BACTERIA IN RELATION TO PLANT DISEASES. FERDINAND COHN. 2. ROBERT KOCH. 4. EMILE ROUX. 3. LOUIS PASTEUR. S. EMILE DUCLAUX IACTERIA ix RELATION TO PLANT DISEASES BY ERWIN F. SMITH, In charge of Laboratory of Plant Pathology, Office of Physiology and Pathology, Bureau of Plant Industry, U. S. Department of Agriculture . VOLUME ONE. METHODS OF WORK AND GENERAL LITERATURE OF BACTERIOLOGY EXCLUSIVE OF PLANT DISEASES. WASHINGTON, D. C. : Published by the Carnegie Institution of Washington September, 1905. CARNEGIE INSTITUTION OF WASHINGTON PUBLICATION No. 27 FROM THE PRESS OF THE HENRY E. WtLKENS PRINTING CO. WASHINGTON. 0. C. PREFACE. The subject of bacterial diseases of plants is older than the poured-plate method of Koch, but until recently our knowledge of it has been in a very chaotic state, it having been for the most part for twenty-five years a recognized but uncultivated field. In recent years, however, publications on plant bacteria have multiplied, and they now amount to several hundred titles. The writer's studies of the bacteria themselves and of the diseases which they cause, as distinct from the literature of the subject, began in 1893. At that time there was very little reliable information on this subject. The literature is now more extensive, but it is nowhere gathered together in one place and properly sum- marized. It has seemed, therefore, for a long time, that a work of the scope of the treatise here presented might be clarifying and useful to many people. There have been published, and are still appearing, so many papers on the subject of bacterial diseases of plants by writers ignorant of bacteriological methods and indifferent to the requirements of modern pathological inquiry that this whole subject has been brought into disrepute. This is the only possible explanation of the fact that up to a very recent date writers on pathology and bacteriology have been telling their readers that there is no good evidence of the existence of any such diseases. The following editorial paragraph from the Botanical Gazette, February, 1893, may be cited as indicating the general feeling on this subject at that date: What is especially needed at this stage of advancement is the cpntinuous and systematic examination of the whole ground by one or more well-equipped investiga- tors, and the publication of a critical statement of what may be safely accepted as proven. Even a summarization of the present status of the subject, without critical laboratory study, would be helpful, if well done. That this feeling has become intensified with the progress of time and the multiplication of literature is shown by the following citation from the large Treatise on Bacteriology, by Miquel and Cambier, published in 1902: The list of bacteria capable of attacking the higher plants increases rapidly from day to day ; but whether the experiments of plant pathology offer greater difficulties than those of animal pathology, or whether the authors who have undertaken them have ignored the multiple resources which bacteriology offers to-day, many of the species described must be studied anew, their monography offering regrettable lacunae. By the side of some fruitful and well-conducted labors we find, unfortunately, alto- gether too many which must be done over entirely. It was with the hope of making useful discoveries and clearing up part of this contradiction and uncertainty that the writer began his study of this class of diseases. His first effort in the way of preparation was to supplement his botanical training with a knowledge of bacteriological methods which he obtained from standard literature and competent teachers. His second effort was to gather 257067 IV PREFACE. together and properly digest all of the literature relating to this subject. This resulted in the projection of a critical review of the literature, begun in 1896 in the American Naturalist but left unfinished, owing to pressure of research work, and a card catalogue which is now here published in full with critical remarks. His third endeavor was to carefully work over, in the laboratory, field, and greenhouse, as opportunity offered, all of the so-called bacterial diseases of plants, submitting each supposed parasite to all of the tests of modern pathology. The latter has proved a far larger undertaking than was anticipated, the number of diseases attributed to bacteria having increased rapidly in recent years. It is expected that more than 125 diseases will be treated or touched upon in this monograph, many of which have come under the writer's own observation. An attempt has been made to cover the literature of the whole world and to work over personally every disease so far as material could be obtained. The present volume contains an "outline of methods of work," which was written up in substantially the same form four years ago, in connection with the investigations of the Laboratory of Plant Pathology, Bureau of Plant Industry, United States Department of Agriculture, its publication having been delayed in order to bring the rest of the manuscript into suitable shape. The monograph is published in this form with the approval of the Secretary of Agriculture. The bibliography at the end of this volume covers the general subject of bacteriology, exclusive of plant diseases, and is arranged chronologically by sub- jects. Not every good paper is referred to, but for the most part only such as have fallen under the writer's own observation. It is believed, however, that by consulting these the student will soon be able to get hold of the entire literature of any special branch. The reader who wishes to keep pace with the advance of the science should consult the International Catalogue (R) published by the Royal Society of London. The illustrations, especially those dealing with histology, have been drawn, with very few