The plate has a round aperture in the middle, half an inch in diameter, and is fastened to FIG. 32. SIMPLE WARM STAGE. an ordinary 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 is painted round the edge of the smaller cover-glass to prevent evaporation, and the prepara- tion is placed over the hole in the plate (Fig. 32). The slide bearing the copper plate is clamped to the stage of the microscope (Fig. 33). The flame 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 LIQUID MEDIA. 99 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. For more accurate observations, the apparatus shown in Fig. 34 may be employed. The vessel/", FIG. 33. SIMPLE WARM STAGE SHOWN IN OPERATION. 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 indiarubber tube c to the brass box a. The box is pierced by a tubular aperture to admit light to the object, and has an exit tube c', by which the cooled water from the IOO BACTERIOLOGY, 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. FIG. 34. SCHAFER'S WARM STAGE. Another form in which warm water or steam can be used for heating, and by the employment of u (H a FIG. 35. STRICKER'S WARM STAGE. icea water also used for observing the effects of cold, is shown in Fig. 35. It consists of a hollow rect- angular box, with a central opening (C) permitting LIQUID MEDIA. 101 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. FIG. 36. SECTION OF ISRAEL'S WARMING APPARATUS AVD DROP-CULTURE SLIDE. Israel's Warming Apparatus. It is obvious that in employing very high powers a difficulty will F IG . 37. ISRAEL'S WARMING APPARATUS. be presented by the warm stages just described owing to their interference with the illumination. 102 BACTERIOLOGY. To overcome this an apparatus has been con- structed by which the slide is warmed from above.* 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, FIG. 38. ISRAEL'S WARMING APPARATUS IN OPERATION. 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. 36). The entrance and exit pipes are fixed on at a * Israel, Zeitsch.f. Wiss. Mikrosc, ii., pp. 45963. 1885. LIQUID MEDIA. IO3 right angle to the side (Fig. 37). The former, 2, 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. 38). A mixture of paraffine and vaseline is recom- mended for indicating the temperature of the chamber, and experience has shown that if a tem- FIG. 39. SIMPLE GAS CHAMBER. perature 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 gases or vapours upon micro-organisms, a modi- fication of the simple moist chamber (Fig. 31), may be employed (Fig. 39). A piece of glass tubing is first fixed to the slide by means of sealing wax, and the ring of putty is so placed as to include the end of this, leaving a small interval at the side, or a little notch is made in the putty opposite, so as to afford an exit for the gas or vapour (Fig. 40). 104 BACTERIOLOGY. A more complicated apparatus, combining: both a warm stage and a gas chamber, is shown at Fig. 41. This consists of a rectangular piece of ebonite EE fixed to a brass plate which rests on the stage of the microscope. On the upper surface of the ebonite is 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. 40. GAS CHAMBER IN USE WITH APPARATUS FOR GENERATING CARBONIC ACID. 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 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 LIQUID MEDIA. effect of electricity we may prepare a drop -culture in the moist-chamber (Fig. 42). The cover-glass to be used is provided with two strips of tinfoil, FIG. 41. STRICKER'S COMBINED GAS CHAMBER AND WARM STAGE. which are isolated from the brass of the microscope, and so arranged that a current of electricity may be passed through them (Fig. 43). FIG. 42. SIMPLE MOIST-CHAMBER ADAPTED FOR TRANSMISSION OF ELECTRICITY. 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 I O6 BACTERIOLOGY. firmly down on gold leaf or tin-foil and allowed to dry. When dry, the metal is scraped away, leaving FIG. 43. APPARATUS ARRANGED TOR TRANSMITTING ELECTRICITY. two triangles with a small interval between them, as in Fig. 44. FIG. 44. SLIDE WITH GOLD-LEAF ELECTRODES. The liquid containing the micro-organisms is placed between the electrodes, covered with a cover- glass, and then subjected to the electric current. 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 . I O8 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 109 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 ose 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 I I O 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 sublimate solution, wiped dry, and sterilised in the hot-air steriliser, before they are put away. If these precautions are 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 I I 2 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 ithe 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. I 13 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 an ose, the needle of a Pravaz' syringe, a capillary tube, or the extremity of the capillary neck of a Sternberg's bulb. If the cultivation is contaminated by the presence of putrefactive or other micro-organisms they must be isolated subsequently by carrying out a series of plate-cultivations. ~ I 14 BACTERIOLOGY. Having completed the dissection, the organs of such a small animal as a mouse may be removed en 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 an ose 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 toij^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 I 1 8 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 . . . . . . 604 Ash 4*72 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 \\\e Bacillus GENERAL MORPHOLOGY AND PHYSIOLOGY. I 19 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, i. ...---. 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- FIG. 45. served in the long filaments, which F ^S^ are endowed with a slow vermi- THRACIS, DOUBLE ....... STAINED WITH GEN- cular movement, while rigidity TIAN VIOLET AND accounts for the maintenance of EOSIN \ ^ heS 1 heatl ! was stained pink, and the characteristic form of several the ceil - contents i MI 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 I2O BACTERIOLOGY. present, with the exception of the tubercle bacillus. This difference in the protoplasm of different species is also illustrated by the necessity 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 Closlridium 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 Bacterium prodigiosum 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. 1 2 I 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 to 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 zoogloea. 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 122 BACTERIOLOGY. readily develops on unsterilised potatoes, the zoo- gloea may spread over the cut surface, forming a pellicle which can be raised en masse like a delicate veil. Another bacillus forms a zoogloea, 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. 46). FIG. 46. 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. 123 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. 47, 48, 49). FIG. 47. STREPTOCOCCUS AND FIG. 48. STREPTOCOCCUS IN THE SARCINACOCCUS FROM A BLOOD OF A RABBIT, x 1200. DROP-CULTIVATION, x 1200. These chains may consist of a few four or five individuals linked together, or of a far greater number, in which case the chains are generally curved or twisted. If the division occur in two FIG. 49. STREPTOCOCCUS OF PROGRESSIVE TISSUE NECROSIS IN MICE. [After Koch.] directions, so that four cocci result, a tetrad or meris- mopedia is formed. If the division occur in three directions, one coccus divides into eight, and we get a packet form or sarcinacoccus. Immediately after division the daughter cells are not perfectly 124 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 staphylococcus, 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, spirochceta (Fig. 50), 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. 125 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. 50. 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 comlpete 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 126 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 ddbris, 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. 127 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 undoubtedly provided with a vibratile FIG. 51. 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]. II. 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 Zopt]. 15. Beggiatoa roseopersicina with a triple flagellum at one end; and 16. with a double flagellum at both ends [after Warming]. lash or flagellum at one end, or with one or more at both ends (Fig. 5 i). 128 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. I2Q 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. 52. 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 DeBary.] 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 ant hr acts > Bacillus megaterium, or Bacil- lus subtilis. The protoplasm becomes granular, 9 130 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. 53. 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. considerably wider, but is never longer than the parent cell. Arthro-spore formation is illustrated in Leuco- nostoc mesenteroides. Certain elements in the chain of cocci, apparently not differing from the rest, FIG. 54. A THREAD OF BACILLUS ANTHRACIS WITH SPORES, IN A DROP-CULTIVATION, x 1400. become larger, with tougher walls, and more refrac- tive (Fig. 55). 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. 55. I EUCONOSTOC 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 - V- 132 .., 'BACTERIOLOGY. : i;<;*-V :-.M : V 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 cultivations 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 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. 52 and 53). Spores differ from the parent cells in their be- . ... FIG. 57. SPORE-BEARING THREADS OF BACILLUS ANTHRACIS, DOUBLE-STAINED WITH FUCH- SINE AND METHYLENE BLUE, X 1200. FIG. 56. SPORES OF BACILLUS AN- THRACIS, STAINED WITH GENTIAN VIOLET, AFTER PASSING THE COVER-GLASS TWELVE TIMES THROUGH THE FLAME, 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 3 4 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, granules may be observed which are sometimes terminal. They can be readily demonstrated by taking a photograph with a ^5 in. Horn, imm., and subsequently enlarging the nega- tive to from 2,500 (Fig. 58) to 6,000 diameters. GENERAL MORPHOLOGY AND PHYSIOLOGY. 135 It is not impossible that these ovoid granules are spores, which, in their behaviour towards staining reagents, thus form an exception to the general rule. But there can be ^~" little doubt that a tubercle ^^* \ TV bacillus consists, for the % most part, of a very delicate FIG. 58. TUBERCLE BACILLI IN sheath, with protoplasmic SPUTUM, x 2500 (from photo- 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 FIG. 59. . ^. . , , LEPROSY BA- preparation. This, however, does not CILLI, FROM always occur, for the bacilli at times are A SECTION . . OF SKIN, not beaded, but are stained in their en- 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. 60. of the rod (Fig. 59). Very probably the appearances in the case of the OF A GLANDERS T -11 r i j /T- s \ j NODULE, x 1200. bacillus of glanders (Fig. 60), and the bacterium of chicken-cholera (Figs. 61 and 62) may be similarly explained. 136 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 staining red, while the bacilli FlG - 6l - were stained blue. BACTERIUM OF CHICKEN- j h j definition o f spirilla CHOLERA, FROM BLOOD OF INFECTED HEN, x Zopf gives the spore-formation as absent or unknown. In comma-bacilli in sewage water, the author has often noted appearances very suggestive of refractive 4? 0** ** /^\ ^ * 5? FIG. 62. FIG. 63. 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. 63). The same also may be observed in vibrios, differing by their regular contour from the irregular spaces occasionally observed in stained preparations (Figs. 64 and 65). 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. 137 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. 64. FIG. 65. 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 138 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 oedema 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. 91). 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. 139 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 blood-serum. 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 th 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 maximu'/n, 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 41 C. 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 140 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. 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- 142 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 ianthinus, which produces a striking purple growth ; Bacillus pyocyancus, which secretes pyocyanin, a substance which has been isolated and obtained in a crystalline form ; Bacterium prodigiosum, which produces a pigment allied to fuchsine ; Beggiatoa roseo-persicina, which is characterised by the presence of bacterio - purpurin ; Sarcina lutea> Bacillus cyano genus , and many others. Zymogenic or ferment bacteria produce their changes in non-nitrogenised media. Bacterium aceti y 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 urea. The change produced is represented by the following formula : = (NH 4 ) 2X CO 3 . Clostridium butyricum converts the salts of lactic acid into butyric acid, producing the butyric fer- GENERAL MORPHOLOGY AND PHYSIOLOGY. 143 mentation 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 6 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 2? 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 ) + i2(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 : 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 144 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, according to Klein, in the liver and internal organs, where the tissues have lost their vitality, and the organisms, through some lesion, have been carried into the circulation. GENERAL MORPHOLOGY AND PHYSIOLOGY.. 145 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. 2). 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 tissue cells 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 seize upon the commis- sariat, appropriating the general pabulum, which 10 146 BACTERIOLOGY. is so essential to the life of the tissues. But this would hardly account for so acute and fatal a result as 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 XVI. and XVII.) 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. 147 produce a ptomaine anthracin, which in a certain dose produces death, independent 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 in quantity 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 urea. 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 148 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 debris 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. 149 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. 15! 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 con- 152 BACTERIOLOGY. cerned 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. 153 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 154 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, the so-called Micrococcus prodigiosus, and the bacillus of blue pus. These were culti- vated 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 conclu- sion 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, 155 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 156 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, allow- ing for deterioration by keeping, a solution con- taining 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. 157 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 mercuric chloride ; 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. t ft F 158 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 2,000. 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 properties. 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 was not efficient, there was evidence of a growth ot 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. 159 observations may be relied upon for practical application. Hot Air and Steam. Koch, in conjunction with Wolfhugel, 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 no 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 6O 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. 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 i2oC. 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 1 1 8 C. Guinea-pigs inoculated with tuberculous pus which had been exposed for five minutes to 104 C., ANTISEPTICS AND DISINFECTANTS. l6l 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 G. (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. ii 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 pigs, cats, white rats, and to adult dogs, asses, and horses. 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 with 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. 163 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 prior to the establishment by Jenner of the protective influence of vaccination. 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 aftor non-fatal cases the disease either did not recur, or the severity of a subsequent attack was in inverse proportion to the seventy 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 164 BACTERIOLOQY. 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 165 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 66 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 all 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 vacua, the virulence was found to disappear in twenty- four hours, and by keeping cultivations at a low temperature with free IMMUNITY. 167 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 & JQ of carbolic acid was found to 1 68 BACTERIOLOGY. be inert in guinea-pigs and rabbits. Bichromate of potash added to a cultivation in the proportion f TTOOO -5-000 ave a f ter 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. 169 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 Aspergillm 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 I7O, 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. 171 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 an,d 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. LEEUWENHOECK,* two hundred years ago, recog- nised, and described, microscopic organisms in putrid water and saliva, which probably correspond with organisms, such as vibrios and leptothrix ot modern times. During two centuries these minute beings have afforded histologists a subject for controversy and dispute. Existing as they do upon the very borderland of the vegetable and animal kingdoms, not only have they been transferred from one to the other, but even the question has been raised whether the smaller forms should be con- sidered as living beings at all. 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 * Leeuwenhoeck, O. Omnia (Lugd. Batav., 1722). 1 76 BACTERIOLOGY. 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 Infusoria. In 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 Spirochceie^ and classed them thus : I. Bacterium . filaments rigid, vacillating. II. Vibrio . ' . filaments flexible, undulatory. III. Spirillum. . filaments spiral, rotatory. Up to that time bacteria were still considered as CLASSIFICATION OF BACTERIA. 177 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 the subject through the writings of Hoff- mann, Pasteur, Cohn, Rabenhorst, Hallier, Billroth, Warming, Nageli, Magnin, Marchand, Sternberg, Van Tieghem, Lister, Klein, Koch, Fliigge, De Bary, Zopf, 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, Beitrage zur Biologic der Pflanzen, 1872, et seg. 12 178 BACTERIOLOGY. Cohn noted, in spite of placing them with the Algce, 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 maintained that distinct genera and species existed. The genera Cohn considered to be dis- tinguished 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, deter- mined the various species. Cohn illustrated, by his well-known comparison of a sweet and a bitter almond the appearances of which are similar but the properties very different, that a distinction into species might depend upon a difference in physiological action only. Others strongly sup- port Cohn's views. By cultivating various micro- organisms through several generations, many conclude that a micrococcus cannot be trans- formed into a bacterium, or a bacterium into a bacillus or spirillum. Koch does not believe- and in this he is supported by Klein that a bacillus can change its nature, and be converted CLASSIFICATION OF BACTERIA. 1 79 from a harmless into a pathogenic form, as asserted by Blichner.* The second classification of Cohn (1875) on ty differed from the first in that, instead of keeping the bacteria as a separate group, he placed them, from their close relationship with the Phycochro- macea, under a new group, the Schizophytes, and added the genera Leptothrix, Beggiatoa, Crenothrix, Sarcina, Ascococcus, Streptococcus, Myconostoc, and Strtptothrix. Nageli maintained that Bacteria were allied to Yeasts, and should be included in the class of Fungi. In fact, he divided the fungi producing decomposition into : Mucorini . . . . moulds Saccharomycetes .... yeasts Schizomycetes .... fission-fungi (This last class comprising bacteria.) Fliigge,t following Rabenhorst, maintained the term Schizomycetes, and divided them as fol- lows : * Biichner, Ueber d. exfierim. Erzeugung d. Milzbrandconta- giums aus d. Heupilzen. f Fliigge, Fermente und Mikrofiarasiten. 1883. i8o BACTERIOLOGY. 3 5 o mall but definite mbers, in regular oups. gr co W H W 8 H- 1 ffi U CO O r- U ^^ f-^ <5 U if' *h. & "c "^ *c **\ QJ "o "o ^ ^ U U v >^ en 'c3 CO 3 d. en i 'e . *^ 2 o u, O ^ c g -p rt g 1 ^ .S ^ ll O 3 flj QJ ^ o tuo-o -0 *^O G +- *^M ^"o H ** O G "t^ G " H o *+j ,L O co - r 'So o ^ CO o *j G J3 | ^3 en ^M i cu rd n-J o cu ?3 n -t! r2 ^ G * * , b'-i-i . ~tj ^ > ^ ^ P-l J, 2^ * ^ o P i *"Q ^ ^ O w o en ^en O *^ ^ ro cj _H _^ Cj ^ ^ S 4 G ^ " 'g-B'g 1'i'S'p, Ja c c 2 *"* J-T* ^*^ i i cu 2 ^ o * !^> ^ !> ^ ^ ^PQ i i H o "5 Z o 1-1 ^^ ^ < P5 i-i * -^ ^' x- ^ ^-x -^ v -^r* / U co ; en en * EPPE'S CLASSIFI t_i CU n3 8 o ^ ' H en Td cu o r^- en ' S ll cj crS 15 m 5-5 ogloea indeterminate ogloea united in balls 03 ^ 'g & *g en en O oj rt H H W IH p^ O '"^^ p sl N ^^ ... NN HH H i H H _^^y ^ W ~^ C/5 -i -M 0) en ^^-^ v^ "^ CU .2 g CO +-> en ft CU P en bjQ o3 C ^ rO cu .^ en en 32 C/5 d pi^ ^ bp O ."S q_, ^ 5P L^ bjQ u ^ '53 cu 1-1 ^ o "t^ O c .2 .2 c M o * 2 s_< 0) o r^J r s T3 "^ ^ cu cu en O ^ 4 r^ CU i g 1 b/D 1 s cu rt ,-5 CU V3 'rt S.S2 s~^ C cu oj q3 nj o t3 cu C c 1 ti << ) breaking up into rod-forms, (<:) into cocci [after Kurth]. the intestine of fowls, especially in the contents of the vermiform appendix. Inoculation of rabbits was followed by negative results. Bacterium merismopedioides, Zopf. Forms threads i 1*5 p, in thickness; these subdivide into long rods, short rods, and finally into cocci. The 246 BACTERIOLOGY. cocci divide first in one and subsequently in two directions, forming characteristic groups, which 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).* Bacterium Pfliigeri, Ludwig. Large, round cocci, mostly in zooglcea, and thread-forms com- posed of rods. They can be cultivated on boiled white of egg and potatoes. They were observed to produce phosphorescence in putrid fish and meat. Bacterium photometricum, Engelmann. Cells slightly reddish in colour, motile. The movements are stated to depend on light. Bacterium litoreum, Warming. Cells ellip- soidal 2 6 p, long, i "2 2*4 p. wide, occur singly in sea water, never as chains or zoogloea. Bacterium fusiforme,Warming. Cells spindle- shaped, with pointed ends, 2-5 p long and -5 '8 /x 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 * Zopf, Die Spaltyilze. 1885. SYSTEMATIC AND DESCRIPTIVE. 247 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 /x in length, and are almost as broad as long, and others vary from '94 1*25 p, long and -42 '63 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 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 //, -9 ft. 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. * Hauser, Ueber Fdulniss-Bacterien. 1885. 248 BACTERIOLOGY. Proteus Zenkeri. Cocci, -4 ^ in twos like Bacterium termo, and short rods 1*65 ju 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 terrno, Dujardin. Short cylindrical or oblong cells, 1*5 //, long, '5 7 broad, generally occurring 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 appear- ing 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 pro- duce a turbidity, and on sterilised potatoes a slimy grey layer. Bacterium lineola. Cells 3-8 p 5-2 //, long, 1-5 ^ wide. They occur singly or in pairs, occasionally in zoogloea, 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 zoogloea and pellicles on various infusions. Cultivated on nutrient agar-agar they form a semi-transparent growth (Plate XXIV., Fig. i). SYSTEMATIC AND DESCRIPTIVE. 249 Genus //. Spirillum. SPECIES. ASSOCIATED WITH DISEASE : 'Spirillum Obermeieri . Pathogenic. I" Pathogenic in man (?), pos- In man . . . Spirillum Cholera Asiaticse-j sibly only saprophytic. ( Pathogenic in animals. Spirillum Finkleri . I Saprophytic in man. 1 I Pathogenic in animals. In animals . / Spirillum tyrogenum . . f< Saprophytic. Pathogenic ( Spirillum sputigenum j in animals. UNASSOCIATED WITH DISEASE : Spirillum plicatile Spirillum serpens Spirillum tenue . Spirillum undula Spirillum volutans Spirillum Rosenbergii Spirillum attenuatum . Spirillum leucomelaneum Simple saprophytes, Spirillum Obermeieri (Spiroch&te Obermeieri, Cohn. Spirillum of Relapsing Fever}. Threads similar to the Spirillum plicatile. In length mostly 1 6 40 /*, 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 * Obermeier, Med. Central!) . 1873. 250 BACTERIOLOGY. man,* but inoculations of mice, rabbits, sheep, and pigs gave 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. 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. 35). f The commas are actively motile ; their movements and development into spirilla may be studied in drop-cultivations (Fig. 84). In plate- cultivations, at a temperature of from 16 20 C., the colonies develop as little specks (Fig. 85), which * Carter, Lancet. 1879 and 1880. Koch, Cohn 1 s Beitrdge. t Compare also Van Ermengem, Recherches sur le Microbe du Choi. Asiat. 1885. SYSTEMATIC AND DESCRIPTIVE. 25! 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 FIG. 84. 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]. very faintly yellowish-red tinge, which have lique- fied the gelatine, and sunk down to the bottom of the resulting excavations (Fig. 86). j. nr. FIG. 85. COLONIES OF COMMA BACILLI ON NUTRIENT GELATINE, NATURAL SIZE [after Koch]. In test-tubes of slightly alkaline nutrient gelatine (10 per cent.), the appearance of the growth is very striking. It commences to be visible in 252 BACTERIOLOGY. about twenty-four hours. Liquefaction sets in very slowly, commencing at the top of the needle FIG. 86. COLONIES OF KOCH'S COMMA BACILLI, x 60 ; from a nutrient gelatine plate-cultivation. track around an enclosed bubble of air, and form- ing a funnel continuous with the lower part of the r tfUfc'i w ',// A ',i ii V Fig. 87. Fig. 88. FIG. 87. 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 KochJ. FIG. 88. COVER-GLASS PREPARATION OF CHOLERA DEJECTA IN DAMP LINEN (two days old), X 600. Great proliferation of the bacilli with spirilla (a) [after Koch]. growth (Plate III., Fig. i) ; the latter preserves for several days its resemblance to a white SYSTEMATIC AND DESCRIPTIVE. 253 thread (Figs. 92 and 93).* In about eight days, however, liquefaction takes place along the whole of the needle track. On a sloping surface of agar-agar the cultivation develops as a white, semi-transparent layer, with well- defined margin. In potato-cultivations the microbe '-" F IG . 89. SECTION OF THE Mucous MEMBRANE OF A CHOLERA INTESTINE, X 600. A tubular gland (a) is divided transversely ; in its interior (/>) and between the epithelium and the basement membrane (<:) are numerous comma bacilli [after Koch], will only grow at the temperature of the blood (37C.), forming a slightly brown, transparent layer. Inoculation of a cultivation of the bacillus in the duodenum of guinea-pigs, with t and without $ 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 * from Remarks on the Comma- Bacillus of Koch. Lancet. 1885. t Nicati et Rietsch, Com. a P Academic de Medecine. 1884. J Van Ermengem, Le Microbe du Cholera Asiatique. 1885. BACTERIOLOGY. cent, solution of potash were injected into the stomach of a guinea-pig, and twenty minutes after ten ccm. of a cultivation of comma-bacilli diffused in broth were similarly introduced. Simultaneously Fig. 90. Fig. 91. Fig. 92. Fig. 93. PURE CULTIVATIONS IN NUTRIENT GELATINE. Fig. 90. Finkler's bacillus, twenty-four hours old. Fig. 91. ,, two days old. Fig. 92. Koch's cholera bacillus, twenty-four hours old. Fig- 93- two days old. with the latter, an injection of tincture of opium was made into the abdominal cavity, in the propor- tion of i ccm. for every 200 grammes' weight of the animal. Those who have had success with SYSTEMATIC AND DESCRIPTIVE. 255 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 dis- puted, the fatal effects of the inoculation attributed to septicsemic 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 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 layer of the intestine, in the mucous flakes and liquid contents of the intestinal canal of cases of Asiatic cholera f (Figs. 87, 88, 89). It is stated that they were also detected in a tank which contained the water supply to a neighbourhood where cholera * Klein, Brit. Med. Journal, and Micro-organism and Disease. 1885. Lankester, Nature, xxxi.; Nineteenth Century. July, 1885. Klein and Gibbes, "An Inquiry into the Etiology of Asiatic Cholera." Bluebook, 1885. t 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 256 BACTERIOLOGY. cases occurred; but comma-shaped organisms are commonly present in sewage-contaminated water (Fig. 94). The comma-bacilli are aerobic, and their development 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 organ- isms by the test of cultivation. FIG. 94. COMMA-SHAPED r^-, t j/-r i ORGANISMS WITH OTHER - 1 n ^ entirely different results (| , obtained in the case of the comma-bacilli of cholera nostras (Figs. 90 to 93), renders a thorough acquaintance with these bacilli of the greatest importance as an aid in diagnosis. 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 Chytridiaceas, it is very 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 ex- pressing a more definite opinion, until the report of these observers is published in full. SYSTEMATIC AND DESCRIPTIVE. 257 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. 50, 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.} Sections of the intestine, which must be well hardened 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. (b) 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. * Brit. Med. Journal, Sept. 1885. t Berliner Klinische Woch., No. 31. \ Cornil and Babes, Les Bacteries, p. 458, 1885. 17 2.S8 BACTERIOLOGY. Spirillum Finkleri (Comma-bacillus in Cholera nostras). Curved rods thicker than the comma- bacillus of Koch, and spirilla. The colonies on plate-cultivations (Plates VI. and VII.) 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 granu- lar appearance. They liquefy nutrient gela- tine 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 Fig. 95. Fig. 9 6. appearances are espe- cially 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 (Figs. 95 and 96). On a sloping surface of nutrient PURE CULTIVATIONS OF THE SPIRILLUM FINKLERI IN NUTRIENT GELATINE. Fig. 95. In twenty-four hours. Fig. 96. In thirty-six hours. SYSTEMATIC AND DESCRIPTIVE. 259 agar-agar a white moist layer forms very quickly. On potatoes they grow at the ordinary temperature 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 sputigenum, Lewis. Curved rods very similar to the comma-bacilli of Koch. Many FIG. 97. SPIRILLUM SPUTIGENUM. Occurring with spirochseta denticola, leptothrix-filaments, micrococci, and bacteria in a scraping from a carious tooth, X 1 200. have failed with repeated attempts to cultivate these bacilli, and, therefore, maintain that they are quite distinct biologically from the spirilla associated with Asiatic cholera. Others assert 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. They occur with other bacteria in saliva, and in scrapings from carious teeth (Fig. 97). * Finkler and Prior, Erganzungshefte zum Centralblatt fur Allgemeine Gesundheitspflege, Erster Band. 1885. 26O BACTERIOLOGY. . Spirillum tyrogenum, Deneke. Curved rods, slightly smaller than Koch's comma-bacilli, with a great tendency to form long spirillar threads .(Fig. 98). 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 liquefaction occurs along the needle track, and on the surface of nutrient agar-agar a yellowish-white FIG. 98. SPIRILLUM TYROGENUM. From a cultivation in nutrient gelatine, X 1200. 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 plicatile, Ehrenberg (Marsh-Spiro- chcete).li\im threads, 2-25 //, in breadth, with numerous narrow windings, 1 10 125 /itlong, occur- ring also in spirulinar forms. The threads have primary and secondary windings ; the former are in each example of equal size, but the latter are often SYSTEMATIC AND DESCRIPTIVE. 26 I 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. 99). 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. 99. SPIRILLUM PLICATILE (Marsh Spirocha-te). From sewage- contaminated water, X 1200. The following may be provisionally described as dis- tinct species, though they are probably the spiral phase- forms of protean species. Spirillum serpens, Miiller (Vibrio serpens). Threads 11 28 //, long, '8 ri //, 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 ij, usually 2 5 spirals. Height of a single screw is 2 3 //,, and the length of spiral, therefore, 4 15 /u.. They are very swiftly motile, and often occur in felted dense swarms in vegetable infusions. Spirillum undula. Threads i- 11-4 /* thick, 9 12 fjL long; spirals 4-5 /x, high; each thread has if 3 spirals. They are actively motile, and possess at each end a flagellum. They occur in various infusions (Fig. 100). Spirillum volutans, Ehrenberg. Threads 1-5 262 BACTERIOLOGY, 2 ^ thick, 25 30 fju long ; tapering towards their extremi- ties, which are rounded off. They possess dark granular contents. Each thread has 2\ 3^ windings or spirals, whose height is 913 /*. They have a flagellum at each end, and are sometimes motile, sometimes not. They are found in various infusions and water of marshes. Spirillum Rosenbergii. Threads with i \\ windings; 4 12 p long; 1*5 2'6 /u, thick. They are colourless, but the contents include strongly refractive sulphur granules. Also spirals 6 7*5 ^ in height, which are actively motile, are found in brackish water. FIG. 100. SPIRILLUM UNDULA, x 1500. Spirillum attenuatum, Warming. Threads much attenuated at the ends, which consist usually of three spirals. The middle spiral is about 1 1 p high, and 6 p in diameter ; and the end ones 10 //- high, and 2 pin 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 IIL Leuconostoc. SPECIES. UNASSOCIATED WITH DISEASE : Leuconostoc mesenteroides . . . Zymogenic saprophyte. Leuconostoc mesenteroides, Cienkowski (Gomme de sucrerie, Froschlaichpilz, Frogspawn fungus). Cells singly, in chains, and in zoogloea, SYSTEMATIC AND DESCRIPTIVE. surrounded by a thick gelatinous envelope (Fig. 101). The life-history has been very thoroughly investigated.* The spores, 1*8 2 JJL in diameter, are of a round or ellipsoidal form, with thick mem- FIG. 101. LEUCONOSTOC MESENTEROIDES. I. Spores. 2. Spores after germination, showing gelatinous envelope. 3, 4, 5, 6. Increase by division. 7. Glomerular form of zoogloea. 8. Section of an old mass of zoogloea. 9. Cocci chains with arthro- spores [after Tieghem and Cienkowski]. 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 ucrerie," Ann. Sc. Nat. 1879. 264 BACTERIOLOGY. 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 pro- cess a chain of cocci results, encased in a cylindri- cal 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 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 arthro- spores (p. 131). 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. 265 Genus IV. Bacillus. SPECIES. ASSOCIATED WITH DISEASE : Bacillus leprse Bacillus in syphilis Bacillus typhosus Bacillus malariae . In man Bacillus of choleraic diarrhosa from meat poisoning . . Bacillus pyogenes fcetidus Bacillus in septicaemia in man Bacillus in gangrenous septicaemia Bacillus tuberculosis Bacillus anthracis Bacillus mallei .... Bacillus of malignant oedema Bacillus of septicaemia of mice Bacillus of ulcerative stomatitis in the calf . . . Bacillus of swine-typhoid Bacillus of swine-erysipelas . Bacillus in tetanus Bacillus alvei Pathogenic. Pathogenic (?) ; possibly only saprophytic. > J5 Pathogenic in man (?), patho- genic in animals. Pathogenic. Saprophytic in man, patho- genic in animals. Saprophytic. Pathogenic. Pathogenic (?) Pathogenic. Chromogenic saprophytes. Zymogenic saprophytes. UNASSOCIATED WITH DISEASE : Bacillus ianthinus _ . . . ) Bacillus pyocyaneus Bacillus cyanogenus Bacillus acidi lactici Bacillus Fitzianus Bacillus subtilis Bacillus figurans . Bacillus of jequirity Bacillus caucasicus Bacillus dysodes . Bacillus Hansenii Bacillus erythrosporus . Bacillus septicus .... Simple saprophytes. Bacillus saprogenes Bacillus fcetidus . Bacillus putrificus coli . Bacillus coprogenus foetidus . Bacillus aerophilus Bacillus mesentericus fuscus . Bacillus mesentericus vulgatus Bacillus leprae, Hansen. Fine slender rods, 4 6 p long, and less than i p. wide, occasionally pointed at both ends, some clearly motile, and others not. In tissue sections they have a beaded 266 BACTERIOLOGY. appearance (Fig. 102). Spore-formation has been described. They have been cultivated artificially on blood serum and alkaline meat extract. In- oculation 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 num- bers in tubercular leprosy in the nodules of the skin (Plate XXIII., Figs, i and 2), and of the mucous membrane of the mouth, palate, larynx, FIG. 102. LEPROSY BACILLI FROM A SECTION OF SKIN, x 1200. etc.")" They occur also in the liver, spleen, testicles, lymphatic glands, and kidneys (Plate XX., 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 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 *Damsch, Virchow* s Archiv, Bd. 92, Heft i. tThin, Med. Chir. Trans. Lond., 1883; Brit. Med. Journal, No. 129, 1884, and Steven, Brit. Med. Journal, No. 1281, 1885. SYSTEMATIC AND DESCRIPTIVE. 26/ preparations may be made.* Cover-glass preparations and sections may be stained by Ehrlich's method (p. 50), or the latter by the following process : M ethod 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. :); Rods re- sembling the bacilli of leprosy and tuberculosis, 3 4 fji long, *8 n 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 stain- ing 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, Compt. Rend, de V Acad. d. Sc. 1883. \ Lustgarten, Die Syphilisbacillen. Mit 4 Tafeln. 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. 2.68 .,, , . _ BACTERIOLOGY. the VecflHrid-fkially the solution is warmed for two hours at 60 C. : Concentrated alcoholic solution of gentian-violet . 1 1 Aniline water . ...... 100 The sections are then placed for a few minutes in absolute alcohol, and from this transferred to I 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 p, broad, and forming filaments up to 50 ju long ; * ort 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 Arch, /. Experimental Pathol. 1880. t Eberth, Virchoivs Archiv, Bd. 83. \ Gaffky, Mittheil. a. d. K. Gesundheitsamte. 1884. SYSTEMATIC AND DESCKJFHVE,K II MT1 2 gelatine which were inoculated spleens. After twenty-four hours the course of the inoculation streak became visible, and in forty-eight hours a distinct whitish growth had developed. With a low power this was found to consist of numerous colonies of a yellow-brownish colour. The gelatine was not liquefied. The rods varied in length FIG. 103. BACILLUS TYPHOSUS FROM A POTATO-CULTIVATION, x 1500. (Fig. 103), were capable of development into threads, and were motile. They can be cultivated on potatoes 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 preparations and sections must be left for several days in the fuchsine solution employed in the method of Ehrlich (p. 163) ; or the solution may be warmed, and in the case of cover- glasses, even raised to boiling-point. They are then decolorised with nitric acid, and after-stained with methylene blue. * Frsenkel and Simmonds, Die Atiolog. bedeutung des Typhus - bacillus. 1886. 27O BACTERIOLOGY. Bacillus malariae, Klebs (Bacillus in inter- mittent 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, peculiar amoeboid bodies and motile filaments are constantly present in the blood of cases of malaria. These organisms, or plasmodia malaria, appear to be closely allied to the flagellated protozoa (see Appendix B). Bacillus of choleraic diarrhoea from meat- poisoning, Klein. J Rods from 3 9 //, in length, 1*3 ft wide, rounded at their extremities, singly or in chains of two. Spore-formation occurs, the spores being i /* 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 * Klebs and Tommasi Crudeli, A rchiv. f. Experimental Pathol. 1879. t Marchiafava, ibid. \ Klein, p. 87. SYSTEMATIC AND DESCRIPTIVE. 27! present in the blood and exudations of the animal. They occurred in the blood and juices, and espe- cially in the glomeruli of the kidneys, of several fatal cases of choleraic diarrhoea. Bacillus pyogenes fcetidus, Passet. Small rods, with rounded ends of about 1*45 p, in length, and '58 fj. 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 steril- ised potato an abundant, shining, brownish culture. From all these media a putrid odour emanates, but no smell is detected from a cultivation 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 p wide, which were observed in the blood-vessels of the swollen lymphatic glands. They are possibly identical with the following: Bacillus in gangrenous septicaemia, Ar- loing and Chauveau. Short rods, possessing * Klein, Micro-organisms and Disease. 1885. 272 BACTERIOLOGY. spores, were observed around wounds in gangre- nous septicaemia, and considered to be the cause of the gangrene. . Bacillus tuberculosis, Koch.* 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. 104), and occur singly, in pairs, or in bundles. They are found in the cells FIG. 104. BACILLUS TUBERCULOSIS, FROM TUBERCULAR SPUTUM, STAINED BY EHRLICH'S METHOD, x 2500. From photographs. of tubercles, especially in the interior of giant cells. In the latter they are often accompanied with grains which exhibit the same colour reaction (Plate XVIII. , Fig. i). They are non-motile. Spore-formation has been described (see p. 134). The best 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 * Koch, Berl. Klin. Woch., No. 15, 1882 ; and MittheiL aus dem KaiserJich. Gesundheitsamte> " ^Etiologie der Tuberkulose." SYSTEMATIC AND DESCRIPTIVE. 273 growth takes place very slowly, and only between the temperatures of 30 and 41 C. In about eight or ten days the growth appears as little whitish or yellowish scales and grains (Plate XL, 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. 52, and Plate XL, Fig. 4). They are then seen to be composed of bacilli, arranged more or less with their long axis corre- sponding 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 ' B 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 18 274 BACTERIOLOGY. scales, and a new generation started. The scales gradually increase in size, and consist entirely of bacilli. In about three to four weeks the cultivation ceases to increase, and it is then necessary to inoculate a fresh tube. The virulence is not weakened by carrying on successive cultivations. A relatively small portion of the 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. 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 enlargement and caseation, followed by tuberculosis of the eyeball and organs generally. The bacilli 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 existence 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 SYSTEMATIC AND DESCRIPTIVE. 275 of man, monkeys, cattle (Perhuchf), birds, and many other animals, and in cases of artificial tuberculosis, in rabbits, guinea-pigs, cats, etc. (Plate XVIII., Fig. 2). In man the bacillus can be detected in the tis- sues, in the sputum, in the blood, and in the urine.* Tuberculosis may also be produced by inhalation and feeding experiments (p. 107). 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 staining the bacillus tuberculosis, each of which will be given in detail Ehrlichs and Gibbes 1 methods may be with advantage employed in staining cover-glass preparations. Gibbes' rapid double stain is obviously the best to employ for clinical purposes. For sections both EhrlicJis and Neehens methods give excellent results. Koctis original method. Cover-glass preparations or sections are laid in Koch's solution (No. 23, c.) 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. * Babes, Centralbl.f. d. Med. Wissensch., 1883, p. 145. 276 BACTERIOLOGY. EhrlicJis 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 solu- tion for about half an hour ; then washed for a few seconds in strong nitric acid (one part commercial nitric 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 XX., 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 * 1 1 Aniline water 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. RindfleiscJis method. Prepare a solution composed of Saturated alcoholic solution of fuchsine 10 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, * Mittheil. aus dem Gesundheitsamte, Zweiter Band, 1884, p. 10 SYSTEMATIC AND DESCRIPTIVE. 2JJ 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. After-stain, if necessary, with bismarck- brown, or methylene-blue. Weigert-Ehrlich method (vide p. 61). Orttis modification of Ehrlictis 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 ma- genta 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 principles, but must be left in the solution for several hours. The crumpling of the sections by the action of nitric acid is avoided. * Gibbes, Practical Pathology. 1883. 278 BACTERIOLOGY. Baumgartcris method. Cover-glass-preparations of sputum are made as already described (p. 48), and immersed in a very dilute solution of potash (i 2 drops 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 re- moved, 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, inasmuch as the tubercle bacilli gradually are decolorised by the clove-oil, (b] 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. Neelserfs 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. SYSTEMATIC AND DESCRIPTIVE. 279 Balmer - Frantzel method. Dissolve two grammes of freshly-powdered gentian-violet in 100 grammes of 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. LichtJieims 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. Franker s method. Sputum preparations are rapidly double-stained by the following method : Prepare a solu- tion by adding concentrated alcoholic methyl-violet or fuchsine solution, drop by drop till opalescence arises, to 5 ccm. of aniline-water heated to 1 00 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 2 SO BACTERIOLOGY. Alcohol . . . . . 70 Nitric acid ... .30 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-Petris 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. Senkewitsctt 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. Kaatzers 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 go per cent. . '..'. 100 ccm. Water . . . ... 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. EhrlicHs 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. 163), 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 SYSTEMATIC AND DESCRIPTIVE. 28 1 balsam. The giant cells are then stained pink, while their nuclei are brown and the bacilli blue (Plate XVIII., Fig. i). Bacillus anthracis (Bacteridie du charbon, Bacil- lus of splenic fever, woohorters* disease, or malignant pustule). Rods, 5 20 JLC 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- 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 in 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. in). The spleen is found to be consider- ably enlarged, and may be removed (p. 112), and examined by making cover-glass preparations, inoculations, and subsequently sections. P Cover-glass preparations. In cover-glass prepara- tions of the blood of the spleen the bacilli are found in enormous numbers. Preparations should be 282 BACTERIOLOGY. 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- FIG. 105. BACILLUS ANTHRACIS, x 1200. From a preparation of blood from the spleen of an inoculated mouse. stained and stained (p. 48). 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 : FIG. 106. BACILLUS ANTHRACIS, X 1200. From a cover-glass preparation stained with Gram's method and eosin. The light part of the rod represents the sheath stained pink, and the dark part the protoplasmic contents stained blue and contracted. angles ; between the segments a clear linear space exists, which gives them a characteristic appearance (Fig. 105). By double staining (p. 49), the rods are seen to consist of a membrane or hyaline sheath with protoplasmic contents (Fig. 106). SYSTEMATIC AND DESCRIPTIVE. 283 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 cul- tures in the incubator develop rapidly, a tempera- ture of 25 40 C. being most favourable for the growth of the bacillus. The spores are eventually set free, and by making a fresh cul- tivation, or by injecting them into a mouse or guinea-pig, they germinate again into the cha- racteristic bacilli, which in their turn grow into filaments and spores. When the spore germinates it swells, the outer layer becomes jelly-like, and giving way at one or other pole, the contents escape and grow into a rod. With the precautions previously described (p, 112) cultivations should be established in nutrient gelatine, nutrient agar- agar, and on sterilised potatoes. Test-tube cultivations in nutrient gelatine. Typically characteristic appearances are obtained by inocu- lating a 5 to 8 per cent, nutrient gelatine. A whitish line develops in the track of the inoculating needle, and from it fine filaments spread out in the 284 BACTERIOLOGY. gelatine * (Fig. 107). Occasionally a little isolated spot develops, from which rays extend in all direc- tions, like the silky filaments of thistle-down. The filaments are more easily observed with a magnifying glass. In a more solid nutrient- gelatine the growth appears only as a thick white thread. As liquefaction of the gelatine progresses, these appearances rapidly disappear, and the growth subsides as a white flocculent mass (Plate V., Fig. 3). In exhausted culture-media, and sometimes in the blood, filaments are seen in a state of degeneration. This has also been observed in sections of the kidney, etc., of a rabbit inoculated with the anthrax bacillus, which had died of septicaemia the morning. Test-tube cultivations in nutrient agar.- Cultivated upon a sloping sur- face of nutrient agar-agar a viscous snow-white layer is developed (Plate puRE C curivA- XIV., Fig. i). Without access of air BACILLUS AN- no cultivation can be obtained, the NUTRIENT bacilli being aerobic. This can be GELATINE. (] emons trated by embedding a piece of lung or spleen pulp containing bacilli in nutrient agar-agar (p. 137)- No growth of the bacilli takes place. * The Authorj Lancet, 1885. following SYSTEMATIC AND DESCRIPTIVE. 285 Potato -cultivations. A very characteristic growth results from the inoculation of sterilised potatoes. The damp-chamber containing the potatoes is placed in the incubator, and in about thirty-six to forty-eight hours a creamy, very faintly yellow- ish layer forms over the inoculated surface, with usually a peculiar translucent edge (Plate XV., Fig. i). On removing the cover of the damp- chamber a strong, penetrating odour of sour milk is encountered. FIG. 108. COLONIES IN A PLATE-CULTIVATION, X 70. Plate-cultivations. From the spleen or blood of the heart, cultivations must be established in nutrient gelatine on plates. The colonies develop in about two days, according to the temperature of the room. They appear to the naked eye as little white spots or specks, which, on examination with a low power of the microscope and small diaphragm, exhibit two distinct forms. One form, on careful focussing, has the appearance of a little compact ball of twisted thread ; in the other, liquefaction of the gelatine has commenced, and the thread 286 BACTERIOLOGY. bundles are spreading out like locks or plaits of hair in the neighbouring gelatine. These appear- ances are perfectly characteristic (Fig. 108). Cover-glass impressions. The plate-cultivations should be also examined as soon as the colonies appear, by making cover-glass impressions (p. 52), and staining them with aniline dyes. The filaments, examined with a high power, will then be seen to consist of a number of rods or segments (Plate I., 3)' O n tne other hand, filaments from a FIG. 109. COVER GLASS IMPRESSION-PREPARATION, x 70. tube cultivation in a solid medium will be found to be composed, not only of rods, but here and there of torula-like involution- forms (Plate I., Fig. 30). In a cover-glass impression from a potato - culture (Plate I., Fig. 29) the individual segments have a great tendency to be isolated one from the other, and there is copious spore-formation. Preservation of spores. Spores may be preserved simply by allowing anthracic blood to dry and sealing it in a tube. The spores from a potato SYSTEMATIC AND DESCRIPTIVE. 287 cultivation are treated as follows : The inocu- lated surface containing the creamy cultivation is sliced off in a thin layer, and is mashed up with distilled water in a glass capsule. Sterilised silk-thread is cut up into lengths of about a quarter of an inch, and allowed to soak in the paste for some hours, under a bell-glass. The threads are then picked out with a pair of forceps, and laid upon a sterilised glass plate, covered with a bell-glass, and allowed to dry. From the plate, when perfectly dry, they are transferred to a small test-tube, which can be plugged with cotton-wool, or sealed in the Bunsen burner. Examination in the tissues. The organs must be hardened in absolute alcohol, cut and stained, (pp. 54, 291). The method of Gram is the most instructive, and eosin a very satisfactory contrast stain. The capillaries all over the 'body, lungs, liver, kidney, spleen, skin, mucous membrane, etc., will be found to contain bacilli. In some cases the bacilli are so numerous (e.g., in the capillaries of the kidney, Plate XVI., Fig. 2), that examination with a low power gives the appearance of an injected specimen. Inoculation of animals. A thread containingspores, a drop of blood from an infected animal, or a minute portion of a cultivation, introduced under the skin of a mouse or guinea-pig, causes its death, as a rule, in from twenty-four to forty- eight hours. Sheep fed upon potatoes which have been the 288 BACTERIOLOGY. medium for cultivating the bacillus, die in a few days. Goats, hedgehogs, sparrows, cows, horses, are all susceptible. Rats are infected with difficulty. Pigs, dogs, cats, white rats, and Algerian sheep have an immunity from the disease. Frogs and fish have been rendered susceptible by raising the temperature of the water in which they lived. Dissemination of the disease and mode of infection. It has been stated that when carcases of animals which have died of anthrax are buried under the soil, the development of the bacilli into spores can take place. The spores were supposed to be taken up by earth worms, carried to the surface, and deposited in their castings ; animals then grazing or sojourning on the soil are thus liable to be infected.* This has not been borne out by experiment.! Bacilli, however, occur in large numbers in the blood and discharges from the nose and mouth of the moribund animals, and in the urine and faeces. They find a nourishing soil in decaying vegetable and animal matter, and having free access of oxygen form copious spores, so that the grass is extensively contaminated. In warm and marshy districts the spore-formation is still more active, and the spores may be carried by floods over adjacent meadows. As to the mode of infection, the animals may be directly infected through buccal wounds caused by siliceous grasses, or by wounds of insects ; the intestinal and pul- * Pasteur, Bulletin de VAcademie de Medecine. 1880. t Koch, Mittheil. a.d. Gesundheitsamte. 1881 SYSTEMATIC AND DESCRIPTIVE. 289 monary mucous membranes are also regarded as pathways of infection. In animals the disease is known as " splenic fever." In man the mode of infection is by inhalation of spores, and ingress by the pulmonary or intestinal mucous membrane, or by direct inoculation of a wound or abrasion. The spores are derived from the wool or hides of animals which have died of anthrax, and the resulting disease is known as 4 * wool- sorter's disease," and " pustula maligna." Bacilli are found in the serum of the pustule, and in sputum, urine, faeces, and sweat ; and if the disease prove fatal, in the capillaries throughout the body. Attenuation of the virus. By cultivating the bacillus in neutralised bouillon at 42 43 C. for about twenty days, the infecting power is weakened, and animals inoculated with it {premier vacciri) are protected against the disease.* To obtain a still more perfect immunity, they are inoculated a second time with material (deuxieme vacciri) which has been less weakened. The animals are then pro- tected against the most virulent anthrax, but only for a time. From such a culture, however, new cultures of virulent bacilli can be started, and a culture that is " vaccin " for sheep kills a guinea- pig, and then yields bacilli that are fatal to sheep. f Exposure to a temperature of 55 C., or treatment with *5 to i per cent, carbolic acid, deprives the * Pasteur, Comfit. Rend., 1861, and Revue Scientifique, 1883. t Klein, Micro-organisms and Disease. 1 885 . 19 2 QO BACTERIOLOGY. bacilli of their virulence. The virulence of the bacillus is also altered by passing the bacillus through different species of animals. The bacillus of sheep or cattle is fatal when re-inoculated into sheep or cattle ; but, if inoculated in mice, the bacilli then obtained lose their virulence for sheep or cattle ; only a transitory illness results, and the animals are protected for a time against virulent anthrax.* The possibility of mitigating the virus KIG. no. SPORES OF BACILLUS ANTHRACIS UNSTAINED, x 1500. depends upon the species of animal ; rodents cannot be rendered immune by any known " vaccin." METHODS OF STAINING THE BACILLUS ANTHRACIS. Cover-glass preparations of blood, etc., can be stained with a watery solution of any of the aniline dyes. They may be rapidly stained with a drop of fuchsine or gentian violet (p. 48), but more satisfactorily by floating the cover- glasses for twenty-four hours. The preparations may be dried and mounted in Canada balsam, but the typical appearances are best observed in freshly-stained specimens examined in water. The sheath and protoplasmic contents can be differen- tiated by staining with eosin after the method of Gram. * Klein, Reports of the Medical Officer of the Local Government Board. 1882. SYSTEMATIC AND DESCRIPTIVE. 2QI The spores (Fig. 1 10) are not stained by the ordinary methods. The cover-glass preparations must be raised to a high temperature in the incubator, or treated with sulphuric acid (p. 309), or passed about twelve times through the flame of the Bunsen burner, or floated on hot solution of the dye. FIG. in. SPORES OF BACILLUS ANTHRACIS, x 1200; stained with gentian violet, after passing the cover-glass twelve times through the flame. To double-stain spore-bearing bacilli. Float the cover- glasses for about twenty minutes on hot alcoholic solution of fuchsine. Decolorise in weak hydrochloric acid, and after-stain with methylene blue (Fig. 112). Tissue sections are best stained by the method of Gram, * *& OEEBSI 2 r d,_ ; ; 5 .... * % '! FIG. 1 12. --FROM A DOUBLE-STAINED PREPARATION OF BACILLUS ANTHRACIS, x 1200. and after-stained with eosin, picrocarminate of ammonia, or picro-lithium-carmine. A more rapid double stain is obtained by immersing the sections in a watery solution of gentian-violet, rinsing in alcohol, and then staining by the method of Orth (p. 61). Weigerfs Method. Place the sections for two to five minutes in a I per cent, watery solution of gentian violet 2Q 2 BACTERIOLOGY. Wash in alcohol, rinse in water, and transfer to picro- carmine solution (Weigert) for from half an hour to an hour. Treat with alcohol till the colour is almost washed out, and finally clear in oil of cloves and mount in Canada balsam. Bacillus mallei (Bacillus of glanders). Rods about the size of tubercle bacilli (Fig 113). When cultivated on solid sterile blood serum at 38 C., the growth appears in the form of minute, transparent drops consisting entirely of the characteristic bacilli. On sterilised potatoes they form, in a week to ten days at 37 C., a brown gelatinous layer. Pure cul- FIG. 113. BACILLUS MALLEI, x 1200; from a section of a glanders' nodule. i tivations after several generations produce the fol- lowing results when inoculated into horses, rabbits, guinea-pigs, and field-mice. A spreading ulcer with indurated base appears at the site of inoculation, while smaller ulcers break out in its vicinity. The lymphatics become swollen, and general infection follows in the form of nodules in the internal organs, and nodules and ulcers on the nasal septum. In guinea-pigs a characteristic tumour of the testis, or ovary and vulva, frequently results, and should be prepared for microscopical sections. The bacilli are found in the nodules of the nasal mucous mem- SYSTEMATIC AND DESCRIPTIVE. 2 93 brane, the lung, spleen, liver, and other organs in horses and sheep affected with glanders. METHODS OF STAINING THE BACILLUS OF GLANDERS. The bacilli of glanders are extremely difficult to demon- strate. The most satisfactory results are obtained as follows : Method of Schuts. The sections are placed for twenty- four hours in a mixture ot Potash solution (i in 10,000); {Equal Concentrated alcoholic methylene-blue solution;] parts. Wash the sections in a watch-glass with water acidulated with four drops of acetic acid. Transfer for five minutes to 50 per cent, alcohol, fifteen minutes to absolute alcohol, clarify in clove-oil, and mount in Canada balsam. Bacillus cedematis maligni, Koch (Pas- teur's Septicczmid). Rods from 3 3*5 /* long and i i 'i IL wide; they mostly lie in pairs, and then appear to be double this length. The rods are rounded at their ends, and form threads which are sometimes straight, but more commonly curved. In stained preparations they have a somewhat granular appearance. The bacilli are distinguished from anthrax bacilli by their being somewhat thinner, by their rounded ends, and by their being motile. Anthrax bacilli also never appear as threads in fresh blood, and are differently distributed throughout the body. They are anaerobic, and can be culti- vated on blood-serum and on neutral solution of Liebig's meat extract in an atmosphere of carbonic 2Q4 BACTERIOLOGY. acid. By embedding" material containing bacilli in nutrient agar-agar and nutrient gelatine, charac- teristic cultivations are obtained. The following process may be adopted to obtain a pure cultiva- tion.* A mouse inoculated subcutaneously with dust, as a rule, dies in one to two days. It is then pinned out, back uppermost, on a slab of wood (p. 113), and the hair singed with a Paquelin's cautery from one hind leg up to the neck, across the latter, and down again to the opposite hind leg. Following the cauterised line, the skin is cut through with sterilised scissors, and the flap turned back and pinned out of the way. With curved scissors little pieces of the subcutaneous cedema- tous tissue, in the neighbourhood of the inoculated spot, are cut out, and sunk with a platinum needle in a i per cent, nutrient agar-agar, or 5 per cent, nutrient gelatine. Fragments of tissue may also be embedded by the method already described (P- 137). The inoculated tubes are placed in the incubator. In a few hours a whitish turbidity spreads out from the piece of tissue, and upwards in the needle track. Examined microscopically, the turbidity is found to be due solely to the development of bacilli of oedema. The surface exposed to the air exhibits no trace of the bacilli. To investigate the tubes microscopically, a steri- lised glass tube with a capillary end may be used, * Hesse, Deutsch. Med. Woch., No. 14. 1885. SYSTEMATIC AND DESCRIPTIVE. 295 with its neck plugged with sterilised cotton wool, and provided at the mouth with a suction ball. The capillary end is thrust into the cultivation, and a small fragment removed by aspiration. In the course of the first day the bacilli spread throughout a great part of the agar-agar in such a way that a more or less equally diffused cloudi- ness of the medium ensues, with subsequent ap- pearance of strongly marked clouds or lines of turbidity. At the same time gas bubbles develop along the needle track, and a collection of liquid takes place, while spore-formation also commences. The following day these appearances are more marked, the opacity is more pronounced, the develop- ment of gas increases, and the liquid contains more spore-forming bacilli and numerous free spores. The nutrient gelatine cultures during the first day show no macroscopic change, but after a few days the piece of tissue is surrounded with a white halo. This gradually spreads in all directions, and is ap- parently beset with hairs. The gelatine liquefies, and the fragment of tissue, degenerated bacilli, and spores, sink to the bottom. The cultivation is also very characteristic in \ per cent, nutrient agar-agar. If placed in the incubator, in a few hours a cloudi- ness forms around the piece of embedded tissue, which is caused by bacilli gradually spreading in all directions in the nutrient medium. Mice inocu- lated from these cultivations die more quickly than from the original infection from dust. On potatoes 296 BACTERIOLOGY. they are cultivated by introducing a piece of liver or other tissue containing" the bacilli, into the interior of a sterilised potato (p. 113), incubated at 38 C. The bacillus is not deprived of its virulence by cul- tivation. The spores of the oedema-bacilli appear to be very widely distributed. They are found in the upper cultivated layer of the soil, in hay dust, in decomposing liquids, and especially in the bodies of suffocated animals, which are left to decompose at a high temperature. From any of these sources animals can be successfully inoculated. If a guinea- pig, for example, be subcutaneously inoculated with earth, putrid fluid, or hay dust, death frequently occurs in from twenty-four to forty-eight hours. At the autopsy the most characteristic symptom is a widespread subcutaneous oedema, which originates from the point of inoculation, accompanied with air- bubbles, and contains a clear reddish liquid full of motile and non-motile bacilli. The internal organs are little changed, the spleen is enlarged and of a dark colour, and the lungs are hypersemic, and have hsemorrhagic spots. Examined immediately after death, few or no bacilli are detected in the blood of the heart, but in that of the spleen, liver, lungs,and other organs, in the peritoneal exudation, and in and upon the serous coating of abdominal organs they are present in large numbers. If, on the other hand, the animal is not examined until some time after death, then the bacilli are found in the blood of the heart, and distributed all over the body. SYSTEMATIC AND Bacillus of septicaemia of mice, Koch. Extremely minute bacilli, '8 i JJL long", and *i *2 broad, often in pairs, seldom in chains of four. On cultivation they do not appear to make threads, but the bacilli lie together in masses. Spores have been observed. The bacilli are probably non- motile. They are most commonly in the interior of white blood corpuscles. In these they increase, and in many cases a white cell is only represented by a mass of bacilli. The bacilli, or rather their spores, occur in putrid liquids. If a number of mice are inoculated with a minimum quantity of putrid fluid, about a third of them die of septi- caemia. They rapidly sicken, their eyes inflame, their eyelids stick together, they become soporific, and die in from about forty to sixty hours. At the autopsy one finds slight oedema at the seat of inocu- lation, and enlargement of the spleen ; the bacilli are found both free and lodged in the white cor- puscles, in the oedetnatous tissue, and in the blood capillaries. A minimal quantity of this blood produces the disease if inoculated in house-mice or sparrows. Field-mice have an immunity. Rab- bits and guinea-pigs inoculated in the ear surfer from only a local erythema, which disappears after- five or six days, and renders them for a time im- mune. Rabbits inoculated in the cornea suffer from an intense inflammation of the eyes. The bacilli are easily cultivated outside the body on a mixture of aqueous humour and gelatine, and. 298 BACTERIOLOGY. especially on nutrient gelatine rendered slightly alkaline with sodium phosphate. They grow also very well on the ordinary nutrient gelatine, form- ing in plate-cultivations scarcely perceptible cloud- like specks, and in a test-tube of nutrient gelatine they form a delicately clouded cultivation along the needle track (Figs. 1 14 and 1 15). A small quantity of pure culti- vation carried through many generations re- produces the disease when inoculated into mice. The organs should be hardened in absolute alcohol, and sections stained prefer- ably by the method of Gram (Plate XXII., Figs, i and 2). Bacillus of ulcer- ative stomatitis in the calf, Lingard and Batt. Rods 4 ^ 8 /i, mnrp in length I LL or mor< ,ngin , ip in width. Spores are frequently present. In- jected into the rabbit or mouse they produce a fatal result. They were observed in ulcerations on the tongue and mucous membrane of the mouth of calves. Fig. ii 4 . Fig. ii 5 . PURE-CULTIVATIONS OF THE BACILLUS OF SEPTICAEMIA OF MICE IN NUTRIENT GELATINE. Fig. 1 14. In two days. Fig. 115. in five days. SYSTEMATIC AND DESCRIPTIVE. 299 METHOD OF STAINING THE BACILLI OF ULCERATIVE STOMATATIS. Sections through the ulcerations .of the calf's tongue, or of the inoculated tissue of the rabbit, were stained by immersion in a mixture of magenta and methylene-blue. They were then washed in spirit, cleared in clove- oil, and mounted in Canada balsam. Bacillus of swine-typhoid (Bacillus of swine- plague or swine-fever. Bacillus of pneumo-enteritis of FIG. 116. FROM A PREPARATION OF BRONCHIAL Mucus OF A PIG [after Klein]. the pig, Klein*). Rods 23 /x, long, actively motile (Fig. 116); spore -formation described (Fig-. 118). They can be cultivated in broth and hydro- cele-fluid, and carried on through successive generations. A drop of any of these cultivations produces the disease in pigs, mice, and rabbits (Fig. 117); the animals die with a characteristic swelling of the spleen, coagulative necrosis of tracts of the liver tissue, and inflammation of the * Klein, Report to Med. Ojfic. Loc. Govt. Board. 18771878. 3OO BACTERIOLOGY. lungs ; pigs inoculated with artificial cultures are protected against a fatal attack. The bacillus was observed in the diseased organs FIG. 117. BLOOD OF FRESH SPLEEN OF A MOUSE, AFTER INOCULATION WITH SWINE FEVER [after Klein]. of pigs that had died of swine fever, and of animals that had died from the inoculated disease. FIG. 1 18. BACILLI FROM AN ARTIFICIAL CULTURE, WITH SPORES [after Klein]. fc Bacillus of swine-erysipelas (Bacillus ctes erysipelas malignum, of Rothlauf, or Rouget die pore). Extremely minute bacilli, bearing a close re- semblance to the bacilli of septicaemia of mice.* In test-tubes of nutrient gelatine they develop a cloudy growth in the needle track (Plate XXVI., * Loffler und Schiitz, Arbeiten aus dem Kaiser lichen Gesund- heitsamtey. vol. i. 1885. SYSTEMATIC AND DESCRIPTIVE. 301 Fig. i), and in plate-cultivations characteristic, thread-like, branching, or star-like colonies are formed (Plate XXVL, Fig. 2). Inoculated into mice and rabbits, a fatal result is produced ; but experiments with pigs were unsuccessful. Pigeons were also susceptible, and the bacilli were detected in their blood (Plate XXVL, Fig. 3). Bacillus in tetanus, Nicolaier. Rods, some- what longer but scarcely thicker than the bacillus of mouse-septicsemia. Occasionally thread-forms result, but they are collected mostly in irregular masses. They exhibit a characteristic spore-for- mation. They were found associated with other bacteria in abscesses resulting from the inoculation of mice and rabbits with garden earth. Inoculation of earth subcutaneously in these animals induces fatal tetanus. A cultivation of the mixture of micro-organisms on blood serum also produced the same disease. Bacilli stated to be identical with the bacilli of earth-tetanus have been ob- served in a case of tetanus in man. Further researches are required to establish their patho- genic properties. Bacillus alvei, Cheshire and Cheyne.* Rods varying in size, and forming large oval spores. When cultivated in nutrient gelatine in test-tubes a delicate, ramifying growth appears on the sur- face, and irregular whitish masses arise along the * Cheshire and Cheyne, Journ. Royal Microscopical Society, 1885, pp. 582-601. 302 BACTERIOLOGY. needle track. Processes shoot out from these masses, and extend through the gelatine for long- distances. They are thickened at points in their course, and clubbed at the ends. The gelatine is gradually liquefied, and the bacilli form a loose, white, flocculent deposit at the bottom of the tube. The liquid in the tube becomes yellowish in colour after a time, and gives off an odour of stale but not ammoniacal urine. The colour and odour are distinctive also of the disease attributed to the bacilli. In plate-cultivations, the bacilli grow out in series of rods in single file, or in rows of several side by side. The processes which are formed, tend to curve, and at a short distance from the track of the needle-streak form a distinct circle, from which another process grows out, and a fresh circle is developed. The gelatine in the vicinity of the bacilli gradually liquefies, and channels are formed in the gelatine in which the bacilli move backwards and forwards. On nutrient agar-agar a whitish layer develops, consisting of bacilli arranged side by side, which in a few days are replaced by rows of spores similarly arranged. On potatoes they form a dryish yellow layer, and in milk a tremulous jelly. A cultivation of the bacillus in milk, sprayed over a honeycomb containing a healthy brood of bee larvse, produced the disease known as " foul -brood." Adult bees fed on ma- terial containing bacilli became affected ; inocula- tion of mice and rabbits with the bacillus gave SYSTEMATIC AND DESCRIPTIVE. 303 doubtful results. The bacilli were isolated from the diseased larvae of bees. Bacillus pyocyaneus (Micrococcus pyocyaneus, Gessard. Bacterium aruginosum, Schroter. Ba- cillus fluorescens). Slender rods sometimes linked in twos or threes, or collected in irregular masses. Spore-formation present. On plate-cultivations white colonies with indistinct contour appear in twenty-four hours, and the whole of the gelatine has a greenish shimmer. In test-tube cultivations the gelatine is liquefied, and coloured green by reflected light, and a deep orange by transmitted light (Plate V., Fig. i). On nutrient agar-agar they form a white layer, and colour the medium a pea-green. The pigment formed by the rods is a definite principle, pyocyanin.* It can be ex- tracted with chloroform from pus, and from washings of bandages ; it is soluble in acidulated water, which it colours red. In neutral solution it becomes blue. It crystallises in chloroform in long needles ; and forms sometimes lamellae and prisms. The rods occur in the pus of those cases in which the wounds and pus-stained bandages exhibit a greenish-blue colour. Bacillus ianthinus (Bacterium ianthinum, Zopf. Bacillus violaceus). Slender rods, about four times their width in length, with rounded ends. They also form threads, and are actively motile. Spore- * Gessard, De la Pyocyanine et de Son Microbe. Paris these. 1882. 304 BACTERIOLOGY. formation present in the rods. On plate cultiva- tions the colonies occur as circumscribed liquefied areas, in the centre of which is a collection of the coloured growth. In test-tubes a funnel-shaped liquefaction takes place, while a granular-looking violet mass subsides to the bottom. On agar-agar and potatoes a beautiful violet growth rapidly develops. They were observed on pieces of pigs' bladder floating on the surface of water rich in bacteria. They occurred only on the surface of the bladder exposed to the air, and never on the part under water. They occasionally occur in common tap water. The colouring matter is soluble in alcohol. Bacillus cyanogenus, Fuchs. (Bacterium syncyanum. Bacillus of Blue Milk}. Motile rods, 2-5 3*5 p in length, and "4 ^ wide (Fig. 119). The rods after division may remain linked together, and form chains. Non-motile rods enveloped in a gelatinous capsule, and involution- forms, have also been described. Spore-formation present. Cultivated in a test-tube of nutrient gelatine, the bacilli grow principally upon the free surface, in the form of a white layer. The surface of the gelatine becomes cupped, and a peculiar greenish - brown colour develops in the medium, especially in proximity to the growth. On a sloping surface of nutrient agar-agar, they grow as a white layer, and colour the SYSTEMATIC AND DESCRIPTIVE. 305 upper part of the medium a smoky brown (Plate II., Fig. 2). The bacilli can also be cultivated in milk and on various other substrata, as potatoes (Plate XXL), boiled rice, and starch. A pure cultivation in neutralised sterile milk develops a weak alkaline ': ! FIG. 119. BACILLUS CYANOGENUS, x 650. A. Active rods. B. Rods in zoogloea. C. Chain of short rods. D. Chain of cocci. E. Cocci stage. F, G. Spore -forming rods. H. Involution-forms [After Neelsen]. reaction. The colouring matter which is formed, varies with the nourishing medium ; for example, in milk a slate-blue coloration is produced, but if the milk has become acid by the growth of the Bacillus acidi lactici, then the colour is an intense blue The micro-organism occurs occasionally in 20 306 BACTERIOLOGY. cow's milk, producing a blue colour. It has been observed, especially in the north of Germany, during the warm months ; and where milk is kept in hot rooms, in the winter also. The blueing was originally attributed to a diseased condition of the cows, or to their eating certain meadow plants. Bacillus acidi lactici. Long and short rods, i 2'S p, long, '3 *4 /x thick, and thread-forms; no cocci ; spore-formation.* Cultivated on nutrient gelatine the breadth of the rods is lessened. They grow best between 35 and 42 C., and cease under ioC. Cultivated at a temperature over 45*5, they are no longer able to produce acidity. Probably several micro-organisms are able to produce an acid reaction in milk. They occur with various other bacteria in sour milk, and a pure cultivation, isolated by plate- cultivations, turns sterilised milk sour. Bacillus Fitzianus, Zopf. Cocci, short rods, long rods, and threads. This bacillus, cultivated in meat extract and glycerine at 36 C., causes an active fermentation with the production of ethyl alcohol. Spore-formation occurs in the rods. Observed in unboiled hay infusion, accompanying the hay bacillus. Bacillus subtilis (Hay bacillus}. Cylindrical rods as much as 6 /x, in length, and about three times as long as broad. Single forms grow to * Hueppe, Mittheil a.d. Gestindheitsamt> N. Band. SYSTEMATIC AND DESCRIPTIVE. 307 double their length, and then undergo division. They also form threads which may b6 composed of long rods, short rods, and cocci. They are motile, and provided with a flagellum at each end. If the nourishing medium is impoverished, the multiplication of the rods by division gradually ceases, and spore-formation commences. The rods become motionless, and a dark spot is visible, either in the middle or towards one end. This gradually develops into a shining spore with a dark contour. The rods swell slightly during this process, their contour becomes undefined, and soon disappears entirely, so that the spores are set free in about twenty-four hours. The spores are i'2 //> long, and '6 p, broad. They develop into rods in the following way. On one side of the spore a swelling appears, at the summit of which an opening in the spore- membrane results, and the germ escapes. This lengthens into a rod, and remains for, a time attached to the empty spore-membrane. These spores are widely distributed, and occur in the air, soil, dust, etc. On the excrement of herbivorous animals the bacilli form a white efflorescence, and on infusion of horse-dung a thick crumpled skin. They flourish equally in liquids and upon damp, solid, nourishing media. On potatoes they grow as a yellowish-white skin ; on ordinary nutrient liquids they develop a thin, and subsequently .a thick, dense, crumpled pellicle, with copious spore-forma- tion. They are aerobic ; deprivation of oxygen 308 BACTERIOLOGY. causes the growth of the bacilli to cease, and the rods degenerate. They may be cultivated in various other nourishing media, such as blood- serum, nutrient gelatine, and nutrient agar-agar (Plate XIV., Fig. 2). The simplest way to obtain a culture of the bacillus is to make a decoction of hay. The hay is chopped into small pieces, and boiled with dis- tilled water in a flask for a quarter of an hour ; it is then filtered into a beaker, which must be covered with a glass plate, and set aside in a warm place. In two or three days the liquid swarms with the bacilli, the spores of which exist in great numbers in ordinary hay. A more sure method for obtaining a pure cultivation is as follows : (a) Add only a small quantity of water to some finely chopped hay, and set aside for four hours at 36 C. (&) Pour off the extract, and dilute it to the Sp. Gr. 1-004. (c) Boil gently for one hour in a bulb plugged with cotton wool. (d} Set aside 500 ccm. of the extract at 36 C. In about twenty- four hours, as a rule, a pellicle has commenced to develop upon the surface of the liquid. If the reaction is definitely acid, carbonate of soda solution must be added to the decoction. SYSTEMATIC AND DESCRIPTIVE. 309 METHODS OF STAINING HAY BACILLUS. To demonstrate the flagella of the bacilli, they may be stained with haematoxylin solution (Koch). The linking together of cocci, long rods, and short rods in the threads, is shown by treating with alcoholic solution of fuchsine, or with iodine solution (Zopf). To stain the spores the cover-glass preparations must be heated to a very high temperature (210 C.), in the hot-air steriliser for half an hour, or they may be exposed for a few seconds to the action of concentrated sulphuric acid (Biichner), or floated for twenty minutes on hot solution of the dye. Bacillus ulna, Cohn. Cocci, short rods, long- rods, and threads. Diam. of the cocci 1*5 2*2 /x. Spore-formation in both short and long rods. No septic odour is produced by this bacillus in a nourishing liquid. Cloudy masses are found on the surface of the liquid, which later form a thick dry pellicle. The latter consists of bundles of threads matted together. The formation of ellipsoidal spores occurs in the usual way ; they measure 2 '5 2*8 /x long, and more than i /* wide. The bacillus is found in rotting eggs, and can be culti- vated on boiled white of egg. It is closely allied to Bacillus subtilis. Bacillus tumescens, Zopf.* Cocci, long and short rods. They form a jelly-like disc, '5 i cm. in diam., on slices of boiled carrot, with the appear- ance of a rather tough crumpled skin of a whitish colour. Examination of this pellicle shows that it * Zopf, Die Spaltpilze. 1885. 3 io BACTERIOLOGY. is formed of rows of rods lying closely together. These rods can be observed to divide into short rods and cocci. Spore-formation occurs in two stages of development, viz., in the cocci and in the short rods. A cultivation is obtained by exposing slices of boiled carrot, slightly moistened, to the air at the temperature of the room. Bacillus megaterium, De Bary. Large rods 2*5 /u< wide, and four to six times as long. They FIG. 120. BACILLUS MEGATERIUM. a. A chain of rods, X 250. The rest X 600. b. Two active rods. d\.of. Successive stages of germination. h and /. Successive stages of germination. [After De Bary.] are usually somewhat curved. Transverse division occurs, each segment attaining the length of the original rod. In the fresh state they appear non- arti- culated, but when treated with a dehydrating agent (tincture of iodine, alcohol), they are seen to be composed of short segments. The rods are motile, and form irregular chains, of a disjointed appear- SYSTEMATIC AND DESCRIPTIVE. ance. They can be cultivated on nutrient agar- agar and nutrient gelatine. The latter is slowly liquefied, but the appearances are not characteristic. Spore-formation occurs in the usual f F^\ way (Fig. 120). It was first observed on boiled cabbage. Bacillus figurans (Wurzel Ba- cillus 'j Bacillus my co ides, Fliigge). Rods, with rounded ends, varying in length. Spore-formation present. In plate-cultivations they cause a cloudy growth, spreading from various points; if a cover-glass impression is made, this is found to consist of the regularly- arranged parallel rods. The chains of rods become twisted at intervals into curious convolutions, from which off- shoots are continued in various direc- tions. These long shoots or processes are again twisted at intervals into varying shapes and patterns (Plate XXV., Figs, i and 2). Cultivated in nutrient gelatine, the bacilli form on the surface visible windings, from which fine filaments grow down into the gelatine. They spread out also in PURE CULTIVA- J r TION OF BACIL- almost parallel lines transversely from LUS FIGURANS IN NUTRIENT the needle track. On an oblique sur- AGAR-AGAR. face of nutrient agar-agar the filaments spread downwards into the substance of the jelly, and FIG. 121. 3 I 2 BACTERIOLOGY. outwards from the central streak on the surface, forming a feather-like cultivation* (Fig. 121). They are present in garden earth, and have also been cultivated from the air when charged with dust raised from the soil. Bacillus tremulus. Rods shorter and thinner than those of Bacillus subtilis. They are provided with a flagellum at both ends, and exhibit charac- teristic 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 ju, long and -58 p, thick. They can be cultivated on nutrient gelatine and blood serum. Infusion of je- quirity containing the bacilli, or an artificial cultiva- tion of the bacilli, inoculated into the conjunctiva of healthy rabbits produces severe ophthalmia. The poisonous principle is, however, believed to be a chemical ferment, a6rm,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 * Described by the author, " Notes from a Bacteriol. Laboratory," Lancet. 1885. t Klein, Micro-organisms and Disease. 1885. SYSTEMATIC AND DESCRIPTIVE. 313 Bacillus subtilis. They occur in the form of whitish lumps in company 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, long and 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 pep- permint and turpentine. A great loss may result to bakers if the fungus is introduced with the yeast. Bacillus Hansenii, Rasmussen. Rods 2-8 6 IJL long, *6 *8 p, wide. Cultivated on sterilised potato in four days they form a chrome-yellow la}er with an agreeable 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 ^ long, 1*1 IJL wide. The colouring matter is insoluble in most reagents. The bacilli occur on nourishing solutions, malt infusion, broth, wine, which have been kept at 31 to 33 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. BACTERIOLOGY. Bacillus septicus, Klein.* Rods varying in size, non-motile. They form threads or leptothrix filaments, and are rounded at the ends. They are an'serobic, and form spores independently of access of air. In a nourishing" fluid they are overcome by the presence of micrococci. Bacterium termo or Bacillus sub filis. 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- formed organisms have been described by Rosen- FIG. 122. BACILLUS SAPROGENES, No. i. [After Rosenbach.] bach as intimately associated with putrefactive processes. No. i. Large rods (Fig, 122), which form an irregular sinuous streak with a mucilaginous ap- pearance, 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 * Klein, Micro-organisms and Disease. 1885. SYSTEMATIC- AND DESCRIPTIVE. 315 transparent drops, which become grey. They were isolated from a patient suffering from profusely- sweating feet. The cultivations yield a character- istic odour similar to the last. They are pathogenic in rabbits. They appear to be identical with Bacillus foetidus {Bacterium foetidum. Thin). No. 3. See Bacterium saprogenes. Bacillus fcetidus (Bacterium fatidum, Thin). Cocci, short rods, long rods, and leptothrix. The cocci, 1*25 1*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 saprogenes fcetidus, Schottelius. Rods with rounded ends, shorter, but about same width as the hay- bacillus. Immotile; spore-forma- tion 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 3 1 6 BACTERIOLOGY. 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. Power- fully 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 cultivations 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, Fliigge (Potato bacillus). Rods, longer and thicker than the above, and sometimes thread - form s ; spore-for- mation 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. SYSTEMATIC AND DESCRIPTIVE. 317 Genus V. Vibrio. SPECIES. UNASSOCIATED WITH DISEASE : Vibrio rugula .... Zymogenic saprophyte. Vibrio rugula, Miiller. Rods and threads, 6 16 JJL long, about -5 2-5 thick. The rods are either simply bowed, or possessed of one shallow spiral (Fig. 123). They bear a flagellum at each FIG. 123. 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.] 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 3 * 8 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. SPECIES. ASSOCIATED WITH DISEASE IN ANIMALS : Clostridium of symptomatic anthrax Pathogenic. UN ASSOCIATED WITH DISEASE : Clostridium butyricum . . . Zymogenic saprophytes. Clostridium polymyxa . * . ,, ,, Clostridium butyricum, Prazmowski (Bacillus amylobacter. Van Tieghem ; Bacillus butyricus. Bacillus of butyric acid fermentation}. Rods 3 10 IJL long, and under i p wide, often indistinguish- able from Bacillus subtilis. They grow out into long, apparently unjointed threads. They are mostly actively motile, but also occur in zoogloea. 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. 124). 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 SYSTEMATIC AND DESCRIPTIVE. 319 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. 124. CLOSTRIDIUM BUTYRICUM. A. Active stage, (a, b) Bent rods (vibrio-form) and threads. (V) 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. 320 BACTERIOLOGY. 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 Bitlrothii. 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. Clostridium of symptomatic anthrax (Ramch- 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- * Arloing, Cornevin et Thomas, Bull, de V Acad. de Med. 1881. SYSTEMATIC AND DESCRIPTIVE. 32.1 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 t Injection into the veins in small quantity produces a febrile disorder, in larger quantities death. Animals in the former case suffer an abortive ill- 1 ness, which protects them against further inocula^ tion. The micro-organism is the cause of a disease in cattle, "blackleg" " quarter- evil" or " Rausch- brand" At the autopsy the micro-organisms are found in the subcutaneous 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 hsemorrhagic effusion, occur- ring in the extremities, impedes the animal's move- ments. The cattle infected die in thirty-six to forty-eight hours. GROUP III. LEPTOTRICHE^E. Genus I. Crenothrix. Threads articulated ; cells sulphurless ; habitat water. Genus II. Beggiatoa. Threads unarticulated ; cells with sulphur granules ; habitat water. 21 322 BACTERIOLOGY. Genus III. Phragmidiotlirix. Threads jointless ; suc- cessive subdivision of cells is continuous ; cells sulphurless ; habitat water. Genus IV. Leptothrix. Threads articulated or unar- ticulated ; successive subdivisions of cells not continuous ; cells sulphurless. Genus I. Crenothrix. SPECIES. UNASSOCIATED WITH DISEASE : Crenothrix Kiihniana . . Simple saprophyte. Crenothrix Kiihniana, Rabenhorst. Cocci, rods, and thread-forms. The cocci are globular, i 6 p, in diam. The threads are colourless, 1^5 5 p. thick, and club-shaped at the extremity, reach- ing a diam. of 6 9 /*. 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 ar- ticulated, and ensheathed. The segments are set free when the sheath bursts, and develop into new threads. In other cases the segments remain en- closed, 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. 125). 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. 323 FIG. 125. CRENOTHRIX KUHNIANA. a, b t c, d, f. Cocci in various stages of fission, X 600. /. Zoogloea of cocci, X 600. g. Various forms of zoogloea, natural size. h. Colony of threads composed of rods grown out of a zoogloea of cocci. i r. Thread-forms ; some straight, others spiral, with more or less differ- entiation between base and apex, (r) is composed of short rods at the base and above these of cylindrical segments, and at the apex these segments have divided into cocci, X 600. [After Zopf.] 324 BACTERIOLOGY, Genus II. Beggiatoa. SPECIES. UNASSOCIATED WITH DISEASE : Beggiatoa alba .... Simple saprophyte. Beggiatoa mirabilis . . . Beggiatoa roseopersicina . . Chromogenic saprophyte. Beggiatoa alba, Vauch. Cocci, rods, spirals, and threads (Fig. 1 26). The threads are longer and thicker than leptothrix, indistinctly articulated, actively oscillating, and colourless ; their protoplasm contains numerous strongly refractive granules consisting of sulphur. They occur as greyish- or chalk- white gelatinous threads, 3 3*5 p, thick, in sulphur springs and marshes. Beggiatoa mirabilis, Cohn. Threads dis- tinguished from others of this genus by their breadth, which may reach 30 /A. They are motile, bent and curled in various ways, and rounded at the ends. Around the threads isolated cells have been observed, " macrococci," but spiral forms are as yet unknown. The threads are filled with sulphur granules. They occur on sea water, form- ing a white gelatinous scum on decomposing algae, etc. METHODS OF EXAMINING SPECIES OF BEGGIATOA. The articulation of the threads is best demonstrated by staining with an alcoholic solution of methyl-violet, fuchsine, or vesuvin ; or by treating with sodic sulphate, or warm glycerine. SYSTEMATIC AND DESCRIPTIVE. 325 Beggiatoa roseo-persicina (Cohnia roseo-persi- cina. Bacterium rufescens, or Peach -coloured bacterium , Lankester). Cocci, rods, spirals, and threads (Fig. FIG. 126. BEGGIATOA ALBA. A. Threads, at base distinctly linked, partly spiral. B. A thread, spiral in its whole length. C, D. Fragments detached from threads ; immotile. E. Active spirillum-forms, with a flagellum at either end. F. G. Thin and short spiral forms. H. A spiral showing the individual links. X 540. [After Zopf.] 127). The cocci, globular or oval, reach 2-5 p. in diam. They form at first solid families, bound to- 326 BACTERIOLOGY. gether by gelatinous substance. Later they become larger, globular or ovoid in shape, and hollow, con- taining watery fluid in their interior. The families reach a diameter of 660 /x, in which the cocci form simply a peripheral layer. The hollow families or vesicles are often perforated, presenting a delicate reticulated appearance, which finally may become broken up into irregular structures. The red colour- FIG. 127. SEVERAL PHASE-FORMS OF BEGGIATOA ROSEO-PERSICINA. [After Warming.] ing matter can be distinguished from other red pigments, and it is designated by the name bacterio- purpurin. It is quite distinct from the pigment produced by Bacterium ptodigiosum, being peach- blossom red, and insoluble in water, alcohol, etc. Examined spectroscopically it shows a strong ab- SYSTEMATIC AND DESCRIPTIVE. 3^7 sorption in the yellow, and a weaker band in the green and blue, as well as a darkening in the more refrangible half of the spectrum. In the cocci, especially of the older vesicles, dark granules are to be seen, which consist of sulphur. The micro- organisms occur on the surface of marshes, or on water in which algae are rotting. They form a rose-red, blood-red, violet-red, or violet- brown scum ; and sometimes in such quantity that whole marshes and ponds may be coloured blood-red by them. All the following hitherto described as distinct species, are probably only phase-forms of Beggiatoa roseo-per- sicina. Spirillum sanguineum, Cohn (Ophidomonas san guinea). Threads 3 JJL and more in thickness with 2 2\ spirals, each 9 12 /z- high, with their ends provided with rlagella. Their colour is due to the presence of red- dish granules contained in the cells. They were observed in brackish water with putrefying substances. Spirillum rosaceum, Klein Resembles Spirillum undula, but is reddish in colour ; the colouring matter is insoluble in water, alcohol, or chloroform. Spirillum violaceum, Warming. Threads, cres- cent-shaped, or possessing i-J- or I spiral, with their ends broad, rounded, and provided with flagella. The colour is due to the contents, which are violet. Monas vinosa. Round or oval cells of about 2*5 ^ in diameter, often united in pairs. Their motion is slow and tremulous, and the cell substance pale-red, with dark grains interspersed. Flagella have not been observed. They were observed in water with decaying vegetable matter. 328 '" ** ' >-' BACTERIOLOGY. MonaS Okenii. Short cylindrical cells, 5 //, wide, 8 15 fi long, with rounded ends. They undergo lively movements, each end being provided with a flagellum twice as long as the cell itself. They have pale- red cell- substance with dark grains. They occur in stagnant water. Rhabdomonas rosea. Spindle-form cells, 3-8 5*0 //- broad, 20 30 //, long. They exhibit slow, trembling movements, having at each end of the cell a flagellum. The cell substance is very pale, with dark grains inter- spersed. In brackish water. MonaS Warmingii. Cylindrical cells, rounded at the ends; 15 //. long, 5 8 Abroad. They are possessed of a flagellum at each end of the cell, and exhibit rapid, irregular movements. The cell substance is pale* red, and studded at the rounded ends with dark-red grains. Genus III. Phragmidwihrix. SPECIES. UNASSOCIATED WITH DISEASE : Phragmidiothrix multiseptata . . Simple saprophyte. Phragmidiothrix multiseptata. Cocci and threads. The latter, 3 6 ^ in breadth, are separated by transverse partitions into short cylindrical discs, whose height is a fourth or sixth of their breadth. Repeated transverse and longitudinal division takes place in the discs, resulting in the formation of cocci. The cocci have not been observed isolated from the threads in a free state, but they develop in situ into slender threads. In addition to this continuous subdivision, .Phragmidiothrix differs from Beggiatoa SYSTEMATIC AND DESCRI in the absence of sulphur, and from Crenothrix by its wanting a sheath. They occur attached to crabs (Gammarus locus fa) in sea water. Genus IV. Leptothrix. SPECIES. ASSOCIATED WITH DISEASE : Leptothrix buccalis . . Saprophytic. Leptothrix gigantea ... ,, Leptothrix buccalis, Robin. Long, thin threads, *7 i p, broad, colourless, often united in thick bundles or felted together. Masses of cocci occur with the threads, and the threads themselves are composed of long rods, short rods, and cocci. The threads may break up into spiral-, vibrio-, and spirochseta-forms. The last-named occur in large numbers in the mouth, and have been named Spiro- chccte buccalis. The Leptothrix buccalis is found in teeth slime, and is believed to be intimately connected with dental caries. The threads pene- trate the tissue of the teeth, after the enamel has been acted upon by acids generated by the fer- mentation of food. The short rods, long rods, cocci, leptothrix-forms, and screw-forms are found in the dental canals. METHODS OF STAINING THE LEPTOTHRIX BUCCALIS. The threads of Leptothrix buccalis have a special staining reaction (Leber). They become coloured if in an acid medium with iodine; if the medium is alkaline, it must be 330 BACTERIOLOGY. acidified with very dilute hydrochloric acid or acetic acid, and the filaments then stained with iodine. The contents are stained violet, and contrast with the sheath and septa, which remain uncoloured. Leptothrix gigantea, Miller. Long rods, short rods, and cocci, can be observed in the same thread. There are also screw-threads, which may take the form of spirals, vibrios, or spirochsetse. The threads increase in diameter from base to apex, and corresponding with the thickness of the threads, the rods and cocci show different dimensions. They have been observed in the diseased teeth of dogs, sheep, cats, and other animals. GROUP IV. CLADOTRICHE^E. Genus L Cladothrix. SPECIES. UN ASSOCIATED WITH DISEASE : Cladothrix dichotoma . , . Saprophytic. Cladothrix Foersteri . . ., ,, ASSOCIATED WITH DISEASE : Actinomyces .... Pathogenic. Cladothrix dichotoma, Conn. 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 SYSTEMATIC AND DESCRIPTIVE, 331 formerly described as Leptothrix parasitica, or it" coloured by impregnation with iron, as Leptothrix FIG. 128. CLADOTHRIX DICHOTOMA. A. Branching schizomycete : (a) Vibrio-form ; (b} Spirillum-form [slightly magnified]. B. A screw-form with (a) Spirillum-form ; (b) Vibrio-form. C. Long spirochaeta-form. D. Fragment with spirillum-form at one end. vibrio-form at the other. E. Screw-forms ; (a) continuous ; () composed of rods ; (V) composed of cocci. F. Spirochseta-form : (a) continuous ; (b} composed of long rods ; (c) short rods ; (d) cocci. [After Zopf.] ochracea. Later they form false branches by single rods turning aside, which by repeated 3 3 2 BACTERIOLOGY. division lengthen into threads. A thread appears to be first composed of long rods, then of short rods, and lastly of cocci. The iodine reac- tion must be applied to distinguish these forms, especially when 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 spiro- chseta-forms (Fig. 128). 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. Artificially they can be cultivated on infusions -of rotting algse and animal substances, forming on these media small tufts, about i 3 /x, 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. SYSTEMATIC AND DESCRIPTIVE. 333 Here we may add some little-known species, which possibly belong to this group. Sphserotilus natans. Cells 4 9 /* long, 3 p thick, united in a gelatinous sheath to form threads. The cells comprise rods and cocci-forms ; the cocci are set free, and 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 //, 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 fju long. Rapidly motile and revolving round a longitudinal axis. They occur in marsh water and putrefying infusions. Spiromonas Cohnii. Colourless cells, consisting of i^ spirals, with both ends acutely pointed and provided with a flagellum. Breadth of the cells 1-2 4 p. They occur in water containing decomposing matter. The following species is described last, so that it may stand between the cladothrix group of bacteria and the hyphomycetous fungi given in the appendix which follows. It is attached provisionally to the former for reasons stated below, but there is need for further inves- tigation before its position is established, as there is still some doubt as to the true life-history of the fungus. 334 BACTERIOLOGY. There is also reason for believing that there are different forms of actinomycosis in animals, and that the rare disease in man differs etiologically from the not so uncommon disease in cattle. It is only then pro- visionally that the different forms are here, described under one heading. Actinomyces. Actinomycosis is a disease occurring in animals * and occasionally in man.f It is caused by a parasite known as Actinomyces, or the " ray-fungus." The parasite appears in the form of a rosette of pyriform or club-shaped ele- ments (Plate XXTX., Fig. i). The little masses are colourless, pure white, or of a yellowish or yellowish- green tinge, and visible to the naked eye. The fungus is believed to effect an entrance to the animal by the mouth, being taften 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 neighbourhood, resulting in the formation of a neoplasm, composed chiefly of round cells, resembling a tuberculous nodule. The nodules may break down and suppurate, or may go on increasing in size. Fibrous tissue develops between the nodules, and large tumours eventually result containing purulent cavities and excavations. * Bellinger, Centralbl. f. Med. Wiss. 1877. t Israel, Virchow' s Archiv, vols. 74 78. Pontick, Die Actino- mycose des Menschen. 1882 ; and Beitr. z. Kenntn. der Actino- mycose des Menschen. 1882; Lancet, May 2nd, 1885 ; Conti, Gaz. Med. Hal. Lombordia, 1885 ; Acland, Trans. Path. Soc. 1886. SYSTEMATIC AND DESCRIPTIVE. 335 In 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 " wooden tongue," and also of diseases 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 fistulse and sinuses, with the clinical character of empyema. In one case there were the symptoms of chronic bronchitis with foetid expectoration. In other cases the dis- ease, originating in the lung, spread to the prse- vertebral tissues. If the actinomyces invade bones, as has been especially observed in the bodies of the vertebrae, caries results. In another group of cases the organism has been said 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 sper- matic duct of the horse.* The disease has been transmitted from cattle to cattle by inoculation,! and a rabbit has been in- fected by means of a piece of actinomycitic tumour * Johne, Bericht tibr das Veterindrwesen im Konigreich Sachsenfurdasja.hr. 1884. t Johne, Deutsche Zeitschr. /. Thiermedicin . 1881, 336 BACTERIOLOGY. from a human subject, introduced into the peri- toneal cavity. Until quite recently actinomyces has been classed as a hyphomycete, and the flask-shaped structures regarded as gonidia. By certain * cultivation- experiments we are led to regard the latter as a result of a degenerative stage in the life-history of the fungus accompanied by the development of involution - forms. Inoculations of nutrient gelatine, in the form of plate-cultivations, and inoculations on blood serum and nutrient agar- agar were made, it is stated, with success. The cultures developed on the latter in from five to six days, growing best at a temperature of 33 37 C. Nutrient gelatine was not liquefied. The appear- ances of the cultivation were described as quite characteristic ; it has at first a whitish, granular appearance, followed after a few days by little yellowish-red spots which coalesce in the centre, and finally a whitish downy layer results with a golden- red centre ; in time the periphery also becomes dotted with little yellow-centred masses. The fungus thus cultivated has been described as corresponding on examination with the form found in man and animals, and at one stage to consist of thread-forms, short rods, and cocci. As a result of these observations actinomyces has been relegated to the bacteria, forming one of the cla- * Bostrom, " Ueber Actinomycose," Verhandlungen des Con- gresses fur Inn. Med. 1885. SYSTEMATIC AND DESCRIPTIVE. 337 dothrix group, and possibly closely allied to the Streptothrix Fcersteri of Cohn. METHODS OF EXAMINING AND STAINING ACTINOMYCES In the fresh state a little of the tissue of a tumour, or the purulent detritus, may be transferred to a clean glass slide, and teased out with needles. The little specks are easily recognised, and can be isolated with the needles and transferred to a drop of glycerine upon a fresh slide. A cover-glass must then be gently pressed down upon the preparation, which is then examined. To stain the fresh tufts, the little fungus masses are teased out, and transferred to a watch-glass containing alcohol, to which a few drops of concentrated alcoholic solution of eosin are added. They can be mounted and preserved in glycerine. Sections can be stained by either of the following methods. Weigerfs Methcd : Immerse the sections for one hour in orseille, rinse with alcohol, and after-stain with gentian violet (Plate XXVIIL). Platris Method: Sections are left for ten minutes in Gibbes' solution (No. 22), warmed to 45 C. They are then rinsed in water, and after-stained in concentrated alcoholic solution of picric acid for 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 (Plate XXVI I.). 22 Plate 29. YEAST-FUNGI OR SA.CCHAROMYCETES MD MOULD-FUNGI OR HYPHOMCETES. DESCRIPTION OF PLATE XXIX. FIG. i. Actinomyces,toastd. out in the fresh state and stained with eosin. 2. Torula cerevisice (after Rees). 3. Saccharomyces mycoderma^ or oidium albicans, from an artificial cultivation (after Grawitz). 4. Saprolegnia (after Sachs). 5. Oidium lactis (after Fliigge). 6. Fungi of favns, or oidium lactis (after Neumann). 7. Penicillium glaucum (after Fliigge). 8. Aspergillus niger, from a preparation mounted in glycerine. 9. Aspergillus niger, from the same preparation (Zeiss A o- i)- 10. Aspergillus glaucus (after De Bary). I i. Botrytis Bassiana (after De Bary). APPENDIX A. YEASTS AND MOULDS. Yeast -fungi and mould-fungi, like bacteria or fission- fungi, are achlor aphyllous TJiallopliytes. They belong to two separate orders, the Saccharomycetes and Hyphomy- cetes, 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 SACCIIAROMYCETES. Saccharcmyces cerevisise (Totula Cells round or oval, 8 9 JJL long, singly or united in small chains. Spores occur three or four together in a mother-cell, 4 5 p in diameter (Plate XXIX., Fig. 2). Sacch. ellipsoideus. Elliptical cells, mostly 6 fji long, singly or united in little branching chains. Two to * Sachs, Text-book of Botany. 1882. APPENDIX. 341 four spores found in a mother-cell, 3 3'5 p, in diam. It is widely distributed, and is the principal agent in accidental 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 p, long, and reaching 2' 5 p. 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, 1 8 22 yu, long, which build lateral, secondary round or oval daughter-cells, 5 6 p, 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 p, long, 2 3 p, wide. Budding 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 p, long, 5 p, thick; the others round, 5 6 p, in diam. United in ramified families. Spores unknown. Sacch. mycoderina (Mycodenna cerevisia et vini). Cells oval, elliptical, or cylindrical, 6 7 p, long, 2 3 p, thick, united in richly-branching chains. Spore-forming cells reaching 20 p, 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 allicans). Cells partly 342 APPENDIX. round, partly oval or cylindrical, 3 5 5 JJL 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 epithelium, bacteria, yeasts, and the mycelia of various moulds. They can be easily cultivated in a nutrient solution containing sugar and ammonic tartrate.* The cells germinate accord- ing 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 XXIX., Fig. 3). Sacch. glutinis. Cells round, oval, or short cylinders, 5 11 JJL long, 4 JJL 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 10 /A in diam. Forms a coral-pink growth in nutrient gelatine, nutrient agar-agar (Plate XIII., Fig. 3), or on sterilised potatoes (Plate X., 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 *Sf., Fig. 3). MOULD-FUNGI OR HYPHOMYCETES. The mould-fungi have been divided into five orders :f Hypodermii, Phy corny cetes, Ascomycetes, Basidiomycetes^ and *Grawitz, Virch. Arc/av, vol. 70. f Fliigge, Fermente u. Mikrofiarasiten. 1883. APPENDIX. 343 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 disagreeable smell. Urocystis OCCulta. The spores consist of several cells united together ; partly large, dark-brown cells in the interior, and outside several flat semi-circular, 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, -on 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 toru- loid cells, which multiply by germination, and are dissemi- nated in the blood throughout the body of the fly. These cells again grow into hyphse, which penetrate the skin, each forming a conidium, which is cast off with consider- able force. The parasite is fatal to flies, especially in the 344 APPENDIX. autumn. They are often observed attached to the walls or window-panes, surrounded by a powdery substance, consisting .of the extruded conidia. Kmpusa 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 myce- lial threads, attacking caterpillars (Agrotis segetuni}. PHYCOMYCETES. . Saprolegnia. Colourless threads, forming dense 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 ofT 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 XXIX., Fig. 4). In the sexual mode of reproduction, a spherical bud, the oogonium, deve- lops 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 XXIX., 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 produces the com- mon disease of salmon. Peronospora infestans. Mycelium, -005 mm. in APPENDIX. 345 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. Hyphse, 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. Hyphae, 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 hyphse can be observed in a few hours, if the fungus be cultivated in a decoction of horse dung. Mucor racemosus. Hyphse, at most 1-5 cm. long 346 APPENDIX. 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 phycomyces, Lichtheim. Mycelium thick- walled, olive-green fruit-hyphse, 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 Mticor rhizopodiformis and Mucor corymbifer, when introduced into the vascular system of rabbits, can germinate in the tissues, especially in the kidneys, where they set up hsemorrhagic inflamma- tion. 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. APPENDIX. 347 ASCOMYCETES. OidiumTuckeri. Fruit hyphae, bearingsingle 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 hyphae, simple, erect, and colourless, bearing at their ends a series or chain of conidia (Plate XXIX., 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 (AcJwrion ScJioenleinii], Herpes tonsurans (Tri- cophyton tonsurans) and Pityriasis versicolor ( Microsporon furfur) (Plate XXIX., Fig. 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')" 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 aspergillns 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 XXIX., 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 *oo8 mm. long, colourless or pale to grey-green. * Grawitz, Virckoiv's Archiv, vol. 70. + Morris and Henderson, Journ. RoyalMicrosc. Society. 1883. 348 APPENDIX. 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 . . . . . 1 500 Sugar-candy. . . * . 70* Tartaric acid . . -* ,'-, 4* Nitrate of ammonia ; . 4 Phosphate . . . . . '6 Carbonate of potassium ... '6 magnesium . . -4 Sulphate of ammonia . . . '25 zinc ... -07 iron ... -07 Silicate of potassium . . . "07 It was also found that the fungus grew best when the liquid was spread out in a layer 2 or 3 cm. in depth in a * Duclaux, Health Exhibition Handbook, London, 1884. APPENDIX. 3-1 9 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 25 (the average) to 2 grammes, and the presence of TFWITTR? part of nitrate of silver, or srnjoo- part of 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 hyph f -e 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 hyphse 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 XXIX., 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 dewlike drops of liquid may appear (Plate IX., 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 '00071 mm., 35O APPENDIX. 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 XXIX., Fig. 7). It is the commonest of all moulds. Botrytis Bassiana. Hyphae and spores colour- less. Hyphae usually simple, but sometimes united in arborescent stems (Plate XXIX., Fig. n), It is the cause of muscardine, a fatal disease of silkworms, and occurs also in various other caterpillars and insects. UNCLASSED. Chionyphe Carter!. 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. 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 they appear to be more commonly found in the blood of apparently perfectly healthy animals. Flagellated organisms in the blood of rats and hamsters. Lewis')" described peculiar organisms in the blood of healthy rats in India. When first noticed * Abstract of paper by the Author, Journ. Roy. Micros. Soc., read November loth, 1886. See also papers on the Micro-parasites of Malaria by Laveran, Marchiafava and Celli. \ 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. 351 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 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. 129). After fixing with osmic acid they measured O'8 I ^ 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 /x. Lewis detected these parasites in 29 per cent, of the species Mus decumanus and MILS FIG. 129. PARASITES IN THE BLOOD OF RATS [after Lewis]. 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*f* later met with the same organisms. * " Spirillen im Blut von Hamstern," Centralbl. f. Med. Wiss. 1881, No. 4. t Mittheilungen aus dem Kaiser lick. Gesundh. Ami. 1881. . 8. 352 APPENDIX. 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* observed the presence of a parasite in the blood, and by means of subcutaneous inoculation, and by the introduction into the stomach of blood containing the parasites, the disease was transmitted to healthy animals. Steel,f 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. Flagellated organisms in the blood of fish. In the blood of mud-fish (Cobitis fossilis) MitrophanowJ observed the presence of peculiar micro-parasites (Fig. 130). As a i per cent, salt solution had been added to the blood under examination, it occurred to Mitrophanow that they were possibly the cytozoa described by Gaule ; but this idea was dismissed by the fact that they were found in blood to which no salt solution was added. Their size varied from 30 to 40 p, in length, and i to I *5 //, in width. At first their rapid movements baffled examina- tion, but as the rapidity lessened there was the appearance of a curling movement in the body portion, and a swinging movement of the lash. The organism moved in the direction of the lash, the anterior end of the body being * Evans, Report published by the Punjab Government Military Department, No. 439. 1880. t Steel, A.V.D., An Investigation into an Obscure and Fata Disease among Transport Mules in British Burma. 1885. \ " Beitrage zur Kenntniss der Hamatozoen," Biol. Centrabl., iii., 1883, pp. 35-44. APPENDIX. 353 more pointed than the posterior, and gradually fining off into the lash. When the body seemed to rest, the lash might be seen to whip out in all directions. As the movement of the body gradually diminished, it appeared to have a complicated screw-form, the axis of the screw corresponding to the body, to which an undulating mem- brane is fastened spirally. This could be distinguished when the organism was dying, because the body in death contracted, and the membrane then looked like a spiral FlG. 130. H^EMATOMONAS COBITIS. ff t First variety ; d, second variety ; c, third variety. d, First variety in a state of diminished activity. e, The same after treatment with osmic acid. [After Mitrophanow.] addition. Thus the organism consisted of a body, a spiral membrane, and a flagellum. With higher magnification the organism appeared to consist of a refractive, strongly-contractile, protoplasmic substance, which, when death occurred, formed a shape- less mass. In the same blood two other forms were observed, one without a membrane, but having two highly- 23 354^ \\ APPENDIX. refractive spherules in the protoplasm, and another with neither membrane nor flagellum, consisting of very granular protoplasm with several refractive spherules, and capable of protruding processes like pseudopodia. ' In the blood of the German carp (Cyprinus Carassius) Mitrophanow describes a parasite which is perceptibly larger, and possesses an undulating membrane fastened along the edge of the long body (Fig. 131). When the body bent first towards one side and then to the other, a FIG. 131. ORGANISMS IN THE LOOD OF THE CARP. a, b, c t Hamatomonas carassii. d, e,f, , h, Other organisms in the same blood [after Mitrophanow]. wave-like movement was observable at the free edge of this membrane. In Cobitis fossilis these parasites were found in all the fish examined except one, and in greater numbers in the hot months. In Cyprinus Carassius they were only found occa- sionally. Mitrophanow described other varieties, which he considered were possibly not complete organisms, but deve- lopmental forms. He considered that these organisms were APPENDIX. infusoria between the genera Cercomonas and Trichomonas, with great similarity to the Trickomonas described in the Lieberkiihn's glands of fowls and ducks (Eberth*). On account of their special habitat Mitrophanow suggested a new genus (Hcematomonas), defining this genus as follows : Parasites of normal fish-blood, worm-like, actively-moving organisms, with indistinct differentiation of body parenchyma. Bodies pointed at both ends, 30 to 40 ju, long and i to 1*5 /A wide. May possess in front a flagellum, and on one side an undulating membrane. Species : Hczmatomonas cobitis. Body provided with a spiral membrane and a flagellum at the fore-end. Parenchyma of body homogeneous. Second variety, body and flagel- lum only. Movement undulatory, body containing highly refractive spherules. Third variety, plasma-like body, without membrane or flagellum ; quickly changes form by sending out processes laterally, and contains two to four refractive spherules. Blood of Cobitis fossilis. Hczmatowonas carassii. Long bodies, with narrow membrane attached along the whole length ; less actively motile. Several forms also observed strikingly smaller than the above ; many disc-shaped. Often seen attached to a red corpuscle, setting them in motion by their move- ments. Blood of Cyprinus carassius. Quite recently the author has investigated the parasites found in the disease known as Surra, and came to the following conclusions : In stained preparations the somewhat tapering central portion, or body, of the parasite is found to be continuous at one end with a whip-like lash, and at the other end to terminate in an acutely-pointed stiff filament, or spine-like process. Here and there, possibly from injury or want of development, the spine-like process appears to be blunted * Vide Leuckart, The Parasites of Man, translated by Hoyle, p. 248. 356 APPENDIX. or absent By very careful focusing on the upper edge of the central portion, the author discovered the existence, much more markedly in some of the parasites than in others, of a longitudinal membrane with either a straight or undulating margin (Fig. 132). The membrane is attached along the body, arising from the base of the rigid filament, and becomes directly continuous at the opposite end with the flagellum. In some cases the edge only is deeply stained, giving the appearance of a thread continuous with tJie flagcllum^ so that one might be easily led to overlook the membrane, and imagine that the FIG. 132. "SURRA" PARASITES OCCURRING SINGLY AND FUSED. From preparations stained with magenta, X 1200. flagellum arose from the opposite end of the body, at the base of the spine-like process. Close to the base of the spine-like process a clear unstained spot is, in many parasites, easily distinguished, and at the opposite end there is, in some, the appearance of the deeply-stained protoplasmic contents having con- tracted within the faintly-stained cell-wall. Where the longitudinal membrane has a wavy outline the undulations are much more marked in some cases than in others. Here and there the wavy outline appears first on one side of the central portion, and then on the other, but there never is any waving outline on both sides of the same part of the body, and this is explained by careful exami- APPENDIX. 357 nation, which shows that in dying the somewhat ribbon- like parasite has become doubled on itself. Owing to the somewhat curved and twisted shape of the parasite and the curling of the flagellum, in the stained preparations, it was difficult to make exact measurements ; but the average width, according to whether the membrane was visible or not, varied from i to 2 ft, and the length of the body from 20 to 30 ft. The flagellum was about the same length as the body. From these observations (especially the discovery of the undulating longitudinal membrane) the author recognised a very close resemblance to Mitrophanow's descriptions, and concluded that, if we followed the classification adopted by Mitrophanow, the genus Hcsmatomonas must FIG. 133. A MONAD IN RAT'S BLOOD, x 3000. The organism is represented at partial rest, with its posterior filament im pinging on a corpuscle, and showing the undulating longitudinal membrane, the long flagellum, and the refractive spherules in the granular protoplasm. not be restricted to organisms in fish-blood. It must be expanded to include this parasite of mammalian blood, which should in that case be named Hcsmatomonas Evansi, rather than Spiroch&td Evansi, as proposed by Steel. In the course of this investigation the author was led to examine the blood of rats obtainable in this country. Organisms were discovered in the blood of about 25 per cent, of common brown rats ; and, after examining them with various objectives, from a \ dry to a ^ oil- immersion of Powell and Lealand, the following con- clusions were arrived at : That they are polymorphic, presenting for the most part slightly tapering bodies 358 APPENDIX. which terminate at one end in a stiff, immotile, acutely- pointed, flexible filament or spine-like process, and at the opposite end are provided with a long flagellum, while longitudinally attached a delicate undulating, fin-like membrane can be traced, which starts from the base of the posterior filament, and becomes directly continuous with the flagellum (Fig. 133). With careful illumination the body is found to be distinctly granular, with one or more highly-refractive spherules. When the rapid movement is arrested the undulating membrane is distinctly visible. The best opportunity occurs for seeing this when the organism FIG. 134. MONADS IN RAT'S BLOOD, x 1200. , 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/, the same seen edgeways. comes to partial rest with its stiff filament against a corpuscle, as if to obtain a point d'appui, while lashing its flagellum in all directions (Fig. 134, ). At other times, when the parasite has impinged with its posterior extremity against a corpuscle, or the stiff filament is apparently entangled 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 APPENDIX. 359 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 in one direction, is suddenly observed working in the opposite direction. Then suddenly the organism makes progression, and it can be distinctly seen to move in the direction of tJte flagellum, thj 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 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 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 may be learnt (Fig. 135). 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 360 APPENDIX. speck is very striking ; and in other cases again the posterior filament is only faintly tinged, or an unstained spot occurs near its base. 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 Hcztnatomonas. 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 FIG. 135. MONADS IN RAT'S BLOOD, showing membrane under different aspects, blood-corpuscles some crenated, and stained discs, X I2co. the characteristic features of these organisms are the characteristic marks of the genus Trichomonas^ They are, therefore, embraced by the genus Trichomonas, 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 * Vide Leuckart, The Parasites of Man, translated by Hoyle, 1886. APPENDIX. 361 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 the 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. APPENDIX C. EXAMINATION OF AIR. 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 alu- minium, 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 debris 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 dis- covered in the air of hospitals for diseases of the skin, and achorion in wards with cases of favus. The tubercle-bacil- 362 APPENDIX. lus is said to have been detected in the breath of patients suffering from phthisis. These points indicate that, in addition to the interest for the microbiologist, considerable importance from a hygienic point of view must be attached to the sys- tematic examination of the air. Especially a knowledge of the microbes which are found in the air of marshy and other unhealthy districts, and in the air of towns, dwel- lings, hospitals, workshops, factories, and mines, will be of practical value. Miquel,* who has particularly studied the bacteria 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 as 142, winter quarter 49, spring quarter 85, and summer quarter 105. In air col- lected 2,000 to 4,000 metres above the sea-level, not a single bacterium 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 microscopic 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. 81) 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. 82) may be similarly exposed. In both the last mentioned methods separate colonies develop, which may be isolated as already described, and pure cultivations carried on in various other nutrient media (P- 79)- 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 * Miquel, Organismes rivants de r atmosphere. APPENDIX. 36 3 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. 136). The ad vantage of FIG. 136. HESSE'S APPARATUS. 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 connected by means of india-rubber tubing with an J 64 APPENDIX. aspirating apparatus. This apparatus consists of a couple of litre-flasks, suspended by hooks from the tripod stand which supports the whole apparatus. The cylinder, caps, and plug, are washed with solution of corrosive sub- limate, and then with alcohol. After being thus cleansed, 50 ccm. of nutrient gelatine are introduced, and the whole sterilised by steaming for half an hour for three successive days. 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. 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 school-room just vacated by the scholars gave thirty-seven colonies of bacteria and thirty-three moulds. Various forms of " aeroscopes " and " aeroniscopes " 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 con- nected by india-rubber tubing with an aspirator. The air passing down the funnel strikes upon the glycerine, APPENDIX. 365 which arrests any solid particles. For a description of the more exact apparatus employed by Maddox, Cun- ningham, and Miquel reference should be made to the writings of these authors, and particularly to the treatise published by the last-named. APPENDIX D. EXAMINATION OF SOIL. SURFACE-SOIL, or mould, is exceedingly rich in bac- teria. Miquel, e.g., has computed that there exists in a gramme of soil an average of 750,000 germs at Mont- souris, 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 from the earth the bacillus of anthrax, and sheep, sojourning upon a plot of ground where animals which have died of 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 culti- vated a bacillus from earth which produced 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, however, much more satisfactory : A sample of earth is collected, dried, and triturated, and a small quantity sprinkled over the surface of nutrient gelatine prepared for a plate-culti- vation. In another method the gelatine is liquefied in a 366 APPENDIX. test-tube, the powder added, and, in the usual way, dis- tributed 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 substances in powder, may be distributed over nutrient gelatine, and the micro-organisms which develop studied, both as to their morphological and biological characteristics. APPENDIX E. EXAMINATION OF WATER. 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 presence of countless bacteria. This test is of no real value, for it does little more than indi- cate that bacteria were present, which we may accept as being present in ice and all ordinary water. On the other hand, the bacteriological test of Professor 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 charac- * Parkes, Manual of Practical Hygiene. 1 883 . APPENDIX. 367 teristics. 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 dis- tinguished from other comma-shaped organisms, and inasmuch as its presence may be an indication of con- tamination 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-cultivations 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 steriliser. When required for use, the plug is removed and held between the ringers, 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 con- tact 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 (p. 31). 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 368 APPENDIX. is removed from the flask, and the cotton-wool plug singed in the flame to prevent contamination from adventitious germs on the outside 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 plug of the flask and tube quickly replaced. If the water is 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 throughout the gelatine (p. 75), and the gelatine finally poured on a plate. FIG. 137. APPARATUS FOR ESTIMATING THE NUMBER OF COLONIES IN A PLATE-CULTIVATION. When the gelatine has set, the plate is transferred to a damp chamber, which should be carefully labelled and set aside in a place of moderate temperature. In about two or three days the cultivation 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 sub- divided into ninths, is so arranged on a wooden frame that it can cover the nutrient gelatine-plate without touching it (Fig. 137). A lens is added to assist in discovering minute colonks. If then the colonies are very numerous, APPENDIX. 369 the number in some 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 liquefying colonies should be also made, and their number, as well as the total number of colonies present in I ccm. of the sample, re- corded. Any peculiar macroscopical appearances, colour, etc., should be noted, and then the microscopical appear- ances of the colonies studied. Lastly, examination of the individual organisms should be made by cover-glass- preparations, and by inoculation of nutrient gelatine, pota- toes, 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. 75,) 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. 94), 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 examina- tion 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 carbonic acid of water containing many bacteria diminishes the number of the latter. The ex- amination of waters before and after filtration, or after addition of chemical substances, are matters which require further investigation. APPENDIX F. 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 I. ACTINOMYCOSIS. II. ACUTE YELLOW ATROPHY. III. ANTHRAX. IV. CATTLE PLAGUE. V. CEREBRO-SPINAL MENINGITIS. VI. CHICKEN-CHOLERA. VII. CHOLERA. 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Die Faulniss des Blutes. 1878 SELMI. Chemische Ben, Bd. 6, 7, 12, Sulle Ptomaine ad Alcaloide Cadaverici. KAUFMANN. Zersetzung des Blutes durch Bacillus Subtilis. Journ. f. Prakt. Chemie, Bd. 17. APPENDIX. 383 1879 KILLER. Die Lehre von der Faulniss. 1880 HUSEMANN. Arch. d. Pharmac. 1881 KoNlG. Massenerkrankung von Menschen nach dem Genuss von Fleisch einer an Putrider Metritis Verendeten Kuh. Ber. ub d. Veteri- narwesen im Konigreich Sachsen. TANRET. Compt. Rend., T. 92. BROUARDEL ET BOUTMY. Compt. Rend., T. 92, p. 1056. 1882 HUSEMANN. Arch. d. Pharmac. SCHIFFER. Arch. f. Anat. u. Physiol., Physiol. Abtheil. Bocci. Centralbl. f. d. Med. Wiss. GROEBNER. Beitrage z. Kenntniss der PtomaTne. BERGMANN u. ANGERER. Das Verhaltniss der Fermentintoxication zur Septicamie. Wtirz- burger Jubil. Festschr. BOUCHARD. Compt. Rend, de Biol. NENCKI. Journ. f. Pract. Chem., Bd. 26. GAUTIER. Compt. Rend., T. 94. GAUTIER ET ETARD. Compt. Rend , T. 94. ROSENBACH. Giebt es Verschiedene Arten von Faulniss? Deut. Zeitschrift fur Chir., XVI., S. 342. ETARD ET OLIVIER. De la Reduction des Sul- fates par les Etres Vivants. Compt. Rend. 1883 HUSEMANN. Arch d. Pharmac. GUARESCHI ET MOSSO. Arch. Ital. de Biolog. BRIEGER. Zur Kenntniss der Faulnissalkaloide. Zeitschr. f. physiol. Chemie, Bd. 7. 1884 MAAS. Ueber Faulnissalkaloide des gekochten Fleisches und des Fischfleisches. Fortschr. d. Med., II., 729. BRIEGER. Ber. d. Deutsch. Chem. Ges., Bd. 17. VANDEVELDE. Les Ptomaines. Arch, de Biol. par van Beneden. WILLGERODT. Ueber Ptomaine. 384 APPENDIX. 1884 BRIEGER. Ueber Giftige Producte der Faulniss- bakterien. Berl. Klin. Woch., Nr. 14. OTTO. Anleitung zur Ausmittelung der Gifte. 1885 TAPPEINER. Med. Centralbl. BLUMBERG. Experimenteller Beitrag zur Kennt- niss der Putriden Intoxication. Virch. Arch., Bd. 100, S. 377. OFFINGER. Die Ptomaine. BRIEGER. Ueber Ptomaine. BRIEGER. Weitere Untersuchungen uber Ptomame. BACKLISCH. Ber. d. Deutsch. Chem. Gesellsch., Bd. 1 8. HAUSER. Ueber Faulnissbakterien. 1886 BRIEGER. Ueber Ptomaine. Berl. Klin. Woch. (H) ANTISEPTICS AND DISINFECTANTS. 1875 ElDAM. Einwirkung Versch. Temperaturen a. d. Entwicklung von Bacterium Termo. Cohn's Beitr. zur. BioL, Bd. I., Heft 3. SCHROTER. Cohn's Beitr. zur Biol. der Pflanzen, Bd. I, Heft 3. IS/6 KOCH. Cohn's Beitr. zur Biol. der Pflanzen, Bd. II, Heft 2. BUCHHOLZ. Ueber das Verhalten von Bakterien zu einigen Antiseptics. 1877 NAGELI. Die Niederen Pilze. BUCHHOLZ. Arch. f. Exp. Pathol., Bd. 7. FRISCH. Ueber den Einfluss nied. Temp. auf. d. Lebensfahigkeit der Bakterien. Sitzungsber. d. Wiener Akad, Bd. 75 u. 80. TVNDALL. Philos. Transact, of the Roy. Soc. VALLIN. Ann. d'Hyg. 1879 SOYKA. Ber. d. Bayr. Akad. d. Wissensch. SCHWARTZ. Sitzungsber. d. Dorpater Naturf. Ges. W'ERNICKE. Diss. Dorpat. APPENDIX. 385 1879 HABERKORN. Das Verhalten von Harnbakterien gegen einige Antiseptica. Dissert. Dorpat. WERNICH. Die Aromatischen Faulnissproducte in ihrer Einwirkung auf Spalt und Sprosspilze. Virchow's Archiv, Bd. 78. 1880 MERKE. Virchow's Archiv, Bd. 8 1. M6RSCHELL. -Deut Med. Woch. LASSAR. Deut. Med. Woch. PASTEUR. Ann. d'Hyg. WERNICH. Grundriss der Desinfectionslehre. MEYER. Ueb. d. Milchsaureferment u. sein Ver- halten gegen Antiseptica. TOUSSAINT. Bull, de 1'Acad. 1881 WOLFFHUGEL. Ueber den Werth der schwefligen Saure als Desinfectionsmittel. Mittheilg. a. d. Kais. Ges. Amt, Bd. I., S. 188. WOLFFHUGEL u. KNORRE. Zu der verschiedenen Wirksamkeit von Carbolol u. Carbolwasser. Mitth. a. d. Kaiserl. Ges. Amt, Bd. I., S. 352. KOCH. Ueber Desinfection. Mitth. a. d. Ges. Amt, Bd. I., S. 234. DE LA CROIX. Das Verhalten der Bakterien d. Fleischwassers gegen einige Antiseptica. Arch. f. Exp. Pathol, Bd. 13. KOCH, GAFFKY u. LOFFLER. Versuche iiber die Verwerthbarkeit heisser Wasserdampfe zu Des- infectionszwecken. Mitth. a. d. Kaiserl. Ges. Amt, Bd. I., S. 322. KOCH u. WOLFFHUGEL. Unters. iib. die Desin- fection mit heisser Luft. Mitth. a. d. Kaiserl. Ges. Amt, Bd. I., S. 301. HUEPPE. Ueber das Verhalten ungeformter Fer- mente gegen hohe Temperatur. Mittheilg. a. d. Kaiserl. Ges. Amt, Bd. I., S. 341. 1882 LEBEDEFF. Desinfectionsversuche am Malignen Oedem. Arch, de Physiol. Norm, et Pathol. W. CHEYNE. Antiseptic Surgery. 25 386 APPENDIX. 1882 LEBEDEFF. Arch, de Phys. Norm, et Pathol. HUEPPE. Ueber die Hitze als Desinfectionsmittel. Deut. Militararztl. Zeitschr. 1883 FISCHER. Unters. iib. d. Wirkung des Naphtalins. Berl. Klin. Woch. PERRONCITO. Sur la Tenacity de Vie du Virus Charbonneux. Arch. Ital. de Biol. VALLIN. Traite* des Disinfectants et de la Desin- fection. MlQUEL. Antiseptiques et Bacteries. Semaine Me"dicale. CHAUVEAU. Compt. Rend. CHAMBERLAND ET Roux. Compt Rend. LAILLIER. Du Gaz Acide Sulfureux. Ann. d' Hygiene. ARLOING, CORNEVIN, ET THOMAS. Lyon Med. VALLIN. Les Nouvelles Etuves a Desinfection. Revue d'Hygiene. LARRIVE". L'Eau Oxygenee. These de Paris. MALY U. EMICH. Antisept. Wirkung der Gallen- sauren. Sitzungsber. d. Kais. Akad. d. Wiss. zu Wien. Jan. SCHULTZ. Die Antiseptischen Eigenschaften der Citronensaure. Deut. Med. Woch., Nr. 17. 1884 SCHILL U. FISCHER. Ueber die Desinfection des Auswurfs der Phthisiker. Mitth. a. d. Kaiserl. Ges. Amt, Bd. II. PLAUT. Desinfection der Viehstalle. STEINMEYER. Ueber Desinfectionslehre. COLIN. Compt. Rend., T. 99. CHAIRY. Action des Agents Chimiques sur les BacteVies du Genre Tyrothrix. Compt. Rend. CHAUVEAU. Compt. Rend. MlGNET. Annuaire de 1'Observatoire de Mont- souris. . SCHNETZLER. Les Proprie'tes Antiseptiques de I 1 Acide Formique. Archiv. de Gdneve. APPENDIX. 387 1884 ROSSBACH. Einfluss des innerl. Naphthalinge- brauchs auf die Harnfaulniss. Bed. Klin. Woch. HOFFMANN. Experimentelle Untersuchungen iiber die Wirkung der Ameisensaure. Diss. Greifswald. FISCHER u. PROSKAUER. Mitth. a. d. Kaiserl. Ges. Amt, Bd. II. HEYDENREICH. Sur la Sterilisation des Liquides au moyen de la Marmite de Papin. Compt. Rend., T. 98. PlCTET ET YOUNG. De 1'Action du Froid sur les Microbes. Compt. Rend., T. 98. VALLIN. Ann. d'Hygiene. ROCHEFORT, HERSCHER. Revue d'Hygiene. DujARDIN-BEAUMETZ. Bull.de 1' Acad.de Med. de Paris. MARIE-DAVY. Revue d'Hygiene. 1885 M. WOLFF. Zur Desinfectionsfrage. Centralbl. f. d. Med. Wiss., Nr. 1 1. REINL. Zur Theorie der Heilwirkung des Fran- zensbader Moores. Prager Med. Woch., Nr. 10 u. 1 1. THOL. Ueber d. Einfluss nicht aromat organ. Sauren auf Faulniss u. Gahrung. Diss. Greifs- wald. SCHULZ. Die Ameisensaure als Antisepticum. Deut. Med. Woch., Nr. 24. SCHEDE. Die Antiseptische Wundbehandlung mit Sublimat Sammlung Klin. Vortrage, Nr. 25. KoNIG. Sublimatdampfe. Chirurg. Centralbl. FRANK. Ueber Desinfection von Abtrittsgruben. GARTNER u. PLAGGE. Deut. Med. Woch. 1886 HANDFORD. Brit. Med. Journ. KOCH U. GAFFKY. Arbeit, a. d. K. Gesundh. Amt. WINTER BLYTH. Studies of Disinfectants by New Methods. Proc. Roy.. Soc. 388 APPENDIX. (I) IMMUNITY. 1876 OEMLER. Arch. f. Wiss. u. Pract. Thierheilk. 1879 OLLIVE. Sur la Resistance des Moutons de la Race Berberine a 1'Inoculation du Charbon. Compt. Rend., T. 89. CHAUVEAU. De la Predisposition et de ITmmu- nite Pathologique. Compt. Rend., T. 89. 1880 PASTEUR. Bull, de 1'Acad. de Med. and Gaz. He'd. de Paris, Nr. 18. SEMMER u. KRAJEWSKI. Centralbl. f. d. Med. Wiss. TOUSSAINT. Bull, de 1'Acad. de Med. ; and Compt. Rend. 1881 TOUSSAINT. Compt. Rend. LOEFFLER. Zur Immunitatsfrage. Mitth. a. d. Ges. Amt, Bd. I. SEMMER. Virchow's Arch., Bd. 83. GRAWITZ. Die Theorie der Schutzimpfung. Virchow's Arch., Bd. 84. TOUSSAINT. Gazette Medicale de Paris, Nr. 32. OEMLER. Arch. f. Wiss. u. Pract. Thierheilk. 1882 R6SZAHEGYI. Pester Med.-Chir. Presse. KOCH. Ueber die Milzbrandimpfung. 1883 FRANK. Jahresber. d. K. Thierarzneischule in Miinchen. ARLOING, CORNEVIN ET THOMAS. Du Charbon Bacterien; Pathogenic et Inoculations Pre- ventives. CHAUVEAU. Compt Rend., T. 96, Nr. 9. CHAUVEAU. Compt. Rend., T. 96, Nr. 10. CHAUVEAU. Du Role de 1'Oxygene de 1'Air dans 1'Attenuation quasi-instantanee des Cultures Virulentes par 1'Action de la Chaleur. Compt. Rend., T. 96, Nr. n. APPENDIX. 389 1883 PASTEUR. Sur la Vaccination Charbopneuse. Compt. Rend. PASTEUR. La Vaccination Charbonneuse. PASTEUR. Reponse au Doct. Koch. Revue Scientifique. PASTEUR. Bull, de TAcad. de Med. BUCHNER. Eine neue Theorie iiber Erzielung v. Immunitat gegen Infectionskrankheiten. PERRONCITO. Sull' Attenuazione del Virus Carbonchioso. Atti R. Ace. d. Lincei. CHAMBERLAND ET Roux. Compt. Rend., T. 96, Nr. 15. CHAMBERLAND. Le Charbon et la Vaccination Charbonneuse d'apres les Travaux Recents de M. Pasteur. MASSE. Des Inoculations Preventives dans les Maladies Virulentes. 1884 FELTZ. De la Dure"e de I'lmmunit^ Vaccinale Anticharbonneuse chez le Lapin. Compt. Rend., T. 99, p. 246. CHAUVEAU. De T Attenuation des Cultures Viru- lentes par I'Oxigene Comprime'. Gaz. Heb- dom. de Me\i et de Chir., 22. KOCH, GAFFKY u. LOEFFLER. Exper. Studien iiber d. Kunstl. Abschwachung der Milzbrand- bacillen. Mitth. a. d. Ges. Amt, Bd. II. BLAZEKOVIC Zur Praventiv-Inoculation Pasteur's. Oesterr. Monatschr. f. Thierheilk. PUTZ. Vortrage f. Thierarzte, Ser. 7, Heft I. 1885 HESS. Vorl. Mitth. ii. die Schutzimpfung gegen Milzbrand im Kanton Bern nach der Methode Chauveau. Schweiz. Arch. f. Thierheilk., Bd. 27. STREBEL. Zur Rauschbrandimpfung. Schweiz. Arch. f. Thierheilk. BOULEY. L'Inoculation Preventive de la Fievre Jaune. Compt. Rend., T. 100. 39 APPENDIX. (J) BACTERIA ASSOCIATED WITH DISEASES IN MAN AND ANIMALS. (I.) Actinomycosis. 1877 BOLLINGER. Centralbl. f. d. Med. Wiss. 1878 ISRAEL. Virchow's Arch., Bd. 74. 1879 ISRAEL. Virchow's Arch., Bd. 78. 1881 PONFICK. Die Actinomykose. Berlin. JOHNE. Deutsche Zeitschr. f. Thiermed. 1882 HlNK. Centralbl. f. d. Med. Wiss. PFLUG. Centralbl. f. d. Med. Wiss. GANNET. Boston Med. and Surg. Journ. 1883 PUSCH. Arch. f. Wiss. u. Pr. Thierheilk. BANG. Tidskrift far Veterinaerer. ZEMANN. Wien. Med. Jahrb., S. 477. FLEMING. Actinomycosis. 1884 ISRAEL. Virchow's Arch., Bd. 96. MiTTELDORPF. Deut. Med. Woch. KARSTEN. Deut. Med. Woch. CHIARI. Prager Med. Woch., Nr. 10. TREVES. Lancet. FlRKET. Rev. de Med. 1885 ISRAEL. Kenntniss der Actinomykose des Menschen. Klinische Beitrage. BOSTROM. Verh. d. Congr. f. Inn. Med. Wiesbaden. BAUMGARTEN. Berl. Klin. Woch. JOHNE. Bericht u. d. Veter.-Wesen i. K. Sachsen. MURPHY. New York Med. Journ. PONFICK. Breslauer Aerztl. Zeitschr. SOLTMANN. Breslauer Aerztl. Zeitschr. MAGNUSSEN. Beitrage zur Diagnostik u. Casui- stik der Actinomykose. Diss. Kiel. 1886 HERTWIG. Archiv. f. Wiss. u. Prakt. Thierheilk. O'NEILL. Lancet. ACLAND. Brit. Med. Journ. and Trans. Path. Soc. ROSER. Deut. Med. Woch. ISRAEL. Archiv f. Klin. Chir. APPENDIX. 391 (II.) Acute Yellow Atrophy 1875 EPPINGER. Prag. Viertelj. 1882 HLAVA. Prag. Med. Wochenschr. BALZER. Archiv de Phys. Norm. et. Path. (III.) Anthrax. 1855 POLLENDER. Viertelj ahrschr. f. Ger. Med., Bd. 8. 1857 BRAUELL. Virchow's Arch., Bd. n. 1858 BRAUELL. Virchow's Arch., Bd. 14. 1863 DAVATNE. Compt Rend. Paris. T. 57. Ibid., T. 59- 1865 DAVAINE. Compt. Rend. Paris. T. 60. 1866 DAVAINE. Compt. Rend. Paris. T. 61. 1872 BOLLINGER. Centralbl. f. d. Med. Wiss., Bd. 10. 1873 COLIN. Bull. Acad. de Med., Paris. T. 2, DAVAINE. Compt Rend. Paris. T. 77. 1876 KOCH. Beitrage zur Biologic der Pflanzen, Bd. II., Heft 2. 1877 PASTEUR. Compt. Rend. Paris. T. 84. TOUSSAINT. Compt. Rend. T. 85. DAVAINE. Rec. de Med. Vet., T. 4. DAVAINE. Compt. Rend. BERT. Compt. Rend. Soc. de Biol., T. 4. PASTEUR. Bull. Acad. de Med. 1878 COLIN. Bull. Acad. de Med. Paris. T. 7. TOUSSAINT. Compt. Rend. Paris. OEMLER. Archiv f. Wiss. u. Pract. Thierheilk., Bd. 4. EWART. Quart. Journ. of Microsc. Sc. BERT. Compt. Rend. Soc. de Biol., T. 5. KOCH. Wundinfectionskrankheiten. 1879 SCHMIDT. Milzbrand bei Wildschweinen. Deut. Zeitschr. f. Thiermed. u. Vergl. Pathol. ? - 3Q2 APPENDIX. 1879 TOUSSAINT. Recherches Experimental sur la Maladie Charbonneuse. BERT. Compt. Rend. Soc. de Biol, T. 6. GREENFIELD. Quart. Journ. Micr. Sc. London. OEMLER. Archiv. f. Wiss. u. Pract. Thierheilk. COLIN. Bull. Acad. de Med. Paris. T. 8. PASTEUR. Bull. Acad. de Med. Paris. i860 PASTEUR. Compt. Rend. Paris. T. 90 and 91. CHAUVEAU. Compt Rend. T. 90 and 91. TOUSSAINT. Compt. Rend. COLIN. Bull. Acad. de Med. Paris. T. 9. PASTEUR. Bull. Acad. de Med. Paris. BOULEY. Bull. Acad. de Med. Paris. T. 9. WACHENHEIM. Etude Experimented sur la Septicite et la Virulence du Sang Charbonneux. BiJCHNER. Ueber die Exper. Erzeugung des Milzbrandcontagiums aus den Heupilzen. BiJCHNER. Versuche iiber die Entstehung des Milzbrands durch Einathmung. Sitzungsber. d. K. Bayer. Akad. d. Wissensch. FOKKER. Centralbl. f. d. Med. Wissensch. Bd. 1 8. SEMMER. Centralbl. f. d. Med. Wiss.. Bd. 18. OEMLER. Archiv f. Wissensch. u. Pract. Thierheilk. SZPILMAN. Zeitschr. f. Physiol. Chemie. Strass- burg. Bd. 4. GREENFIELD. Proc. Roy. Soc. London. l88l PASTEUR. Compt. Rend. Paris. T. 92. CHAUVEAU. Compt. Rend., T. 92. BOULEY. Compt. Rend. T. 92. Ibid., T. 93. COLIN. Bull. Acad. de Med., T. 10. BOULEY. Bull. Acad. de Med., T. 10. RODET. Contribution a 1'Etude Experimentelle du Charbon Bacteridien. KOCH. Mitth. aus. d. Ges. Amt, Bd. I. FOKKER. Centralbl. f. d. Med. Wiss. BUCHNER. Vortrage im Aerztl. Verein zu Miin- chen. APPENDIX. 1881 HUBER. Deut. Med. Woch., Bd. 7. KLEIN. Rep. of the Medical Officer of the Local Govt. Board. STERNBERG. Am. Monthly Micr. Journ. 1882 CHAUVEAU. Compt. Rend. Paris. T. 94. RODET. Compt. Rend., T. 94. PASTEUR. Compt. Rend. Paris. T. 95. FELTZ. Compt. Rend., T. 95. FOKKER. Virchow's Archiv, Bd. 88. ARCHANGELSKI. Centralbl. f. d. Med. Wiss. SEMMER. Der Milzbrand und das Milzbrandcon- tagium. ESSER u. SCHUTZ. Zur Casuistik des Milzbrands. Mitth. a. K. Preuss. Amtl. Vet. Sanitats- bericht. 1883 CHAUVEAU. Compt. Rend. Paris. T. 96. ARCHANGELSKI. Centralbl. f. d. Med. Wiss. ROLOFF. Ueber, die Milzbrandimpfung und d. Entwicklung d. Milzbrandbakterien. Archiv f. Wissensch u. Pract. Thierheilk., Bd. 9. ROLOFF. Der Milzbrand. Berlin. BUCHNP:R. Die Umwandlung der Milzbrandbak- terien in unschadliche Bakterien. Virchow's Arch., Bd. 91. TOEPPER. Die Neueren Erfahrungen iiber d. Aetiologie d. Milzbrands. KLEIN. Quart. Journ. Micr. Sc. DOWDESWELL. Rep. Med. Off. Local Gov. Board. 1884 BLEULER. Milzbrand beim Menschen. Corre- spondenzbl. d. Schweiz. Aerzte. SCHRAKAMP. Zur Aetiologie des Milzbrandes. Archiv f. Hygiene, Bd. 2. v. CHELCHOWSKY. Zur Charakteristik des Milz- brandvirus. Der Thierarzt. SEMMER. Centralbl. f. d. Med. Wiss., Bd. 22. PRAZMOWSKI. Acad. d. Wissensch. in Krakau. PRAZMOWSKI. Biol. Centralbl., Bd. 4. 394 APPENDIX. 1885 BOLLINGER. Zur Aetiologie des Milzbrands. Sitzungsber. d. Ges. f. Morphol. Physiol. zu Miinchen. KITT. Sitzungsb. d. Ges. f. Morphol. u. Physiol. -zu Miinchen. FRIEDRICH. Zur Aetiologie des Milzbrands. OSOL. Experiment. Untersuch. ii. das Anthrax- gift. Inaug. Diss. Dorpat. 1886 W. KOCH. Milzbrand und Rauschbrand. Stutt- gart. HOFFA. Die Natur des Milzbrandgiftes. Wies- baden. v. FODOR. Deut Med. Woch. JOHNE. Ber. ii. d. Veter.Wesen. i. K. Sachsen. BOLLINGER. Arbeit, a. d. Patholog. Inst. zu Miinchen. FRANK. Zeitschr. f. Hygiene. (IV.) Cattle PJague. 1883 SEMMER u. ARCHANGELSKI. Ueber das Rinder- pestcontagium und dessen Mitigation. Centralbl. f. d. Med. Wiss. (V.) Cerebro-Spinal Meningitis. 1883 LEYDEN. Die Mikrokokken der Cerebrospinal- Meningitis. Centralbl. f. Klin. Med., Nr. 10. 1885 LEICHTENSTERN. Deut. Med. Woch., Nr. 23 u. 3 1. (VI.) Chicken-Cholera. 1877 lOANNES ET Mi^GNlN. Journ. d' Acclimatation. 1878 SEMMER. Hiihnerpest. Deut. Zeitschr. f. Thier- med. u. Vergl. Path. APPENDIX. 395 1879 PERRONCITO. Ueber das Epizootische Typhoid der Hiihner. Arch. f. Wiss. u. Prakt. Thierheilk. 1880 PASTEUR. Sur le Cholera des Poules. Compt. Rend., T. 90. 1882 ZURN. Die Krankheiten des Hausgefliigels. CoRNIL. Arch, de Physiol., Bd. 10. 1883 BARTHE"LMY. De I'lncubation des CEufs d'une Poule atteinte du Cholera des Poules. Compt. Rend., T. 96., No, 18. BABES. Arch, de Physiol. BABES. Compt. Rend, de 1'Acad. d. Sc. 1884 PETRL Centralbl. f. d. Med. Wiss. 1885 KITT. Mitth. liber die Typhoidseuche des Gefliigels. Allg. Deut. Gefliigelzeitung. (VII.) Cholera. 1883 STRAUSS, Roux, THUILLIER ET NOCARD. Compt. Rend. Soc. de Biol, T. 4. 1884 FiNKLER U. PRIOR. Ueber den Bacillus der Cholera Nostras und seine Cultur. Naturfor- scherversammlung Magdeburg. PFEIFFER. Der bisherige Verlauf der Cholera in Thuringen, etc. Corresp.-Bl. des Allgem. Aerztl. Ver. in Thuringen, Nr. 9. KOCH. Ueber die Cholerabakterien. Deut. Med. Woch. EMMERICH. Die Cholera in Neapel. Deut. Med. Woch., Nr. 50. FINKLER u. PRIOR. Unters. iiber Cholera Nostras. Deut. Med. Woch., Nr. 36. DOYEN. Mikro-organismen in Leber und Niere von Choleraleichen. Soc. de Biol. de Paris. Dec. 13. BuCHNER. Ueber Cholerabacillen. Munchn. 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Woch., Nr. 2. PFEIFFER. Ueber die Cholera in Paris. Deut. Med. Woch., Nr. 2. DENEKE. Ueber eine Neue den Choleraspirillen ahnliche Spaltpilzart. Deut. Med. Woch., Nr. 3. MILLER. Kommaformiger Bacillus aus der Mundhohle. Deut. Med. Woch., Nr. 9. KLEBS. Ueber Cholera Asiatica. APPENDIX. 397 1885 SCHOTTELIUS. Zum Mikrosk. Nachw. v. Cholera- bacillen in Dejectionen. Deut. Med. Woch., Nr. 14. DRASCHE. Allg. Wien. Med. Zeit. VAN ERMENGEM. Die Ferran'schen Impfungen. Deut. Med. Woch., Nr. 29. GIBIER ET VAN ERMENGEM. Rech. Exper. sur le Cholera. Compt. Rend., T. 101. VAN ERMENGEM. Recherches sur le Microbe du Cholera Asiatique. NlCATI AND RiETSCH. Arch, de Physiol. NlCATI AND RiETSCH. Revue d'Hygiene. NlCATI AND RIETSCH. Revue de Medecin, T. 5. BRUNETTL Fatti Considerazioni Conclusion! sul Colera. KLEIN. Brit. Med. Journ. and Proc. Roy. Soc. London, No. 38. KLEIN and GlBBES. An Inquiry into the Etiology of Asiatic Cholera. Bluebook. BUCHNER. Archiv. f. Hygiene. BUCHNER u. EMMERICH. Munch. Med. Woch. CROOKSHANK. Remarks on the Comma Bacillus of Koch. Lancet. 1886 KOCH. Etiology of Cholera. Berlin Cholera Conference. Translated by Laycock, in Microparasites and Disease (New Syd. Soc.) PETTENKOFER (abstracted by Koplik). Lancet. CANTANI. Deut. Med. Woch. WEISSER. Zeitschrift f. Hygiene. BITTER. Archiv f. Hygiene. PFEIFFER. Deut. Med. Woch. KLEIN. Bacteria of Asiatic Cholera. Prac- titioner. WEISSER and FRANK. Zeitschr. f. Hygiene. APPENDIX. (VIII.) Dental Caries. 1867 LEBER u. ROTTENSTEIN. Unters. iiber Caries der Zahne. Berlin. i860 ARNDT. Beob. an Spirochaeta Denticola. Arch. f. Pathol. Anat, Physiol. u. Klin. Med., Bd. 79. 1882 MILLER. Der Einfluss der Mikroorganismen auf die Caries der Zahne. Arch. f. Exp. Path., XVI. 1883 MILLER. Ueber einen Zahnspaltpilz Leptothrix Gigantea. Ber. d. Deutsch. Bot. Ges., Heft 5. 1884 MILLER. Deutsche Med. Woch., Nr. 25 u. 36. MILLER. Correspdzbl. f. Zahnarzte, Bd. 13. ROSENBACH. Mikroorganismen bei den Wundin- fectionskrankheiten, S. 77. (IX.) Diphtheria. 1867 BUHL. Micrococci of Diphtheria. Zeitschr. fur Biol. 1868 HUETER u. TOMMASI. Centralbl. f. d. Med. Wissensch. 1871 OERTEL. Deut. Arch. f. Klin. Med., Bd. 8. 1872 LETZERICH. Virch. Arch., Bd. 55. BlRCH-HlRSCHFELD. Archiv fur Heilkunde. 1873 EBERTH. Der Diphtheritische Process. Med. Centralbl., XL, Nr. 8. 1874 LETZERICH. Mikrochemische Erscheinungen des Diphtheritispilzes. Berl. Klin. Woch., XI. 1875 KLEBS. Arch. f. Exp. Pathol., Bd. 4. 1876 LETZERICH. Virch. Arch., Bd. 68. . 1878 ZAHN. Beitrage zur Pathol. u. Histol. der Diph- theric. 1879 FRIEDBERGER. Ueber Croup u. Diphtheritis beim Hausgefliigel. Deut. Zeitschr. fur Thiermed. u. Vergl. Pathol. APPENDIX. 399 1879 NlCATl. Compt. Rend., T. 88. 1880 NEUMAYER. Neue Thesen zur Diphtheritisfrage. 1881 EVERETT. Med. and Surg. Reporter. TALAMON. Bull, de la Soc. Anat. de Paris. T. 56. CORNIL. Arch, de Physiol. OERTEL. Zur Aetiologie der Infectionskrank- heiten. FORSTER. Wien. Med. Woch. LUMNER. Aerztl. Int. Bl., No. 31. 1882 WOOD AND FORMAD. Bull. Nat. Board of Health, Wash, and Med. Times and Gazette. SALISBURY. Gaillard's Med. Journ. New York. GERHARDT AND KLEBS. Verhandlung des Con- gresses fur Innere Med. 1883 FiJRBRINGER. Virch. Arch., Bd. 91. HEUBNER. Die Experimented Diphtheric. GERHARDT u. KLEBS. Verhandl. d. Congresses f. Inn. Med. FRANCOTTE. La Diphtheric. 1884 EMMERICH. Deut. Med. Woch. EMMERICEI. Compt. Rendus et Memoires du V. Congres Internat. d'Hygiene. RlVOLTA. Die Diphtheric der Hiihner im Ver- gleich zu der des Menschen. Giornale de Anat. Fisiol. e Patol. delli Anim. LOEFFLER. Mittheil. a. d. Kais. Ges. Amt, Bd. II.. 1886 LOEFFLER. Microparasites and Disease [New Syd. Soc.]. (X.) Erysipelas. 1868 HiiTER. Med. Centralbl, Nr. 34. 1873 RAYNAUD. Union Med. ORTH. Archiv. f. Exper. Pathol. u. Pharmacol., Bd. I. 4OO APPENDIX. 1874 RECKLTNGHAUSEN u. LANKOWSKI. Ueber Ery- sipelas. Virchow's Arch., Bd. 60. LUKOMSKY. Virchow's Archiv, Bd. 60. 1875 TROSIER. Bull. Soc. Anat. de Paris. BAADER. Zur Aetiologie des Erysipels. Schweiz. Naturf. Gesellsch. KLEBS. Archiv f. Exper. Pathol. u. Pharmacol., Bd. 4. 1878 TlLLMANNS. Verhandl. d. Deutsch. Ges. f. Chi- rurgie. 1879 TlLLMANS. Experimentelle u. Anatomische Unters. etc. Archiv f. Klin. Chirurgie, Bd. 23. 1880 WOLFF. Virchow's Arch., Bd. 8 1. 1881 DUPEYRAT. Recherches Cliniques et Experimen- tales sur la Pathogenic de 1'Erysipele. FEHLEISEN. Ueber den Erysipelaspilz. Wiirz- burger Phys. Med. Ges. 1882 FEHLEISEN. Deut. Zeitschr. f. Chir., Bd. 16. 1883 FEHLEISEN. Die Aetiologie des Erysipels. Berlin. 1884 JANICKE U. NEISSER. Exitus Lethalis nach Ery- sipelas. Centralbl. f. Chir., Nr. 25. RHEINER. Beitr. z. Path. Anat. des Erysipels bei Gelegenheit der Typhusepid. in Zurich. Vir- chow's Arch., Bd. 100, Heft 2. 1885 NEPVEN. Des Bacteries dans 1'Erysipele. DENUCE. Etude sur la Pathogenic et 1'Anatomie pathologique de 1'Erysipele. (XI.) Endocarditis. 1875 KOESTER. Virchow's Arch., Bd. 72. 1876 BlRCH-HlRSCHFELD U. GERBER. Archiv d. Heil- kunde. 1877 WEDEL. Berl. Klin. Wochenschr. APPENDIX. 4O I 1878 KLEBS. Archiv f. Exper. Pathol., Bd. 9. O. ROSENBACH. Ueber Artificielle Herzklappen- fehler. Archiv fur Exper. Pathol., Bd. 9. 1880 HAMBURG. Ueber Acute Endocarditis. Berlin. Inaug.-Diss. GOODHART. Trans. Path. Soc., Vol. XXXI. 1881 OBERBECK. Casuistische Beitrage zur Lehre von der Endocarditis Ulcerosa. Inaug.-Diss. LEYDEN. Zeitschr. f. Klin. Med. WEIGERT. Virchow's Arch., Bd. 84. KOCH. Mittheil. a. d. Kais. Ges. Amt, Bd. I. OSLER. Trans. Int. Med. Congress. 1882 WiLKS. Brit. Med. Journ. 1883 KUNDRAT. Sitz.-Ber. d. Kais. Acad. d. Wissensch. zu Wien. 1884 BRAMWELL. Diseases of the Heart. BRISTOWE. Brit Med. Journ. GlBBES. Brit. Med. Journ. 1885 WYSSOKOWITSCH. Centralbl. f. d. Med. Wis- sensch., Nr. 33. COUPLAND. Brit. Med. Journ. ORTH, J. Versammlung Deutscher Naturf. zu Strasburg. NOCARD. Recueil de Med. Vet. OSLER. Brit. Med. Journ. WEICHSELBAUM. Wien. Med. Woch. 1886 RIBBERT. Fortsch. d. Med. (XII.) Glanders. 1882 LoFFLER U. SCHUTZ. Ueber den Rotzpilz. Deut. Med. Woch., Nr. 52. BOUCHARD, CAPITAN ET CHARRIN, Bull, de 1'Acad.d. Sc., Nr. 51. 1883 ISRAEL. Bed. Klin. Woch., Nr. 1 1. WASSILIEFF. Deut Med. Woch., Nr. n. 26 402 APPENDIX. 1883 MOLKENTIN. Zur Sicherstellung der Diagnose von Rotz. Inaug.-Diss. STRUCK. Deut. Med. Woch., Nos. 51 u. 52. VULPIAN AND BOULEY. Bull, de TAcad. de Me"d. FROHNER. Rotzige Elephantiasis des Kopfes beim Pferde. Rep. d. Thierheilk. 1884 KlTT. Versuche iiber d. Ziichtung des Rotzpilzes. Jahresber. d. Miinchen. Thierarzneisch. GRUNWALD. Zur Differentialdiagnose des Rotzes. Oesterr. Monatsschr. fur Thierheilk., Nr. 4. WEICHSELBAUM. Zur Aetiologie der Rotzkrank- heit des Menschen. Wiener Med. Woch., 2 1 24. l886 LOEFFLER. Arbeit, a. d. K. Gesundh. Amt. (XIII.) Gonorrhoea. 1879 NEISSER. Centralbl. f. d. Med. Wiss., Nr. 28. RETER. Centralbl. f. d. Med. Wiss. 1880 BiJCKER. Ueber Polyarthritis Gonorrhoica. Diss. BOKAI. Ueber das Contagium der Acuten Blen- norrhce. Allgem. Med. Centralzeitung, Nr. 74. BOKAI AND FlNKELSTEIN. Prager Med. Chir. Presse. WEISS. Le Microbe du Pus Blennorrhagique. 1881 AUFRECHT. Pathologische Mittheilungen. HAAB. Der Mikrokokkus der Blennorrhcea Neonator. HlRSCHBERG U. KRAUSE. Zur Pathologic der Ansteckenden Augenkrankheiten. Centralbl. f. Pract. Augenheilk. 1882 MARTIN. Rech. sur les Inflamm. Mdtast. a la suite de la Gonorrhde. KRAUSE. Die Mikrokokken der Blenorrhcea Neonator. LEISTIKOW. Ueber Bakterien bei den Venerischen Krankheiten. Charite-Annalen, 7 Jahrg., S. 7 5 o APPENDIX. 4O3 1882 BOCKHART. Sitzungsbericht d. Phys. Med. Ges. zu Wiirzburg. 1883 BOCKHART. Beitrag z. Aetiologie und Pathol. des Harnrohrentrippers. Viertelj. f. Dermatol. und Syph. ARNING. Gonokokken bei Bartolinitis. Viertelj. f. Dermatol. u. Syph., S. 371. ESCHBAUM. Beitr. zur Aetiologie der Gonorrh. Secrete. Deut. Med. Woch., S. 187. NEWBERRY. Maryland Med. Journ. CAMPONA. Italia Medica. AUFRECHT. Mikrokokken i. d. Inneren Organen bei Nabelvenenentziindung Neugeborener. Cen- talbl. f. d. Med. Wiss., Nr. 16. PETRONE. Sulla Natura dell'Artrite Blenorragica. Rivista Clin., No. 2. 1884 CHAMERON. Progre~s Medical, 43. STERNBERG. Med. News, Vol. 45, Nr. 16. KAMMERER. Ueber Gonorrh. Gelenkentziindung. Centralbl. f. Chirurgie, Nr. 4. WELANDER. Gaz. Med. de Paris. KRONER. Zur Aetiol. der Ophthalmoblennorrhoea Neonator. Naturforschervers. in Magdeburg, Arch. f. Gyn., XXV., S. 109. SANGER. Ueber Gonorrh. Erkrankung der Uterusadnexe. Naturforschervers. in Magde- burg. Ibid., S. 126. OPPENHEIMER. Naturforschervers. in Magdeburg. Ibid, S. 51. 1885 FRANKEL. Deut. Med. Woch, S. 22. BUMM. Der Mikroorganismus der Gonorrhoischen Schleimhauterkrankungen. LUNDSTROM. Studier ofver Gonokokkus. Diss. Helsingfors. E. FRANKEL. Mikrokokken bei Colpitis. Deut. Med. Woch, Nr. 2. 1886 SMIRNOFF. Vrach. 404 APPENDIX. (XIV.) Hydrophobia. 1881 COLIN. Bull. Acad. de Med. Paris, T, 10. DciL^RIS. Gaz. Med. de Paris, T. 3. Tribune Med. Paris, T. 14. PASTEUR. Compt. Rend. 1882 PASTEUR, CHAMBERLAND, Roux ET THUILLER. Compt. Rend. BERT. Compt. Rend. 1883 GIBIER. Compt. Rend. 1884 PASTEUR. Nouvelle Communication sur la Rage. Ann. de Med. VeteVin. PERCHERON. La Rage et les Experiences de M. Pasteur. PASTEUR. Compt. Rend. FOL. Acad. des Sciences. 1886 BABES. In Les BacteVies. DOWDESWELL. Journ. Roy. Micro. Soc. and Lancet. DOLAN. Hydrophobia. M. Pasteur and his Methods. PASTEUR. Compt. Rend. VlGNAL. Brit. Med. Journ. LANKESTER. Nineteenth Century, No. 1 1 4. KERR. Brit. Med. Journ. BAUER. Munch. Med. Woch. FRISCH. Wien. Med. Woch. HlME. Experimental Researches concerning Pas- teur's Prophylactic. Lancet. (XV.) Leprosy. 1879 NEISSER. Breslauer Aerztl. Zeitschr. NEISSER. Jahresber. d. Schles. Ges. fur Vaterl. Cultur. APPENDIX. 405 1880 HANSEN. Virchow's Archiv. GAUCHER ET HILLAIRET. Progres Med. 1881 NEISSER. Virchow's Archiv, Bd. 84. CORNIL ET SUCHARD. Ann. de Dermat. et Syph, VossiUS. Uebertragungsversuche von Lepra auf Kaninchen. Ber. iiber d. Ophthalmologencon- gress in Heidelberg. JOHN HILLS. On Leprosy in British Guiana. 1882 KoBNER. Uebertragungsversuche von Lepra auf Thiere. Virchow's Arch. KOBNER. Virchow's Arch. HAUSEN, ARMAUER. Virchow's Arch., Bd. 90. 1883 DAMSCH. Uebertragungsversuche von Lepra auf Thiere. Virch. Arch., Bd. 92. Centralbl. f. d. Med. Wissensch., Bd. 2 I . KAPOSI. Wiener Med. Woch. HlLLlS. Trans. Path. Soc. BABES. Etude Comparative des Bact^ries de la Lepre et de la Tuberculose. Compt. Rend. MORETTI. II Primo Caso di Lebbra nelle Marcho Confermato dalla Presenza del Bacillus Leprae. MULLER. Deut. Archiv f. Klin. Med., Bd. 34. 1884 VlDAL. La Lepre et son Traitement. ARNING. Ueber das Vorkommen des Bacillus Leprae bei Lepra Anaesthetica s. Nervorum. Virchow's Archiv, Bd. 97. THIN. Brit. Med. Journal. 1885 ViRCHOW. Berl. Klin. Woch., Nr. 12. UNNA. Ueber Leprabacillen. Deut. Med. Woch., Nr. 32. STEVEN. Brit. Med. Journ. 1886 TOUTON. Fortsch. d. Med. UNNA. Deut. Med. Woch. NEISSER. Virchow's Archiv. UNNA u. LUTZ. Dermatologische Studien, Heft I. MELCHER u. ORTMANN. Berl. Klin. Woch. 406 APPENDIX. (XVI.) Malaria. 1879 KLEBS u. TOMMASI-CRUDELI. Arch. f. Exp. Pathol., Bd. 2. 1880 TOMMASI-CRUDELI. Der Bacillus Malariae im Erdboden von Selinunte u. Campobello. Arch, f. Exp. Pathol., Bd. 12. CUBONI AND MARCHIAFAVA. Neue Studien iib. d. Natur der Malaria. Arch. f. Exp. Pathol., Bd. 13. 1881 STERNBERG. Bull. Nat. Board of Health. Washington. CUBONI AND MARCHIAFAVA. Atti della R. Acad. dei Lincei. LAVERAN. Compt. Rend. 1882 MARCHAND. Zur Aetiologie der Malaria. Virch. Archiv, Bd. 88. ZiEHL Deutsch. Med. Woch., Nr. 48. ROSZAHEGYI. Von der Ursache des Wechsel- fiebers. Biol. Centralbl., Bd. 2. TOMMASI-CRUDELI. Die Malaria von Rom. RICHARD. Bestatigung der Laveranschen Beob. Compt. Rend., No. 8. LAVERAN. Les Parasites du Sang dans 1'Impalu- disme. Compt. Rend., No. 17. BARDELS. Gaz. des H6pit. 1883 TORELLI. La Malaria in Italia. MAUREL. L'Etiologie et la Nature du Paludisme. Ann. d'Hygiene. 1884 KOTELMANN. Der Bacillus Malariae im Alter- thum. Virchow's Arch., Bd. 97. GERHARDT. Zeitschr. f. Klin. Med., Bd. 7. SEHLEN. Fortschr. d. Med., Bd. II. LEONI. Gazetta Medica di Roma. MARCHIAFAVA AND CELLI. Atti della R. Academia dei Lincei. APPENDIX. 4O7 1884 MARIOTTI E CIARROCCHI. Lo Sperimentale, T. 54- TOMMAST-CRUDELI. La Production Naturelle de la Malaria. Conference faite a la 8. Sess. du Congr&s Intern. Med. a Copenhague. LAVERAN. Trait< des Fievres Palustres. 1885 MARCHIAFAVA u. CELLI. Neue Untersuchungen iiber die Malariainfection. Fortschr. d. Med., Bd. 3. 1886 CAMILLO GOLGI. Archivio per le Scienze Mediche. (XVII.) Malignant CEdema. 1862 DAVAINE. Bull, de 1'Acad. de Me"d. 1877 PASTEUR. Vibrion. Septique. Bull, de 1'Acad. de MeU 1881 KOCH. Mitth. aus dem Ges. Amt, I., S. 54. GAFFKY. Mitth. as. d. K. Ges. Amt. PASTEUR. Vibrion. Septique. Bull, de 1'Acad. de MeU 1882 BRIEGER u. EHRLICH. Berl. Klin. Wochenschr., Nr. 44. LEBEDEFF. Versuche iiber Desinfection bei Malig- nen Oedembacillen. Arch, de Phys. Norm, et Path. 1884 LUSTIG. Zur Kenntniss Bakteriamischer Erkran- kungen bei Pferden (Malignes Oedem). Jahresber. d. K. Thierarzneischule zu Han- nover. KlTT. Unters. iiber Malignes Oedem und Rausch- brand bei Hausthieren. Jahresber. der K. Thierarzneischule in Munchen. 1885 HESSE, W. U. R. Ueber Ziichtung der Bacillen des Malignen Oedems. Deut. Med. Woch. 4O8 APPENDIX. (XVIII.) Measles. 1882 KEATING. Phil. Med. Times. 1883 CORNIL ET BABES. Archiv de Phys. (XIX.) Ophthalmic Diseases. 1872 R6TH. Retinitis Septica. Virchow's Archiv, Bd. 55- l879 BALOGH. Sphaerobakterien in der entziindcten Hornhaut. Med. Centralbl., XIV. 1882 KAHLER. Ueber Septische Retinitis. Prager Zeitschr. f. Prakt. Heilk., Bd. I. SATTLER. Unters. iiber das Trachom. Ber. lib. d. Ophthalmologen Congress zu Heidelberg. MICHEL. Graefe's Archiv f. Augenheilkunde. 1883 SCHLEICH. Verh. des Ophthalmologen Congr. zu Heidelberg. BOCK. Ueber die Miliare Tuberkulose der Uvea. Virchow's Arch., Bd. 91. KUSCHBERT U. NEISSER. Zur Pathologic und Aetiologie der Xerosis Conjunctivas. Bresl. Aerztl. Zeitschr., Nr. 4. SATTLER. Ueber d. Natur der Jequirity-Ophthal- mie. Zehender's Klin. Monatsblatt ; and Wien. Med. Woch., Nr. 1 7. SATTLER ET DE WECKER. L'Ophthalmie Jequi- ritique. CORNIL ET BERLIOZ. Sur 1'Empoisonnement par le Je'quirity. Compt. Rend, de 1'Acad. d. Sc. 1884 v - REUSS. Pilzconcretionen i. d. Thranenrohrchen. Wien. Med. Presse. GOLDZIEHER. Streptothrix Fcersteri im unteren Thranenrohrchen. Centralblatt f. Prakt. Augen- heilk. APPENDIX. 409 1884 KRONER. Zur Aetiologie der Ophthalmoblen- rrhce. Verb. d. Naturforscher-Vers. Magdeburg. KUSCHBERT. Deutsch. Med. Woch., Nr. 21. VOSSIUS. Berl. Klin. Wochenschr., Nr. I 7. KLEIN. Centralbl f. d. Med. Wiss., Nr. 8. VENNEMANN ET BRUYLANTS. Le J^quirity et son Principe Pathogene. NEISSER. Fortschr. d. Med., Bd. 2, S. 73. SALOMONSEN. Fortschr. d. Med., Bd. 2, S. 78. 1885 WlUMARK. Hygeia. Stockholm. DEUTSCHMANN. v. Graefe's Archiv, Bd. XXXI. 1886 GlFFORD. Archiv f. Augenheilkunde. v. ZEHENDER. Bowman Lecture. Lancet (XX.) Osteomyelitis. 1873 COLLMANN. Bakterien im Organismus eines an einer Verletzung am Oberschenkel verstorbenen Madchens. Gottingen. 1875 EBERTH. Virchow's Arch., Bd. 65. 1876 FRIEDMANN. Fall von Primarer Infectioser Osteomyelitis. Berl. Klin. Woch. SCHiiLLER. Zur Kenntniss der Mikrokokken bei Acuter Infectioser Osteomyelitis. Mikrokok- kenherde im Gelenkknorpel. Centralbl. f. Chirurgie, Nr. 12. 1882 FEHLEISEN. Phys. Med. Ges. Wiirzburg. 1883 BECKER. Vorl. Mitth. iiber den die Acute Infec- tib'se Osteomyelitis erzeugenden Mikroorganis- mus. Deut. Med. Woch., and Berl. Klin. Woch. 1884 PEYROUD. Compt. Rend. KRAUSE. Ueber einen bei der Acuten Infectiosen Osteomyelitis vorkommenden Mikrokokkus. Fortschr. d. Med., Bd. 2. ROSENBACH. Vorl. Mitth. iiber die die Acute Osteomyelitis beim Menschen erzeugenden Mikroorganismen. Centralbl. f. Chirurgie. 410 APPENDIX. 1884 RODET. Compt. Rendus, T. 99. 1885 GARRE. Fortschr. d. Med. (XXI.) Pleuropneumonia. 1879 SUSSDORFF. Ueber d. Lungenseuche des Rindes. Deutsche Zeitschr. f. Thiermed. u. Vergl. Pathol. 1880 BRUYLANTS ET VERRIERS. Bull, de 1'Acad. Belg. 1882 PASTEUR. NotesurlaPeripneumonie Contagieuse des Betes a Cornes. Recueil de MeU Ve't. 1883 CORNIL ET BABES. Arch, de Physiol. Norm, et Path., T. 2. 1884 POELS U. NOLEN. Centralbl. f. d. Med. Wiss.,Nr. 9. MAYRWIESER. Ueber Infectiosen Bronchialcroup bei Rindern. Woch. f. Thierheilk. u. Viehzucht, 19. 1886 POELS u. NOLEN. Das Contagium der Lungen- seuche. Fortsch. d. Med. POELS. Septische Pleuro-pneumonia der Kalber. Fortsch. d. Med. (XXII.) Pneumonia. 1878 KiJHN. Die Contagiose Pneumonic. Deutsch. Arch. f. Klin. Med. 1880 BRUYLANT ET VERRIERS. Bull, de 1'Acad. Beige. 1881 KiiHN. Die Uebertragbarkeit Endemischer Pneu- monieformen auf Kaninchen. Berl. Klin. Wochenschr., Nr. 38. 1882 FRIEDLANDER. Virchow's Arch., Bd. 87. 1883 SALVIOLI u. ZASLEIN. Ueber den Micrococcus und die Pathogenese der Crouposen Pneumonic. Centralbl. f. d. Med. Wissensch. MATRAY. Wien. Med. Presse, Nr. 23. APPENDIX. 4 1 I 1883 ZiEHL. Ueber das Vorkommen der Pneumonie- kokken im Pneumonischen Sputum. Centralbl. f. d. Med. Wissensch. FRIEDLANDER. Fortschr. d. Med., Bd. I. TALAMON. Progr. Medic. GILES. Brit. Med. J., Vol. II. FRIEDLANDER u. FROBENIUS. Berl. Klin. Woch. GRIFFINI AND CAMBRIA. Centralbl. f. d. Med. Wiss. 1884 FRIEDLANDER. Fortschr. d. Med., Bd. II. ZIEHL. Ueber das Vorkommen der Pneumonie- kokken im Pneumonischen Sputum. Centralbl. f. d. Med. Wissensch. KLEIN. Centralbl. f. d. Med. Wissensch. NAUWERCK. Ueber Morbus Brightii bei Crouposer Pneumonic. Beitr. zur Pathol. Anat. von Ziegler. JiiRGENSEN. Bed Klin. Wochenschr., Bd. 22. MENDELSSOHN. Zeitschr. f. Klin. Med., Bd. 7. PLATONOW. Ueber die Diagnostische Bedeutung d. Pneumoniekokken. Inaug.-Diss. Wiirzburg. KORANYI U. BABES. Pester Med. Chirurg. Presse. A. FRANKEL. Verhandl. d. Congr. f. Innere Med., Fortschr. d. Med. Nov. GERMATN-StfE. Compt. Rend. Acad. de Sc. Paris. AFANASSIEW. Compt. Rend. Soc. de Biol. Paris. T. 5. SALVIOLI ET ZASLEIN. Arch, pour les Sc. Med., T. 8. MAGUIRE. Brit, Med. Journ., Vol. II. 1885 DRESCHFELD. Ueber Wanderpneumonie u. ihre Beziehung zur Epidemischen Pneumonic. Fortschr. d. Med., Bd. 3, 389. SCHOU. Untersuchungen iiber Vaguspneumonie Fortschr. der Mcdicin, Bd. 3, Nr. 15. RlBBERT. Zur Farbung d. Pneumoniekokken. Deut. Med. Wochenschr., Nr. 9. 4 1 2 APPENDIX. 1885 GERMAIN-SE"E. Des Maladies Spe'cifiques du Poumon. DE BLASI. Rivista Internaz. di. Med. e. Chir. PAWLOWSKY. Berl. Klin. Woch. PLATANOW. Mitth. a. d. Wiirzburg. Med. Klinik. STERNBERG. Amer. Journ. Med. Sciences. PETERLEIN. Bericht ii. d. Vet-Wesen. i. K. Sach- sen. FRIEDLANDER. Beste Farbung zur Darstellung der Kapseln der Pneumoniekokken. Fortschritt, Bd. 3, 92. 1886 STERNBERG. Micrococcus Pasteuri. Journ. Royal Microscop. Soc. NEUMANN. Berl. Klin. Woch. PIPPING. Fortsch. d. Med. Nos. 10 and 14. FRANKEL. Zeitschr. f. Klin. Med. Bd. X. and XI. FRANKEL. Deut. Med. Woch. FOA u. BORDONI-UFFREDUZZI. Deut. Med. Woch. WECHSELBAUM.- Wien. Med. Woch. MANFREDI. Fortsch. d. Med. JACCOUD. La France Medicale. LANCEREAUX ET BESAN^ON. Archiv. Gen. de Med. THQST. Deut. Med. Woch. '(XXIII.) Puerperal Fever. 1865 MAYRHOFER. Vibrionen als Krankheitsursache des Puerperalfiebers. Monatsschr. f. Geburtsk. u. Frauenkrankheiten, Bd. 25. 1872 WALDEYER. Ueber Vorkommen von Bakterien bei der Diphtheritischen Form des Puerperal- fiebers. Arch. f. Gynakologie, III. 1873 HEIBERG. Die Puerperalen und Pyamischen Processe. ORTH. Virchow's Arch., Bd. 58. APPENDIX. 4 1 3 1873 RECKLINGHAUSEN u. LANKOWSKI. Ueber Ery- sipelas. Virchow's Arch., Bd. 60. 1879 LIFTER. Gehirnerweichungsherde durch Mikro- kokkenembolieen bei Puerperaler Pyamie. Bresl. Ae'rztl. Ztg. 1880 DOLERIS. La Fievre Puerperale et les Organismes Infect. PASTEUR. Bull, de 1'Acad. de Med., T. 9. 1881 KAREWSKI. Exper. Unters. iiber die Einwirkung Puerperaler Secrete auf den Thierischen Organismus. Zeitschr. f. Geburtsh. u. Gyna- kologie, Bd. 7. 1884 AUFRECHT. Die Exper. Erzeugung der Endo- metritis Diphtherica Puerperalis. Naturforsch. Versamml. (XXIV.) Pyaemia, Septicaemia, and Suppuration. 1866 RINDFLEISCH. Lehrb. der Pathol. Gewebelehre. I. Aufl., S. 204. 1867 WALDEYER. Zur Pathol. Anatomic der Wund- infectionskrankheiten. Virchow's Arch., Bd. 40. LISTER. Lancet. 1870 AlNSTiE. Lancet. 1871 TlEGEL. Ueber d. Fiebererregenden Eigen- schaften des Microsporon Septicum. Bern. Diss. WALDEYER. Mikrokokkencolonien in den Paren- chymatischen Organen. Vortrag i. d. Med. Ges. zu Breslau. 4 Aug. 1872 BURDON-SANDERSON. Trans. Path. Soc. 1873 BlRCH-HlRSCHFELD. Untersuchungen iiber Py- amie. HEIBERG. Die Puerperalen u. Pyamischen Pro- cesse. 414 APPENDIX. 1874 TlEGEL. Virchow's Archiv, Bd. 60. 1875 BARTHOLD. Pyaemisch-Metast. Dissert. Berlin. BURDON-SANDERSON. Brit. Med. Journ. 1877 BALFOUR. Edinb. Med. Journ. LISTER. Med. Times and Gazette. 1878 DOWDESWELL. Quart Journ. Micr. Sc. London, Vol. 1 8. KOCH. Wundinfectionskrankheiten. Leipzig. BASTIAN. Brit. Med. Journ. 1880 FERRET. De la Septicemie. Paris. BECK. Rep. Loc. Govt. Board. DRYSDALE. Pyrexia. 1881 KOCH. Mittheil. d. Kais. Ges. Amts, Bd. I. OERTEL. Zur Aetiologie der Infectionskrank- heiten. OGSTON. Brit. Med. Journ., Vol. I. BtfCHAMP. Compt. Rend. BECHAMP. Trans. Internat. Med. Cong. London. HORSLEY. Rep. Med. Officer Loc. Govt. Board. LISTER. Quart. Journ. Microscop. Science. 1882 OGSTON. Journ. of Anat. and Phys., Vol. 1 7. BRAIDWOOD AND VACHER. Brit. Med. Journ. STERNBERG. Amer. Journ. Med. Sc., John Hop- kins' Univ. Stud. Biol. Lab. 1883 DOWDESWELL. Proc. Roy. Soc. London, Vol. 34. BABES. Compt. Rend. DRESCHFELD. Brit Med. Journ. SUTTON. Trans. Path. Soc. 1884 ROSENBACH. Mikroorganismen bei den Wund- infectionskrankheiten des Menschen. Wies- baden. ARLOING. Recherches sur les Septice*mies. WATSON-CHEYNE. Trans. Path. Soc., XXXV. STEVEN. Glasgow Med. Journal. 1885 PASSET. Ueber Mikroorganismen der Eitrigen Zellgewebsentziindung des Menschen. Fort schritte d. Med., Nr. 2. APPENDIX. 4 I 5 1885 GARRE. Zur Aetiologie acut Eitriger Entziin- dungen. Fortschritte d. Med., 165. PASSET. Fortschritte d. Medicin, Bd. 3. KLEMPERER. Zeitschr. f, Klin. Med. HOFFA. Fortsch. d. Med. (XXV.) Relapsing Fever. 1873 OBERMEIER. Vorkommen Feinster, Eigene Bewegung zeigender Faden im Blute von Recurrenskranken. Med. Centralbl., 1 1. Berl. Med. Ges. Berl. Klin. Wochenschr. ENGEL. Ueber die Obermeier'schen Recurrens- spirillen. Berl. Klin. Woch. 1875 LAPTSCHINSKY. Centralbl. f. d. Med. Wissensch., Bd. 13. 1876 WEIGERT. Deut. Med. Woch. HEYDENREICH. St. Petersb. Med. Woch. MANASSEM. S. Petersb. Med. Woch., No. 18. 1877 HEYDENREICH. Der Parasit des Riickfalltyphus. 1879 CARTER. Lancet. ALBRECHT. St. Petersb. Med. Woch. CORN. Deut. Med. Woch. KOCH. Deut. Med. Woch. 1880 GUTTMANN. Virchow's Arch. MtiHLHAUSER. Virchow's Arch., Bd. 97. JAKSCH. Wien. Med. Wochenschr. Juli. 1885 GiJNTHER. Fortschr. d. Med. (XXVI.) Rhinoscleroma. 1885 CORNIL. B. de la Soc. Anatom. i 5 Fev. CORNIL ET ALVAREZ. Sur les Micro-organismes du Rhinoscle"rome. Acad. de Me*d. et Archiv. de Phys. Norm, et Path. 1886 PALTAUF u. EISELSBERG. Fortsch. d. Med. DAVIES. Brit. Med. Journ. 416 APPENDIX. (XXVII.) Scarlatina. 1872 COZE ET FELTZ. Les Maladies Infectieuses. MCKENDRICK. Brit. Med. Journal. 1876 KLEIN. Report of the Medical Officer of the Privy Council. 1882 HAHN. Berl. Klin. Woch, No. 38. 1883 ROTH. Miinchener Aerztl. Intelligenzbl. POHL-PiNCUS. Centralbl. f. d. Med. Wiss., No. 36. CROOKE. Lancet. 1884 HEUBNER u. BAHRDT. Zur Kenntniss der Gelenkeiterungen bei Scharlach. Berl. Klin. Woch., Nr. 44. 1885 CROOKE. Fortsch. d. Med. 1886 LAURE. Lyon Medical. KLEIN. Nature, XXXIV. (XXVIII.) Swine-Erysipelas. 1881 SALMON. Report Depart. Agricul. Washington. DETMERS. Science. 1882 PASTEUR. Sur le Rouget ou Mai Rouge des Pores. Compt. Rend., T. 95. 1883 CORNIL ET BABES. Arch, de Physiol. PASTEUR ET THUILLIER. Bull, de 1'Acad. de Med. de Paris. Compt. Rend., T. 97. EGGELING. Ueber den Rothlauf der Schweine. Fortschr. d. Med. 1884 SALMON. Rep. Dept. Agricult. Washington. 1885 Lo'FFLER. Experimented Untersuchungen iiber Schweinerothlauf. Arbeiten aus dem Kaiserl. Gesundheits Amt, Bd. I. SCHUTZ. Ueber den Rothlauf der Schweine und die Impfung desselben. Ibid. LYDTIN u. SCHOTTELIUS. Der Rothlauf der Schweine. Wiesbaden. APPENDIX. 4 I 7 (XXIX.) Swine-Typhoid. 1879 KLEIN - Re P- f the Med - ffic - of the Council [1877-1878]. 1884 KLEIN. Virchow's Archiv. (XXX.) Symptomatic Anthrax. 1878 BOLLINGER U. FESER. Wochenschr. f. Thierheil- kunde. 1880 ARLOING, CORNEVING ET THOMAS. Compt. Rend. 1881 ARLOING, CORNEVING ET THOMAS. Bull, de 1'Acad. de Med. and Revue de Med. 1883 ARLOING, CORNEVING ET THOMAS. Du Charbon Bacterien, Charbon Symptomatique, etc. 1884 KiTT. Unters. iiber Malignes Oedem und Rausch- brand bei Hausthieren. Jahresber. der K. Thierarzneisch. in Miinchen. BABES. Journ. de 1'Anatomie. NEELSEN u. EHLERS. Ueber den Rauschbrand. Ber. d. Naturforsch. Ges. zu Rostock. EHLERS. Unters. lib. d. Rauschbrandpilz. Inaug. Diss. Rostock. ARLOING, CORNEVING ET THOMAS. Revue de Med. 1886 ARLOING, CORNEVING ET THOMAS. Chabert's Disease. Transl. by Dawson Williams in Microparasites and Disease. (New Syd. Soc.) HESS. Bericht iiber die entschadigten Rausch- brand u. Milzbrandfalle im Canton Bern. STREBEL. Schweiz. Archiv f. Thierheilk. 4 1 8 APPENDIX. (XXXI.) Syphilis. 1872 LOSTORFER. Arch. f. Dermat. u. Syph. 1879 KLEBS. Arch. f. Exp. Pathol., Bd. I o. 1881 AUFRECHT. Centralbl. f. d. Med. Wissensch., Bd. 19. 1882 BlRCH-HlRSCHFELD. Bakterien in Syphilitischen Neubildungen. Centralbl. f. d. Med. Wissen- sch., Nrs. 33, 34. PESCHEL. Centralbl. f. Augenheilk. MARTINEAU ET HAMONIC. De la Bacteridie Syphilitique. Compt. Rend., p. 443. MORISON. Maryland Med. Journ. 1883 LETNIK. Wien. Med. Wochenschr. MORISON. Maryland Med. Journ. Baltimore. MORISON. Wiener Med. Wochenschr. HEYDEN. Preservation de la Syphilis, etc. Traduit par Roberts. 1884 PETRONE. Gaz. Medica Ital. TORNEY ET MARCUS. Compt. Rend., p. 472. LUSTGARTEN. Wien. Med. Woch., Nr. 47. K6NIGER. Deut. Med. Woch., S. 816. 1885 LUSTGARTEN. Die Syphilisbacillen. DOUTRELEPONT U. SCHUTZ. Deut. Med. Woch., Nr. 19. ALVAREZ ET TAVEL. Bull, de 1'Acad. de Med. et Archiv. de Phys. Norm, et Path. DE GlACOMl. Neue Farbungsmethode der Syphi- lisbacillen. Correspondenzbl. f. Schweizer Aerzte, Bd. 15. GOTTSTEIN. Fortschr. d. Med., Bd. 3, S. 543. KLEMPERER. Deut. Med. Woch. 1886 EVE and LINGARD. Brit. Med. Journ. BlENSTOCK. Fortsch. d. Med. KASSOWITZ u. HOCHSINGER. Wien. Med. Blatter. DISSE u. TAGUCHI. Deut. Med. Woch. APPENDIX. 4 1 9 (XXXII.) Tetanus. 1884 CARLE E RATTONE. Studio Sperimentale sulP Etiologia del Tetano. Giorn. della R. Acad. di Medicina di Torino. VOGEL. Drei Falle von Infectiosem Tetanus. Deut. Med. Woch., Nr. 31. NiGOLAIER. Deut. Med. Woch., Nr. 52. 1886 ROSENBACH. Archiv. f. Klin. Chir. (XXXIII.) Tuberculosis. 1868 VILLEMIN. Etude sur la Tuberculose. KLEBS. Virchow's Arch., Bd. 44. 1873 KLEBS. Arch. f. Exp. Pathol. u. Pharmakol., Bd I. 1877 COHNHEIM. Uebertragbarkeit der Tuberculose. Berlin. 1879 BAUMGARTEN. Berl. Klin. Woch., Bd. 17. 1881 TOUSSAINT. Compt. Rend., T. 93. BAUMGARTEN. Centralbl. f. d. Med. Wiss. Berlin. Bd. 19. 1882 BAUMGARTEN. CentralbL f. d. Med. Wiss. Berlin. Bd. 20. BAUMGARTEN. Deut. Med. Woch., Bd. 8. WAHL. Zur Tuberculosefrage. Deut. Med, Woch., Nr. 46. VAN ERMENGEM. Le Microbe de la Tuberculose. Ann. de la Soc. Beige de Microscopic. HlLLER. Deut. Med. Woch., Bd. 8. BALOGH. Wien. Med, Woch. KOCH. Die Aetiologie der Tuberculose. Berl. Klin. Woch. FORMAD. The Bacillus Tuberculosis. The Philad. Medical Times. GREEN. Lancet. 42O APPENDIX. 1882 CREIGHTON. Trans. Path. Soc. RlNDFLEiSCH. Phys. Med.Ges. zu Wiirzburg, Nr. 8. E WART. Lancet. EHRLICH. Deut. Med. Woch. FRANTZEL u. PALMERS. Bed. Klin. Woch. AUFRECHT. Centralbl. fur d. Med. Wiss. 1883 SCHUCHARDT U. KRAUSE. Fortschr. d. Med. KLEBS. Arch. f. ,Exp. Pathol. u. Pharmakol., Bd. 17. EHRLICH. Deut. Med. Woch., Bd. 9. FRANTZEL u. PALMERS. Berl. Klin. Woch. DE GlACOMI. Fortschr. d. Med., Bd. I, S. 145. MEISELS. Wien. Med. Woch. RAYMOND. Arch. Gen. de Med., T. 1 1. DEMME. Jahresber. d. Jennerschen Kinderspitals. Bern. LYDTIN. Badische Thierarztl. Mittheil. RlBBERT. Ueber d. Verbreitungsweise der Tuber- kelbacillen bei Hiihnern. Deut. Med. Woch., Nr. 28. WECHSELBAUM. Wien. Med. Jahrb. JOHNE. Ber. lib. d. Veterinarwesen im Konigr. Sachsen. BOLLINGER. Munch. Aerztl. IntelligenzbL, Nr, 1 6. JOHNE. Die Geschichte der Tuberculose. PtiTZ. Ueber die Beziehungen der Tuberculose des Menschen zu der der Thiere. CELLI and GUARNERI. Arch, pour les Sciences Mtklic. WEIGERT. Deut. Med. Woch. Nr. 2$tt. NAUWERCK. Deut. Med. Woch. SMITH. Bristol. Med. Chir. Journ. WILLIAMS. Lancet. VERAGUTH. Arch. f. Exp. Path. u. Pharmakol., Bd. 1 6. SCHOTTELIUS. Virchow's Archiv, Bd. 91. APPENDIX. 421 1883 BROUILLY. Note sur la Presence des Bacilles dans les Lesions Chirurgicales Tuberculeuses. Rev. de Chin, T. 3. SCHLEGTENDAL. Fortschr. d. Med., Bd. I. KiJSSNER. Beitr. z. Impftuberculose. Deut. Med. Woch., Nr. 36. AUFRECHT. Centralbl. f. d. Med. Wiss., Bd. 21. BOLLINGER. Centralbl. f. d. Med. Wiss., Bd. 21, S. 600. CHEYNE. Brit. Med. Journ., Vol. I. CHEYNE. Practitioner. London. Vol. XXX. BOCK. Virchow's Archiv, Bd. 91. DETTWEILER. Berl. Klin. Woch., Bd. 21. DAMSCH. Deut. Med. Woch., Nr. 17. BABES. Der erste Nachweis des Tuberkelbacillus im Harn. Centralbl. f. d. Med. Wissensch. BABES. Compt. Rendus. ROSENSTEIN. Centralbl. f. d. Med. Wiss. LEVINSKY. Deut. Med. Woch., Bd. 9. LICHTHEIM. Fortschr. d. Med., Bd. I. WEST. Lancet, Vol. I. WEST. Trans. Path. Soc. ZIEHL. Bedeutung der Tuberkelbacillen fur Diagnose und Prognose. Deut. Med. Woch., Nr. 5. DlEULAFOY ET KRISHABER. Arch, de Physiol. Norm, et Path., T. I. MALASSEZ ET VIGNAL. Ibid., T. II. Compt. Rend., T. 97. KOCH. Kritische Besprechung der gegen die Bedeutung der Tuberkelbacillen gerichteten Publicationen. Deut. Med. Woch., Nr. 10. VIGNAL. Compt. Rend. Soc. de Biol., T. 5. GlBBES. Lancet. KUNDRAT. Wien. Med. Presse. LANDOUZY ET MARTIN. Rev. de Med., T. 3. CHIARI. Wien. Med. Presse. 422 APPENDIX. 1883 COCHET. Compt. Rend. Soc. de Biol., Paris, T. 5. PFEIFFER. Berlin. Klin. Woch., Bd. 21. LEUBE. Sitzungsber. der Phys.-Med. Soc. zu Erlangen. SPINA. Studien iiber Tuberculose. Wien. CRAMER. Sitzungsber. d. Phys. Med. Soc. zu Erlangen. MARCHAND. Deut. Med. Woch., Nr. 15. HERON. Lancet. GREEN. Brit. Med. Journ. BAUMGARTEN. Centralbl. f. d. Med. Wissensch. Berlin. Bd. 2T. BAUMGARTEN. Zeitschr. f. Klin. Med., Bd. 6. SOMARI E BRUGNATELLI. Redii R. Instit. Lombardo. 1884 MALASSEZ ET VIGNAL. Sur le Microorg. de la Tuberculose Zoogloeique. Compt. Rend., T. 99, p. 200. DOUTRELEPONT. Die Aetiologie des Lupus Vul- garis. Vierteljahrschr. f. Dermatologie u. Syphilis. CORNIL ET LELOIR. -- Recherches, etc., sur la Nature du Lupus. Arch, de Physiol. Norm. et Pathol. BABES ET CORNIL. Note sur les Bacilles de la Tuberculose. Journ. de 1'Anat. et de la Physiol. Norm, et Pathol. BOULEY. La Nature Vivante de la Contagion, Contagiositd de la Tuberculose. Paris. DjRINE. Rev. de. Med. Paris. T. 4. ARLOING. Compt. Rend. BlEDERT. Virchow's Archiv, Bd. 98. LEYDEN. - - Klinisches iiber Tuberkelbacillen. Zeitschr. f. Klin. Med, VIII. MtiLLER. Ueber den Befund von Tuberkelbacillen bei Fungosen Knochen u. Gelenkaffectionen. Centralbl. f. Chir., 3. ALBRECHT. Arch. f. Kinderheilk., Bd. 5. APPENDIX. 423 1884 SCHILL u. FISCHER. Mitth. a. d. Kaiserl. Ges. Amt, Bd. II. LUSTIG. Ueber Tuberkelbacillen im Blut bei an Allg. Acuter Miliartub. Erkrankten. Wien. Med. Woch, Nr. 48. VOLTOLINI. Ueber Tuberkelbacillen im Ohr. Deut. Med. Woch., Nr. 31. STRASSMANN. Virchow's Archiv, Bd. 96. KOCH. Mittheilungen aus dem Kais. Ges. Amt, Bd. II. BAUMGARTEN. Centralbl. f. d. Med. Wissensch. Berlin. Bd. 22. GAFFKY. Verhalten der Tuberkelbacillen im Spu- tum. Mitth. a. d. Kaiserl. Gesundheitsamt, Bd.II. NEGRI. Colorations des Spores dans les Bacilles de la Tuberculose. Journ. de Microsc., T. 8. SUTTON. Trans. Path. Soc. London, Vol. XXXV. ANDREW. Lancet. WILLIAMS. Journ. Royal Microscop. Soc. 1885 DOUTRELEPONT. Fall von Meningitis Tuber- culosa nach Lupus, Tuberkelbacillen im Blut. Deut. Med. Woch., Nr. 7. KARG. Centralbl. f. Chir. SPINA. Casopis Lekaru Ceskych, Nr. 4. OBRZUT. Prof. Spina's Neue Farbungsmethode der Faulnissmikroorganismen und ihre Bezie- hung zu den Tuberkelbacillen. Deut. Med. Woch., Nr. 12. CREIGHTON. Brit. Med. Journ. CREIGHTON. Lancet. FtiTTERER. Ueber das Vorkommen u. die Ver- theilung der Tuberkelbacillen in den Organen. Virch. Arch., Bd. 100, Heft 2. JOHNE. Ein Zweifelh. Fall von Congenitaler Tuberculose. Fortschr. d. Med., Bd. 3, 198. BlEDERT. Berl. Klin. Woch. RlBBERT. Deut. Med. Woch. 424 APPENDIX. 1885 HARRIS. St. Barthol. Hosp. Reports. STICKER. Centralbl. f. Klin. Med. WESENER. Fiitterungstuberculose. TSCHERNING. Inoculationstuberculose beim Men- schen. Fortschr. d. Med., Bd. 3, 65. 1886 MlDDELDORPF. Fortsch. d. Med. MULLER. Centralbl. fiir Chin GoLDENBLUM. Vrach., Nos. I. and XL BLACK. Lancet. BAUMGARTEN. Zeitschrift. f. Klin. Med. KIRSTEIN. Deut. Med. Woch. MAX BENDER. Ueber die Beziehungen des Lupus Vulg. zu Tuberc. Deut. Med. Woch. HARRIES AND CAMPBELL. Lupus. London. (XXXIV.) Typhoid Fever. 1874 BlRCH-HlRSCHFELD. Unters. zur Pathologic des Typhus Abdominalis. Zeitschr. f. Epidemio- logie, I. KLEIN. Med. Centralbl., XII. 1876 LETZERICH. Virchow's Archiv; Bd. 68. 1877 FELTZ. Compt. Rend., T. 85. 1878 LETZERICH. Archiv f. Exper. PathoL, Bd. 9. l88o EPPINGER. Beitr. zur Pathol. Anatomic aus d. Patholog. Institut Prag. FlSCHEL. Beitr. zur Pathol. Anat. aus d. Pathol.- Anat. Inst. Prag. 1880 TizzoNI. Studi. di Pat. Sperim. sulla Gen. d. Tifo. Milano. KLEBS. Arch. f. Exper. Pathol. u. Pharmakol. EBERTH. Arch. f. Pathol. Anat, Bd. 81. 1881 LETZERICH. Archiv f. Exper. Pathol., Bd. 10. BRAUTLECHT. Pathogene Bakterien im Trink- wasser bei Epidemieen. Virchow's Arch., Bd. 84. APPENDIX. 425 1881 MEYER. Unters. iiber den Bacillus des Abdomi- naltyphus. Inaug. Diss. RAPPIN. Contrib. a 1'Etude des Bact. de la Bouche, a 1'Etat Normal et dans la Fievre Typhoi'de. 1882 COATS. Eberth's Typhoid Bacillus. Brit. Med. Journ. WERNICH. Zeitschr. f. Klin. Med., Bd. 6. MARAGHANO. Centralbl. f. d. Med. Wiss., Bd. 15. ALMQUIST. Typhoidfeberus-Bakterie. CROOKE. Brit. Med. Journ. 1883 BOENS. Acad Roy. de Med. de Belgique. Bull., 3 Ser, T. 17. EBERTH. Der Typhus- Bacillus und die Inte- stinelle Infection. Volkmann, Klin. Vortrage. LETZERICH. Experimentelle Untersuchungen iiber die Aetiologie des Typhus Abdominalis. Leip- zig. 1884 GAFFKY. Zur Aetiologie des Addominaltyphus. Mitth. a. d. Ges. Amt, Bd. II. TAYCN. Le Microbe de la Fievre Typhoi'de de 1'Homme. Compt. Rend.yT. 99, p. 331. 1885 PFEIFFER. Ueber den Nachweis der Typhus- bacillen im Darminhalt u. Stuhlgang. Deut. Med. Woch, Nr. 29, 1886 MICHAEL, Typhusbacillen im Trinkwasser. Fortsch. d. Med MEISELS. Wien. Med. Woch, SEITZ. Bakteriolog. Studien z. Typhusatiologie, LUCATELLO. BolleL d. R. Accad. Med. di Genova. FRAENKEL u, SIMMONDS. Die Aetiologische Bedeutung des Typhus-Bacillus. NEUHAUSS. BerL Klin. Woch. SlROTINlN. Zeitschr. f. Hygiene. BEUMER AND PEIPER. Zeitschr. f. Hygiene. 426 APPENDIX. (XXXV.) Variola and Vaccinia. 1868 CHAUVEAU. Nature du Virus-Vaccin. Compt. Rend. 1871 WEIGERT. Ueber Bakterien in der Pockenhaut. 1872 COHN. Virchow's Archiv, Bd. 55. ZtJLZER. Berl. Klin. Wochenschr. 1873 LUGINBUHL. Der Micrococcus der Variola. Verhdl. d. Phys. Med. Ges. in Wiirzburg. 1874 WEIGERT. Anat. Beitr. z. Lehre v. d. Pocken. PlSSIN. Berl. Klin. Wochenschr. STROP?. Vaccination u. Mikrokokken. KLEBS. Arch. f. Exp. Path. u. Pharmakol., Bd. i o. 1880 ISCHAMER. Ueber das Wesen des Contag. der Variola Vaccine u. Varicella. Aerztl. Verein.. Steiermark. 1881 TAPPE. Aetiologie u. Histologie der Schafpocken. 1882 POHL-PlNCUS. Vaccination. PFEIFFER. Ueber die Riickimpfung auf Kiihe und Kalber. Jahrb. f. Kinderheilk. MARCHAND. Les Virus-Vaccins. Revue Myco- logique. STRAUSS. Vaccinal Micrococci. Soc. de Biol. de Paris. 1883 WOLF. Zur ImpfTrage. BerL Klin. W T ochenschr., Nr. 4. PLAUT. Das Organis. Contagium der Schafpocken. CORNIL ET BABES. Soc. Medicale des Hopitaux. QUIST. St. Petersburg Medic. Woch., Nr. 46. 1884 HAMERINK. Ueber die sog. Vaccination u. Variola. (XXXVI.) Yellow Fever 1883 BABES. Sur les Microbes trouves dans le Foie et dans le Rein d'Individus morts de la Fievre Jaune. Compt. Rend., 17 Sept. APPENDIX. 427 1884 DOMINGOS FREIRE ET REBOURGEON. Compt. Rend., T. 99, p. 804. DOMINGOS FREIRE. Recherches sur la Cause de la Fievre Jaune. 1885 BOULEY. L'Inoculation Preventive de la Fievre Jaune. Compt. Rend., T., 100, p. 1276. CARMONA Y VALLE. Legons sur 1'Etiol. et la Pro- phylax. de la Fievre Jaune. 1886 CERECEDO. El Siglo Medico. (K.) BACTERIA IN THE AIR, IN SOIL, AND IN WA TER. 1858 POUCHET. Compt. Rend,, T. 47. 1860 PASTEUR. Compt. Rend., T. 50. 1861 PASTEUR. Compt. Rend., T. 52. 1862 PASTEUR. Ann. de Chim. et de Phys., T. 64. 1863 PASTEUR. Compt. Rend., T. 56. LEMAIRE. Compt. Rend., T. 57. 1870 TYNDALL. Med. Tim. and Gaz. MADDOX. Month. Microscop. Journal. 1874 CUNNINGHAM. Micr. Exam, of the Air. Calcutta. TISSANDIER. Comp. Rend., T. 78. 1877 PASTEUR. Compt. Rend., T. 85. MlQUEL. Annuaire de 1'Observat. de Montsouris. TYNDALL. Brit. Med. Journ. 1878 MlQUEL. Compt. Rend., T. 86. MlQUEL. De la Presence dans 1'Air du Ferment de 1'Uree. Bull, de la Soc. Chim. 1879 MlFLET, Unters. u'ber die in der Luft Suspen- dirten Bakterien. Cohn's Beitr. z. Biol. d. Pflanzen, Bd. III. WERNICH. Cohn's Beitrage z. Biol. d. Pflanzen, Bd. III., S. 105. KLEBS u. TOMMASI-CRUDELI. Unters. der Luft auf die Mikroorganismen der Malaria. Archiv f. Exper. Path., Bd. IT. 4 2 S APPENDIX. 1879 MlQUEL. Ann. d'Hygiene. 1881 KOCH. Milzbrandbacillen im Boden. Mitth. a. d. Ges. Amt, Bd. I. SOYKA. Sitz.-Ber der K. Bayr. Akad. d. Wiss. Math. Physik. Classe. SOYKA. Vortrage im Aerztl. Verein in Miinchen. BtiCHNER. Die Bedingungen des Uebergangs von Pilzen in die Luft. Vortrage im Aerztl. Verein zu Miinchen. TYNDALL. Essays on the Floating Matter of the Air. 1882 WlNNACKER. Ueberdie in Rinnsteinen beobacht. Nied. Organisrnen. Gottinger Inaug.-Diss. Frankfurt a, M. v. FODOR. Hygienische Unters. iiber Luft, Boden u, Wasser. NAGELI. Unters. iiber Niedere Pilze. SOYKA. D. Vierteljsch. f. OefT. Ges., Bd. 14. MlQUEL. Annuaire de 1'Observat. de Montsouris. 1883 WOLLNY, Ueber die Thatigkeit Niederer Orga- nismen im Boden. Viert. f. Oeff. Ges., S. 705. LETZERICH. Exp, Unters. iib. die Aetiologie des Typhus mit bes. Berucksichtigung der Trink- u. Gebrauchswasser. ZANDER. Centralbl. f. Allg. Ges. GUNNING. Beitr. z. Hygienischen Untersuchung des Wassers, Arch. f. Hyg., 3. MlQUEL. Les Organismes Vivants de 1'Atmo- sphere. OLIVIER. Les Germes de 1'Air. These. Rev. Scientif. ANGUS SMITH. Sanitary Record. SMART. Germs, Dust, and Disease. TORELLI. La Malaria in Italia. 1884 HESSE. Ueber Quantitative Best, der in der Luft enthaltenen Mikroorganismen. Mitth. a. d. Ges. Amt, Bd. II. APPENDIX. 429 1884 HESSE. Ueber Abscheidung d. Mikroorganismen aus d. Luft Deutsche Med. Wochenschr., 2. HESSE. Weitere Mitth. iiber Luftfiltration. Deut. Med. Woch., Nr. 51. SCHRAKAMP. Archiv f. Hygiene, Bd. II. MIQUEL ET FREUDENREICH. La Semaine Medicale. SEHLEN. Fortschr. d. Med., Bd. II, S. 585. CHAMBERLAND. Sur un Filtre Dormant de 1'Eau Physiologiquement Pure. Compt. Rend., T. 99, p. 247. MOREAU ET PLANTYMAUSTON. La Semaine Medicale. ANGUS SMITH. On the Examination of Waters. Rep. to the Loc. Gov. Board. 1885 CRAMER. Die Wasserversorgung von Zurich. CROOKSHANK. Notes from a Bact. Labor., Lancet. BECKER. Reichsmedicinalkalender. HESSE. Ueber Wasserfiltration. Deut Med. Woch. SOYKA. Prager Med. Woch. FRANKLAND. Removal of Micro-organism from Water. Proc. Roy. Soc. 1886 SOYKA. Fortschr. d. Med. LAURENT. Journal de .Pharmacie et de Chemie. WOLFFHUGEL AND RlEDEL. Arbeit, a. d. K. Ges. Amt. HERyEUS. Zeitschr. f. Hygiene. C. FRANKEL. Zeitschr. f. Hygiene. MEADE BOLTON. Zeitschr. f. Hygiene. HESSE. Zeitschr. f. Hygiene. BEUMER. Deut. Med. Woch. FRANKLAND. Proc. Roy. Soc. BlSGHOF. Journ. Soc. Chem. Industry. PFEIFFER. Zeitschr. f. Hygiene. 430 ?,. APPENDIX. APPENDIX G. TABLE SHOWING THE MAGNIFYING POWER ZEISS' OBJECTIVES. Ocular: 12345 a, a, a 3 a* aa A, AA B, BB C, GC D, DD E F I H J K L 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 OF a, a 2 a 3 Q* A, AA B, BB C, CO D, DD E F G H J K L A INDEX. Abbe condenser, 5 Abrin, 312 Abrus precatorius, 312 Abscesses, 197 Acetate of potash, 16 Acetic acid, 9 Achorion Schoenleinii, 347 Actinomyces, 334 method of staining, 337 Actinomycosis, 334 Acute infectious osteomyelitis, coccus of, 197 Acute yellow atrophy, 214 ^roniscopes, 364 ^roscopes, 364 Agar-Agar, 23 peptone-broth, preparation of, 21, 68 Air, examination of, 361 Aitken's test tube, 31, 93 Alcohol, 7, 9 acidulated, 9 Alopecia areata, 231 Alum carmine, 10 Ammonia, lo Aniline, 10 water, 10 Animals, examination of, in Anthrax, 281 Antiseptics, 150 Apparatus, 5 ASCOCOCCUS, 222 Billrothii, 122, 222 Ascomycetes, 347 Asiatic cholera, 255 Aspergillus albus, 349 * clavatus, 349 flavus, 347 fumigatus, 347 glaucus, 347 niger, 347 ochraceus, 349 repens, 347 Asphaltelac, 16 Attenuation of virus, 166, 234, 289 B Babes', method of examining cultiva- tions, 50 of staining comma bacilli, 257 of staining bacillus of leprosy, 267 Bacillus, 265 acidi lactici, 306 aerophilus, 315 alvei, 301 amylobacter, 318 anthracis, 281 butyricus, 318 caucasicus, 312 cavicida, 238 columbarum, 232 cuniculicida, 235 cyanogenus, 304 dysodes, 313 erythrosporus, 313 figurans, 311 Fitzianus, 306 fluorescens, 303 fcetidus, 315 Hansenii, 313 ianthinus, 303 indicus, 240 in gangrenous septicaemia, 271 in septicaemia of man, 271 in syphilis, 267 in tetanus, 301 lepros, 265 liodermos, 244 luteus, 241 malariae, 270 mallei, 292 megaterium, 310 mesentericus fuscus, 316 vulgatus, 3 1 6 multipediculus, 244 mycoides, 3 1 1 Neapolitans, 228 cedematis maligni, 293 of blue milk, 304 of choleraic diarrhoea from meat- poisoning, 270 432 INDEX. Bacillus, of diphtheria, 230 of glanders, 292 of jequirity, 312 of pneumo-enteritis of the pig, 299 of rhinoscleroma, 229 of septicaemia of mice, 297 of splenic fever, 281 of swine-erysipelas, 300 of swine-typhoid, 299 of ulcerative stomatitis in the calf, 298 oxytocus perniciosus, 238 parvus ovatus, 217 pneumonicus agilis, 237 prodigiosus, 241 pseudo-pneumonicus, 228 putrificus coli, 315 pyocyaneus, 303 pyogenes fcetidus, 27 1 ramosus liquefaciens, 244 saprogeries, No. I, 314 No. 2, 314 -No 3, 231, 314 fcetidus, 315 septicus, 314 agrigenus, 234 sputigenus, 236 subtilis, 306 ^ tremulus, 312 tuberculosis, 272 lumescens, 309 typhosns, 268 urese, 243 violaceus, 303 virens, 117 vitulorum, 231 Bacteria, chromogenic, 142 classification of, 175 distribution of, 148 general morphology of, 117 microscopical examination of, 45 pathogenic, 144 - saprogenic, 143 zymogenic, 142 Bacteriacese, 185, 224 Bacteridie du charbon, 281 Bacteridium cyaneum. 218 Bacterium, 225 aceti, 243 seruginosum, 303 brunneum, 242 cavicida, 238 chlorinum, 117 cholerse gallinarum, 232 coli commune, 238 crassum sputigenum, 237 Bacterium, decalvans, 231 fluorescens liquefaciens, 242 putidum, 242 fcetidum, 315 ftmforme, 246 hyacinthi, 239 ianthinum, 303 indicum, 240 in diphtheria of calves, 231 of man, 230 lactis aerogenes. 239 lineola, 248 liodermos. 244 litoreum. 246 luteum. 741 merismopedioides, 245 multipediculum, 244 navicula, 246 Neapolitanum, 228 of Davaine's septicaemia, 236 of diphtheria of pigeons, 232 of fowl-cholera, 232 of rhinoscleroma, 229 of septicaemia in rabbits, 235 of yellow milk, 239 oxytocum perniciosum, 238 Pasteuri, 236 Pasteurianum, 244 Pfiugeri, 246 photometricum, 246 pneumonioe cruposae, 225 pneumonicum agile, 237 prodigiosum, 241 pseudo-pneumonicum, 228 ramosum liquefaciens, 244 rubescens, 325 rubrum, 241 saprogenes, 231 septicum agrigenum, 234 sputigenum, 236 syncyanum, 303 synxanthum, 239 termo, 248 ureas. 242 violaceum, 242 viricle, 117 xanthinum, 239 Zopfii, 244 Balance and weights, 23 Balmer-Frantzel method, 279 Baumgarten's method, 278 new method, 278 Bees, bacillus in disease of, 302 Beggiatoa, 324 methods of examining species of, 324 alba, 324 mirabilLs, 324 INDEX. 433 Beggiatoa, roseo-persicina, 324 Bergamot oil, 7 Bibliography, 370 Biere malade. 207 Bismarck-brown, 10 Blackleg, 321 Black torula, 342 Bleeding host, 211 Blood, 350 rain, 241 serum, liquid, 90 sterile, 29, 86 Blue milk, bacillus of, 304 Borax carmine, 10 Botrytis Bassiana, 350 Bouillon, 89 Bread-paste, 85 Brush, 29 Bulbed tubes, 32 Butyric acid fermentation, bacillus of, 318 Camera-lucida, 17 Canada-balsam, 17 Caoutchouc caps, 25 Carbonate of soda, 24 Carious teeth, 220 Cattle plague, 204 Cedar oil, 6, II Cell-contents, 119 Cell-wall, 118 Celloidin, 7, 55 Cerebro-spinal meningitis, 203 Charbon symptomatique, 320 Chemical composition, 117 disinfectants. 153 Cheshire's trough, 71 Chionyphe Carteri, 350 Cholera, comma bacillus of, 250 fowl, 233 nostras, comma bacillus in, 258 Choleraic diarrhoea from meat poison- ing, bacillus of, 270 Cladothrix, 330 dichotoma, 330 Fcersteri, 332 Cladotrichese, 186, 330 Classification, 175 Fliigge's, 1 80 Hueppe's, 187 - Zopfs, 185 Clostridium, 318 butyricum, 318 of symptomatic anthrax, 320 polymyxa, 320 Coccaceae, 185 Cocci, methods of staining, 223 Cohn-Mayer fluid, 91 Cohnia roseo-persicina, 325 Collection of water samples, 367 Comma-bacillus, of Finkler, 254 of Koch, 250 in cholera nostras, 258 Compressed air, 140 Cork, 7 Cornil and Alvarez, method of, 230 Cotton- wool, 23 Cover-glass impressions, 52 preparations, 48 double coloration, 49 Crenothrix, 322 Kuhniana, 322 Cutaneous inoculation, 108 Cutting tissues, 53 D Damp-chambers, 26, 74 Decalcifying preparations, 54 Dental caries, 203 Desiccator, 43 Diphtheria, 202. 230 Diplococcus albicans tardissimus, 210 Disinfectants, 150 Dissecting boards, 41 case, 41 Dissection, III Distribution of bacteria, 148 Double coloration. 49 Double-stain spore-bearing bacilli, 291 Double-staining, 51 Drawing apparatus, 17 Dressing-case, 41 Drinking water, 322 Drop-cultures, 94 Drop-culture slides, 32 Ebner's solution, 7 Ehrlich's method of examining culti- vations, 50, 276 and eosin, 280 Electricity, 141 application of, 104 Embedding tissues, 55 Empusa muscae, 343 radicans, 344 Endocarditis ulcerosa, 202 Eosin, ii Erysipelas, 200 malignum, bacillus des, 300 Ether, 11 28 434 INDEX, Eurotium aspergillus glaucus, 347 aspergillus niger, 348 repens, 347 Examination, of plate cultivations, of test-tube cultivations, 71 Experiments on living animals, 107 Farrant's solution, 17 Favus, 347 Filter, making. 66 paper, 24 Fire blight, 218 Flagella, to stain, 52 Flagellated protozoa in blood, 350 Flagellum, 128 Flannel, 24 Flecksucht, 239 Folded filter, 67 Foot and mouth disease, 205 Form, 122 Foulbrood, 302 Fowl-cholera, bacterium of, 232 Frankel's method, 279 Friedlander, method of, 227 Frogspawn fungus, 262 Fuchsine, 1 1 Gangrene. 214, 272 Gas, burners, 35, 36 chambers, 103 pressure regulator, Moitessier's,37 Gases, effect of, 140 Gattine, 239 Gelatine, 8, 23 peptone broth, preparation of, 21, 64 plates, 76 Gelatinous envelope, 120 Gentian violet, 1 1 Gibbes', first method, 277 magenta solution, 13 new method, 277 solution for double-staining, 1 1 Glanders, bacillus of, 292 Glass, bells, 26 benches, 27 capsules, 31 dishes, 26, 28 jar, 29 plates, 27 rods, 27 vessels, 22 Glycerine, gelatine, 8, 57 gum, 17 Glycerine, pure, 12 Gomme de sucrerie, 262 Gonorrhoea, 208 Gram's, method of staining, 59 solution, 12 Growth, circumstances affecting, 139 products of, 141 Gum, 8 H Haematomonas carassii, 355 cobitis,353, 355 Evansi, 357 Haematoxylin solution, 12 Haemophilia neonatorum, 214 Hardening preparations, 54 Hay-bacillus, 306 methods of staining, 309 Heat regulator (Meyer's), 40 (Reichert's), 37 (Schlosing's), 34 Herpes tonsurans, 347 Hesse's apparatus. 363 His' method of examining cultivations, 51 Hollis' glue, 17 Hot air and steam, 159 Hot-air steriliser, 20 Hot-water filter, 21 Hydrophobia, 215 Hyphomycetes, 342 Hypodermii, 343 I Immunity. 162 Impression-preparations, 52 Incubators, 32 - Babes', 36 D' Arson val's, 32 Inoculating test-tubes, 69, 74 cutaneous and subcutaneous, 1 08 of potatoes, 83 protective, 163 Instruments, 41 Intermittent fever, bacillus of, 177 Iodine solution, 12 -- (Gram), 12 Iron box, 26 Isolation of micro-organisms, 114 Israel's, case, 28 warming apparatus, 101 mf Japanese isinglass, 23 Jequirity, bacillus of. 312 INDEX. 435 K Kaatzer's method, 280 Kephir, 313 Kleinenberg's solution, 8 Koch's apparatus for examination of air, 362 method for staining comma- bacilli, 257 method of plate-cultivation, 72 original method for staining the tubercle bacillus, 275 postulates. 27 solution (methylene-blue), 13 solution (methyl-violet), 14 Laboratory requisites, 42 Lactic acid, 24 Leprosy, 266 Leptothrix, 329 buccalis, 329 gigantea, 330 ochracea, 331 parasitica, 331 Leptotricheas, 185, 321 Leuconostoc, 262 mesenteroides, 262 Leukaemia, 197 Levelling apparatus, 73 Lichtheim's method, 279 Light, 141 Liquid media, preparation of, 31, 8: Lister's flasks, 31, 92 Lithium-carmine solution, 12 Litmus papers, 23 Living animal, 107 Lochial discharges, 2IO Loffler's solution, 13 Lustgarten, method of, 267 M Madura-foot, 350 Magenta solution (Gibbes'), 13 Malaria, 270 Malignant- oedema, bacillus of, 293 pustule, 281 Marsh-spirochaete, 260 Measles, 214 Merismopedia, 208 gonorrhoea, 208, 223 Methylene-blue, II, 13 Methyl-violet, 13 Meyer's thermo-regulator, 40 Micrococcus, 213 albicans amplus, 2IO - amylivorus, 218 Micrococcus, aurantiacus, 218 candicans, 219 candidus, 219 chlorinus, 218 choleras gallinarum, 232 cinnabareus, 220 citreus-conglomeratus, 209 coronatus, 207 crepusculum, 220 cyaneus. 218 endocarditicus, 192 flavus desidens, 208 liquefaciens, 220 tardigradus. 220 foetidus, 220 fulvus, 221 haematodes, 219 indicus, 240 in acute yellow atrophy, 214 in gangrene, 214 in haemophilia neonatorum, 214 in measles, 214 in rabies, 215 in scarlatina, 213 in typhus, 214 in whooping cough, 214 lacteus faviformis, 221 luteus, 219 of foot and mouth disease, 205 of progressive suppuration in rabbits, 216 of pyaemia in mice, 217 of pyaemia in rabbits. 216 of septicaemia in rabbits, 215, 224 ovatus, 239 parvus, 217 prodigiosus, 241 pyocyaneus, 303 pyogenes tenuis, 215 radiatus, 208 rosaceus, 219 subflavus, 210 tetragonus, 209, 224 ureas, 242 versicolor, 221 violaceus, 219 viticulosus, 221 Micro-organisms, causa causans of diseases, 2 inhalation of, 107 isolation of, 114 Micro-photographic apparatus, 17 Microscope, 5 Microsporon furfur, 347 Microtome, 6 freezing, 7 Microzyma bombycis, 207 Microzyme test, 366 INDEX. Mikrokokken in faulenden Substraten, 220 Milk, 90, 238, 239, 304, 306 Miquel's bulbs, 94 Moist-chambers, 96 Monas, crepusculum, 220 hasmorrhagicum, 214 Okenii, 327 vinosa, 327 Warmingii, 327 Morphology, 117 Moulds, 340 Mouse-cages, 41 Movement, 124 effect of, 140 Mucor, aspergillus, 346 corymbifer, 346 fusiger, 346 macrocarpus, 346 melittophorus, 346 mucedo, 345 phycomyces, 346 racemosus, 345 rhizopodiformis, 346 stolonifer, 346 Muller's fluid, 8 Mycoderma cerevisiae et vini, 341 Myconostoc gregarium, 333 N Nature of the soil, 139 Neelsen's method, 278 solution, 14 Nicati and Rietsch's method, 257 Nitric acid, 14 Nosema bombycis, 239 Nutrient, agar-agar, 68 gelatine, 64 media, 62 Nutrition, 136 O CEdema, 293 Oidium, albicans, 341, 347 - lactis, 347 - Tuckeri, 347 Ophidomonas sanguinea, 327 Orseille (Wedl), 14 Orth's method, 61 modification of Ehrlich's method, 277 solution, 12, 15 Ose, 25 Osmic ac\d, 8 Osteomyelitis, 197 Page's thermo-regulator, 39 Panhistophyton ovatum, 239 Paper trays, 9 Paraffin e, 9 Parrot disease, 206 Paste-cultivations, 85 Pasteur's apparatus, 93 fluid, 91 septicaemia, 293 Peach-coloured bacterium, 325 Pebrine, 239 Penicillium glaucum, 349 Peptonum siccum, 23 Perlsucht, 275 Peronospora infestans, 344 Peters' method, 279 Pfuhl-Petri's method, 280 Phosphorescence, 246 Photo-micrographic apparatus, 17 Phragmidiothrix, 328 multiseptata, 328 Phycomycetes, 344 Picric acid, 14 Picro-carmine (Ranvier), 15 lithium-carmine (Orth), 15 Pilobolus, 345 Pink torula, 342 Pipette, 29 Pityriasis versicolor, 347 Plate-cultivations, 72, 367 Plate-glass, 26 Platinum needles, 25 Plant's method, 337 Pleomorphism, 182 Pleuritis, 228 Pneumococcus, 226 Pneumo-enteritis of the pig, 299 Pneumonia, 227 method of staining bacteria of, 227 Potash solution, 15 Potato-bacillus, 316 bacterium, 244 cultivations, 77 Potatoes, disease of, 226 Potato-knives, 28 paste, 84 Pouchet's asroscope, 364 Pravaz' syringe (Koch's modification), 45 Protected burners, 35 Protective inoculation, 163 Proteus, mirabilis, 247 vulgaris, 246 Zenkeri, 248 Ptomaines, 144 INDEX. 437 Puerperal fever, 198, 201 Pus, 195. 197, I99> 217, 271 Putrefaction, 247 Pyaemia, 195, 198 in mice, 217 in rabbits, 216 Pyocyanin, 303 Quarter-evil, 321 R Rabies, 215 Ranvier's picro-carmine, 15 Rauschbrand. 320 Ray-fungus, 334 Re-agents, 7, 9, 16 Reichert's thermo-regulator, 37 Relapsing fever, spirillum of, 249 Reproduction, 128 Respiration, 136 Rhabdomonas rosea, 328 Rhinoscleroma, 230 Rindfleisch's method, 276 Rosaniline-hydrochlorate, 1 1 Rothlauf, 300 Rouget du pore, 204, 300 Saccharomyces, albicans, 341 apiculatus, 341 cerevisise, 340 conglomerate, 341 ellipsoideus, 340 exiguus, 341 glutinis, 342 mycoderma, 341 niger, 342 pastorianus, 341 ' ro?aceus, 342 sphsericus, 341 Saccharomycetes, 340 Safety burner, Koch's, 36 Safranine, 15 Salmon, disease of, 344 Salt solution, 15 Saprolegnia, 344 Sarcina, 210 alba, 212 aurantiaca, 211 hyalina, 212 intestinalis, 212 litoralis, 212 lutea, 210 Reitenbachii, 212 urinse, 212 Sarcina, ventriculi, 21 1 Scalpels, 29 Scarlatina, 214 Schizomycetes, 180, 185, 194 Schlosing's membrane-regulator, 34 Schiitz, method of, 293 Senkewitsch's method, 280 Septicaemia, bacillus of gangrenous, 272 bacterium of, 235, 236 consecutive to anthrax, 205 in rabbits, 215 of man, 271 of mice, 297 Serum-inspissator, 30 steriliser, 29 Silkworms. 207, 239 Siphon-apparatus, 42 Soil, examination of, 365 Spermaceti, 9 Sphaerotilus natans, 333 Spirillum, 249 attenuatum, 262 cholerae Asiaticse, 250 Finklerii, 258 leucomelaneum, 262 method of staining, 250 Obermeieri, 249 of relapsing fever, 249 plicatile, 260 rosaceum, 327 Rosenbergii, 262 sanguineum, 327 serpens, 261 sputigenum, 259 tenue, 261 tyrogenum, 260 undula, 261 violaceum, 327 volutans, 261 Spirit-level, 26 Spirochsete buccalis, 329 Obermeieri, 249 Spiromonas Cohnii, 333 volubilis, 333 Splenic fever, 281 Spores, method of staining, 51 preservation of, 286 Sputum, 221 septicaemia. 236 Staining bacteria, 57 methods of, 223 actinomyces, 337 bacilli of ulcerative stoma- titis, 299 bacillus, anthracis, 290 of butyric acid fer- mentation, 320 438 INDEX. Staining methods, bacillus, of glanders, 293 of leprosy, 266 of rhinoscleroma, 230 of syphilis, 267 of typhoid fever, 269 bacteria in diphtheria, 231 of pneumonia, 227 comma-bacilli. 257 hay bacillus, 309 leptothrix buccalis, 329 spirillum Obermeieri, 250 tubercle bacilli, 275 Staphylococcus cereus albus, 199 cereus flavus, 200 pyogenes albus, 198 aureus, 197 citreus, 199 Steam, 159 steriliser, 19 Sterilisation, apparatus, 19 of glass plates, 73 Sternberg's bulbs, 31, 92 Streptococcus, 195 articulorum, 202 bombycis, 207 cereus albus. 199 flavus, 200 coronatus, 207 erysipelatosus, 200 flavus desidens, 208 in cerebro-spinal meningitis, 203 in dental caries, 203 in diphtheria, 202 in endocarditis, 201 in puerperal fever, 201 insectorum, 207 in yellow fever, 203 of cattle plague, 204 - - of foot and mouth disease, 205 of progressive tissue necrosis in mice, 205 of septicaemia consecutive to anthrax, 205 of swine erysipelas, 204 perniciosus, 206 - pyogenes, 195 albus, 198 aureus, 197 citreus, 199 rnalignus, 197 radiatus, 208 septicus, 205 toxicatus, 20 1 variolae et vaccinia;. 203 viscosus, 207 Streptothrix Fcersteri, 332 Sulphuric acid, 15 Surra, 352 Sweat, human, 219 Swine-erysipelas, 204, 217. 300. 315 typhoid, 299 Symptomatic anthrax, 320 Syphilis, 267 Table salt, 23 Tarichium megaspermum. 344 Teeth, carious, 220, 259 Temperature, effect of, 139 Test-tube cultivations, 69, 369 stands, 24 water-bath, 21 Tetanus. 301 Thermometers, 27 Thermo-regulator, 38, 39, 40 Tilletia caries, 343 Torula cerevisiae, 340 Transport of water samples, 367 Trichomonas sanguinis, 360 Tricophyton tonsurans, 347 Tripod levelling-stand, 25 Tuberculosis, bacillus of, 272 Turpentine, 15 Typhoid fever, 269 Typhus, 214 U Ulcerative stomatitis in the calf, 298 Ureihral pus, 210 Urine, 90, 243 Urocystis occulta, 343 Ustilago carbo, 343 Vaccinia, 203 Vaginal secretions, 210, 221 Valentin's knife, 7 Variola, 203 Vaseline, 32 Vegetables, 86 Vegetable infusions, 90 Vesuvin, 15 Vibrio, 317 rugula. 317 serpens, 261 Vin filant, 207 W Warm stages, 97 Warming apparatus, IOI Water, 16 INDEX. 439 Water, bath, 21 examination of, 366 Wedl's orseille, 14 Weigert-Ehrlich method of staining, 6 1 Weigert's method of staining, 58, 291 Whooping cough, 214 Wire cages, 24 Wooden-tongue, 335 Woolsorter's disease, 281 Wurzel-bacillus, 311 Xylol, 9, 19 i Yeast fungi, 340 Yeasts, 340 Yellow fever, 203 Ziehl's method, 279 Zinc- white, 17 LONDON : H. K. LEWIS, 136, GOWER STREET, W.C. DESCRIPTION OF PLATES. PLATE I. PIGS, i to 39. Bacteria, Schizomycetes, or Fission fungi, (Facing Title-page.) For description, see p. xxiv. PLATE II. FIG. i. Bacterium indicum. Micrococcus indicus. Bacillus indicus. Tube inoculated from a nutrient agar-agar plate- cultivation. By plate-cultivation, or by successive cultivation on potatoes, a pure cultivation can be obtained. The growth has then the colour of red sealing-wax, and a peculiar crinkled appearance. After some days the growth loses its bright colour, and becomes purplish like an old cultiva- tion of Bacterium prodigiosum. FIG. 2. Bacillus cyanogenus. Bacterium syncyanum. 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. FIG. 3. Bacterium prodigiosum. Monas prodigiosa. Micrococcus prodigiosus. Bacillus prodigiosus. 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 2. CULTIVATIONS ON "NUTRIENT AGAR-AGAR i'lg 1. Micrococcus indicus. Fig 2 . -Bacillus cyari.ogemis. Fig 3 . Mi cro coccus p--odigiosus . kir.Dcry t< i'^t., / - i PLATE III. FIG. i. Spirillum cholerae asiaticae. Comma-bacillus of Kocli. 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. (Fig. 96.) FIG. 2. Bacterium cholerae gallinarum. Micrococcus cholerce gallinarwn. Microbe du cholera des ponies. 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. FIG. 3. Streptococcus cereus albus. Staphylococcus cereus albus. Tube inoculated from the pus of a subcutaneous abscess in a rabbit. The growth assumed a nodular appear- ance along the needle track. -. Figl. Fig 2. CULTIVATIONS IN NUTRIENT GELATINE fig 1. Spirillum choleras asialicse. Pig 2. Micro co ecus cholerae fSallinarum . Fig 3. Staphylococcus cererus aLbus. -1 U H ! Vincent Bnolct.Dty i,Son..Uth . ->- : ^ ..--. *' .*. <* PLATE IV. FIG. i. 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. FIG. 2. Bacterium pneumoniae crouposae. Micrococcus pneumonia crouposce. Friedlander s pneitmo- coccus. Tube inoculated from pneumonic exudation. The growth, in nutrient gelatine, in the form of a round-headed nail is not by itself distinctive. FIG. 3. Saccharomyces niger. Black torula. Tube inoculated from an old contaminated nutrient gelatine cultivation. The growth, isolated and re- inoculated, formed a black crust on the surface of the gelatine. In some of the tubes little separated centres of growth occurred in the upper part of the track of the needle. 2. Fig 3. CULTIVATIONS IN NUTRIENT GELATINE. Fif5 l.Micrococcus tetragortus. Pi(5 2. Bacterium pneumonise croup osae. Fid 3. SsLCGharomyees niger. ^nl Br^okf,Diy b Son, Litk . 1 In- H.K PLATE V. FIG. i. Bacillus pyocyaneus. Bacterium ceruginosum. Bacillus fluorescens. Tube inoculated from pus. The gelatine was liquefied and appeared green by transmitted and orange by reflected light. FIG. 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. FIG. 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 in Fig. 107. In this figure the appearance after three weeks is represented. The gelatine was completely liquefied, and a floc- culent mass had subsided to the bottom of the tube. PLATE 5. Figl. Fu5 2. Fig 3. CULTIVATIONS IN NUTRIENT GELATINE Pig 1. Bacterium aerugmosum. Tig 2. Sarcinalutea. Htf 3. Bacillus an_thracis . // Cmakfhank I'itur. * PLATE VI. Spirillum Finkleri. Comma-bacillus of Finkler and Prior. This figure represents the appearance of a plate-cultiva- tion 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 Fig. 85). 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 completely isolated one from the other ; but later they became confluent and produced complete liquefaction of the gelatine. * 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. PLATE - CULTIVATION. / 'ir\-l. uttetvuatwn cfUw SpiviUiun FvnJderii, after Uv&ntu-four hours. Londcn.Riblislied b\' H iLewia ,136, Gower Street . Vincent Bryokj.Day SeSon.lilh. PLATE VII. Spirillum Finkleri. 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 CULTIVATION. Second all fit it a turn of the Spirillum Finkleru , cvfier thirty -sue hours. "!i3hedbv H.K.Lewi3,l36.Gower Street. Vincent, BnoTet.Uey 4 Son, IX*.. PLATE VIII. FIG. i. 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. FIG. 2. Streptococcus pyogenes aureus. StapJiylococcus pyogenes aurcus. Micrococcus pyogenes aureus. Tube inoculated from an abscess in a rabbit. FIG. 3. Bacillus pyocyaneus. Bacillus fluoresccns. Bacterium aeruginosum. 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. PLAT CULTIVATIONS ON NUTRIENT AGAR-AGAR. Fid 1 . Sareina lutea . Fig 2. Micrococc-us pyogen.es aureus. Firf 3. Bacillus fhiorescens. H.K.I,e-A'iG .ISe.GCTwer Street , PLATE IX. FIG. i. Bacterium prodigiosum Monas prodigiosa. Micrococcus prodigiosus. Bacillus prodigiosus. Blood rain. Growth on potato after three days. FIG. 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. pirf 1. MicrococciLs prodigiosus. Second aUvnoalicn after three days 2. PeriicilliuTa ^ after three weeks yrvwttv. POTATO CULTIVATIONS Lonaon,ftiMishedbv H.K.LewisJSG.Gower Street . PLATE X. FIG. i. Sarcina lutea. Growth on sterilised potato five days after inoculation from a tube-cultivation. Potatoes, especially old ones, have sometimes a tendency to become dis- coloured, and the brown appearance in this figure has nothing to do with the growth of the organisms. FIG. 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. P1ATE 10. Figl. Sarcinal-otea Growth, five days afar vnoculcubum,. Tig 2. SacchaTomyces rosaneus. OCTvyz/ ?wo d^* ffar inoculation POTATO CULTIVATIONS. London.RihKalied bv H . K. Lewis ,136, Cover Street . */* v< -, ^ ., PLATE XL Bacillus tuberculosis. FIG. i. Pure cultivation of the tubercle-bacillus on blood-serum solidified obliquely. FIG. 2. Pure cultivation on solid blood-serum in a glass cap- sule. FIG. 3. The same as Fig. 2, examined under a low power of the microscope. x 80. FIG. 4. Impression-preparation showing the peculiar serpentine growth of the colonies on blood-serum. x 700. (After Koch, Mittheil. a. d. Kaiser I. Gesundh. PLATE 11. BACILLUS TUBERCULOSIS. Fiq. 1 . Pure, -odtiva,tton> on solid- bloccL serum m a te-sV-tu&e. Fig. Z. Fure-adtivatwn on solid Hood seruxw in cLgUtss-ctvpside,. Fig. 3 . The sume preparation as Fig. Z fJfSO/. Fig. 4 . Cover -glass Lnvpr^ssicit prepdralicr. ofwlonies X 700 (Ehrlidis nuthcd). LondortPublishedbv H.K.Ltfwis.l36,Gcrver Street . PLATE XII. FIG. 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 1,500. FIG. 2. Streptococcus pyogenes aureus. Micrococcus pyogenes aureus. Staphylococcits 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 1,500. PLATE 12. MICROCOCCUS TETRAGONITS fit/./, from, CL scciKon- of l&dnti/ of a rnowsc . G-ram's method, Zeiss ' JtT o. C . Oc. 4. MICROCOCCUS PYOGENES AUREUS. Fig. 2. from tt section of Liver of a rallit Gram's inetkod. Zeiss ' Is. o. i>. Oe. 4. aajr- Cnclcshank ft*, it pirn* . London.Puhli3kedbvH.K.I.ewi3. 136, Cower Street. TfruxntSrooix.Uay I Son. li&. 9 - PLATE XIII. FIG. i. Sarcina lutea. In this tube and the two adjacent ones, the inoculations 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 charac- teristic pigment, while the growth in the track of the needle was scanty and colourless. FIG. 2. Bacterium indicum. Micrococcus indicus. Bacillus indicus. FIG. 3. Saccharomyces rosaceus. Pink torula. -..:... PIP 3. CULTIVATIONS IN NUTRIENT AGAR AGAR Fig 1. S aroma lute a. Fig 2. l^Ecro coccus indicrus. ~Fi03. Sacdhai-omyces rosaceus. PLATE XIV. FIG. i. Bacillus anthracis. Bacteridie du charbon. Bacillus of splenic fever, ivoolsorter* 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. 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. FIG. 3. Streptococcus cereus albus. Staphylococcus cereus albus. Tube inoculated from the discharge of a subcutaneous abscess in a rabbit. PLATE 1A. CULTIVATIONS ON NUTRIENT AGAR-AGAR Fig 1. Bacillus artthr-acis. Fig 2. Bacillus subtilis . Fid 3. Stscphylococeus cereus albus. PLATE XV. FIG. i. Bacillus anthracis. Bacteridie du charbon. Bacillus of splenic fever, woolsorters 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. FIG. 2. Bacterium indicum. Micrococcus indicus. Bacillus indicus. 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 15. Fig 1. Bacillus anthracis. Growth/ at Z] C three days after mooulatoon Fig 2. Micro co ecus in.dicus. Growth/ Ihree days after inoculation'. POTATO CULTIVATIONS Pinx; LondoiuRihlialiedbv H.K.LewiG, 136, Carver Street . Vincent SMJpfc.o.Doy i(Son,Ut\. %* T PLATE XVI. Bacillus anthracis. Bacteridie du charbon. Bacillus of splenic fever, woolsoi ters disease, or malignant pustule. FIG. i. From a section of the mucous membrane of the stomach of a mouse. The glandular capillaries are mapped out by the bacilli. Stained by the method of Gram (gentian violet), and eosin. x 500. FIG. 2. From a section of a kidney of a mouse. Under a low power the preparation has exactly the appearance of an injected specimen. Under higher amplifica- tion, 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. PLATE 16. / -> f \vi/'/\ 'j 1 ; N / i/ / / . / from a section of miicoujf membrane of the stoma~J% 'A, ..s-e^Hf^-J- ^"^^^ BACILLUS ANTHRACIS. /. From a, section, of Liver of a, mouse. Weigerts andOrttis methods. .i. Oc,. Z. BACILLUS ANTHRACIS AKD MICROCOCCUS TETRAGO^fUS. Fig. 2. From a section of Lung of a mouse. Grcvm's method and Eos in . "Weiss' 111. o.v. Oc.Z. .K. L.-A/I - . I " R , Cover Street . V^icentroolcs,J)ayf.Son.,liA- 'I ~ v i .~4 tr PLATE XVIII. Bacillus tuberculosis. FIG. i. From a section of a lymphatic gland of a foetal 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 1,500. 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 Tuber- kulose," Fortsch. d. Med., 1885, No. 7, p. 198. FIG. 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 1,500. PLATE 18. Fig. /. jfrvm a. section of a lymphatic (jltttid from, a case of tuhreulosis in a foetal calf. ShrlieksKoch, methods. letaiid bis rruvrck brown) and Eosin. Weiss' fZ- o.-i. Oc.4-. $Mfkw mm BACILLUS TUBERCULOSIS. Fig. t. From a section of Lung from a case- of artificial twbeirulosis in. a rabbit. Ekrlich-Kock method. (methyl- violet. ) Xeiss'rS o.v. Oc. 4-. I.ondon.RitIished"bv H JLLewia,,136,GoMiw Street. PLATE XIX. Bacillus tuberculosis. FIG. i. From a section of the liver of a tubercular hen. With a moderate power the areas of caseation and the topographical distribution of the bacilli can be studied. Stained with the Ehrlich-Koch method (methyl violet and bismarck brown), x 400. FIG. 2. From the same preparation with high amplification, showing that the parts stained blue consist entirely of bacilli, x 1,500. PLATE 19. . J?ig.2. From cu section of Liver of a> hc^rv. Eh rltch - Koch method ( methyl- v ioltt and bisirutrck - brctvrv) %eiss ' DD. Oo. 4- . BACILLUS TUBERCULOSIS. . Oe. londoiuPubliahedbv H.K.Lews.l36.Go--ver Street . entBrooks, Hay Sa PLATE XX. FIG. i. Bacillus tuberculosis. From a cover-glass preparation of tubercular pus. Stained with Ehrlich's method (fuchsine and methylene blue), x 1,500. FIG. 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. I'L/.YE 20. BACILLUS TUBERCULOSIS. . /. From a. cover-qlass prepa-ra,tiori, of pus from a. tM/OGrcuicur CcLi'ity of t(t& n lung. h/rlicJi's -method. ( fucks vne. and metkylene blue}. Zn&fs'/f. o.i. Oc. BACILLUS Fig. 2. From a section of 'Ruin ey from a CCLSS of leprosy. Elirlic/i's method. ( fuctisine and methylene &Ute,J. Z&iss' DD. Oc. 4 ewis ,13 6, Cower Street . HncentSrookf, Day Son.. Lrik. PLATE XXI. Bacillus cyanogenus. Bacterium syncyanum. Bacillus of blue milk. FIGS, i and 2. 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 21. Fig 1. After three days growth.. i 2. After nine days growth.. POTATO CULTIVATION OF BACILLUS CYAKOGENUS. I.ondon.Puhliahedby H.K.LewiGjSe. PLATE XXII. Bacillus of septicaemia of mice. FIG. i. 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. FIG. 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 1,500. - * ** Fig. I. From a .section ofJLidney of CL mouse. Gram's method aiidFosin. %*iss' J)J). 0&2. BACILLUS OF SEPTICAEMIA OF MICE Fly. Z. Ike scvm* preparojtwrv. Zeiss ' Is. o. i. Oc. 4. .KJ.ewis.lSG.Gowsr Street. PLATE XXIII. Bacillus leprae. FIG i. From a section of the skin of a leper. The section is, almost in its entirety, stained purple, and, with moderate amplification, has a finely granular appearance. Stained with Ehrlich's method (fuchsine and methylene blue). x 200. 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 1,500. PLATE 23. Fuj. I. From a section if skin from a case o/~ leprosy. Elirltcft s method. (Auksine and melAylKedly H.K.;. r ."..-. ] : B. lff*w street. CULTIVATIONS^ ON NUTRIENT AGAR-AGAR Fag l.Bajcterrum lineola. , Fig 2 .Micro co ecus rosaceus . Fig 3 . Stsfihylococcus pyogen.es citreus. Londcn.Pabii3liedtv H.K. Lewis, ISG.Gower Street. PLATE XXV. Bacillus figurans. Bacillus mycoidcs. Wurzel Bacillus. FIG. i. From an impression-preparation of a growth on the surface of nutrient gelatine. x 50. FIG. 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 arrange- ment of the individual bacilli, x 1,500. PLATE 25. fig.f. Cover-glass impression -preparation^ ftvm/ a plate -cultivation 2. BACILLUS FIGURANS. Fig.%. Th same, artioTv. Z&iss'ffr. A,v. Oc. LondoixFablialiefi. ty H.K.Le/vis,lS6,Gi?ver Street. PLATE XXVI. Bacillus of swine-erysipelas. Bacillus oj German swine-fever. FIG. i. Pure cultivation in nutrient gelatine. The growth is stated to be identical with that of the bacillus of septicaemia in mice. FIG. 2. Colonies on a plate-cultivation. FIG. 3. Cover-glass preparation of blood from an inoculated pigeon. (After Schiitz, Arb. a. d. Kais. Gesundh. A tut, Bd. I.) PLATE 26. Fig.Z. JFy.3. BACILLUS OF SWINE-FEVER ( S CHUTZ ). Fig. 1. ftt re - cu Itwatitm in nuirient geialine. Fig. Z. Colonies on a plate - cultivation . Fi(j.3. Cover-glass preparation of blood from an, inoculated pigeon Son, Litk. , tiaVifit!WH.K-T,vifl l.,f) Ooww Street. PLATE XXVII. Actinomyces. FIG. i. From a section of a maxillary tumour in a cow. Stained by Plaut's method (magenta and picric acid). x 90. FIG. 2. Part of the same preparation, with higher amplifica- tion. 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 27. Fig. / . From a. f&stion/ of & maxvUa,ry ixMnorvr in a, oow. Piauts matfiod ( Magenta, and picric a^cid). Zviss' AA. Oc. ACTINOMYCE S. Fig.%. The same -preparation. Z&ifs'fc. o.i. Oo. 2. Edgar CrackshwnJc fec.etp* London.Puhlujhedbv H .K.LewiG.I36,Gowcr Street . PLATE XXVIII. Actinomyces. FIG. i. From a section of a maxillary tumour in a cow. Stained by Weigert's method (orseille and gen- tian violet), x 900. FIG. 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 grow- ing 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 re- sembles miliary tuberculosis, 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 28- g. /. .From a> section, of a- majc-Mcvry tu,mou'M-d bv H ..K. J^.vi.- ,136. PLATE XXIX. FIGS, i to ii. Yeast-fungi or saccharomycetes and mould fungi or hyphomycetes. (Facing page 339.) For description see p. 339. fl 1 BIOL06V LIBRARY G UNIVERSITY OF CALIFORNIA LIBRARY