exceptions, under the direct personal supervision of the writer, every one of them when near completion having been inspected critically and modified in various details to correspond as closely as possible to the actual object. The slides from which the drawings have been made will be placed on file in the Laboratory of Plant Pathology, where they may be consulted. This monograph is not intended to take the place of ordinary text-books of bacteriology, of which there are now many, but rather to supplement the same, giving information where they are silent or misleading. It is hoped that it will be of value not only to plant pathologists, for whom it is primarily intended, but also to physicians and animal pathologists for purposes of comparison. In its preparation the writer has had also an eye to the service of gardeners, fruit-growers, and all who take an intelligent interest in plants. It is presented with a keen sense of its imperfections, but with the hope that it may at least serve its main purpose. While the writer has made every effort to be accurate in statement and just in criticism, it is too much to hope that he has always succeeded, and, therefore, he desires to crave pardon in advance for all errors of omission and commission, taking PREFACE. V shelter behind Lavoisier's well-known and convenient apology: "Man would never give anything to the public if he waited till he had reached the goal of his under- taking, which is ever appearing close at hand and yet ever slipping farther and farther as he draws nearer." Those who dwell in the clearer light of the next generation will build better than we have done and will scarcely realize how slowly and painfully many of us have groped about for what seems to them so plain. In conclusion, I desire to make grateful mention of Dr. Theobald Smith, professor of comparative pathology in Harvard University Medical School, and Dr. Veranus A. Moore, professor of comparative pathology and bacteriology in Cornell University, each in turn in charge of the animal pathological investigations of the Bureau of Animal Industry, United States Department of Agriculture, at a time when the writer was beginning his bacteriological studies and was perplexed in many ways. To friendly advice and helpful suggestions from these distinguished men he owes more than he can well repay. AUGUST 25, 1905. CONTENTS. OUTLINE OF METHODS OF WORK. Page. General Remarks 3 The Disease 4 Previous Literature 6 Geographical Distribution 7 Signs of Disease 7 Pathological Histology 8 Direct-infection Experiments 9 The Organism 9 Pathogenesis 9 Rules of Proof 9 Morphology 18 Size, Shape, etc 18 Capsules 19 Flagella 20 Spores Endospores, Arthrospores 21 Cell-unions Zoogloeae, Chains, Filaments. . 22 Involution-forms 23 General Comment 23 Physiology 25 Motility 26 Growth 27 Chemotropism 27 Reaction to Stains 27 Culture-media 29 Nutrient Gelatin 29 Nutrient Agar 31 Silicate-Jelly 36 Solid Vegetable Substances 39 Plant Juices (with and without the addi- tion of water) , 41 Animal Fluids 45 Beef-broth 45 Milk 46 Litmus Milk 48 Rice cooked in Milk 48 Loeffler's Solidified Blood-serum 48 Egg-albumen 48 Egg-yolk 49 -Synthetic Media and Other Special Media. 49 Relation to Free Oxygen 51 Surface and Deep Growths 51 Fermentation-tubes 5 2 Growth in Hydrogen, in Carbon Dioxide, in Vacuo, and in Nitrogen 54 Luminosity 60 The Organism Continued. Physiology Continued. Fermentation-products 60 Alkalies (Ammonia, Amins, etc.) 61 Reducing Powers 62 Hydrogen Sulphide 62 Mercaptan and Other Odors 62 Indol, Phenol, Leucin, Tyrosin, etc 62 Reduction of Nitrates, etc 63 Fixation of Free Nitrogen, etc 64 Assimilation of Carbon Dioxide 64 Pigments 64 Crystals 66 Enzymes 66 Sensitiveness to Plant Acids 69 Sensitiveness to Alkalies 69 Effect of Desiccation 70 Effect of Direct Sunlight 71 Vitality on Various Media 72 Mixed Cultures and Mixed Infections 72 Reaction to Antiseptics and Germicides 74 Thermal Relations: Maximum, Minimum, and Optimum Temperatures for Growth; Thermal Death-point; Effect of Freezing 75 Other Host-plants 87 Pathogenic or Non-pathogenic to Animals . . 88 Economic Aspects. 90 Losses 90 Natural Methods of Infection 91 Conditions favoring Spread of the Disease. ... 93 Methods of Prevention 93 General Considerations 94 Location of the Laboratory 94 Equipment of the Laboratory 94 Care of the Laboratory 96 Preparation and Care of Culture-media 97 The Cleaning and Sterilization of Glassware and Instruments 100 Making and Transference of Pure Cultures. . . 103 The Final Disposal of Infectious Material 106 Methods of Inoculation 108 The Keeping of Records 109 The Making of Collections 117 Distilled Water 124 VIII CONTENTS. Page. | General Considerations Continued. Microscopes 129 Photography and Photomicrography 130 Some Milestones in the Progress of Bacteri- ology 151 Nomenclature and Classifications 154 Migula's Classification 159 Value of Morphological Characters 176 Value of Cultural Characters 178 Undergraduate Work 181 A Final Caution 184 Page. Formulae 187 Stains: General and Miscellaneous 187 Cleaning Cover-glasses 189 Flagella-staining 189 Capsule-stains 194 Spore-stains 194 Non-synthetic Culture-media 195 Synthetic Culture-media 197 Miscellaneous 200 Fixing Fluids 202 BIBLIOGRAPHY, GENERAL LITERATURE. I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII. XXIII. XXIV. XXV. XXVI. XXVII. XXVIII. Page. Journals 203 Transactions, Beitrage, Jahresberich- ten, Festschriften, etc 204 Manuals 204 Physical, Chemical, Zoological, and Botanical Works of special use to the Plant Pathologist 206 Books and Papers of more or less general interest 210 Important Books and Papers on special human and animal diseases. 212 Predisposition, Conditions Favoring Infection or Immunity 214 Symbiosis, Antagonism 214 Carriers of Infection 215 General Morphology of the Bacteria. 215 Spores 218 Flagella 219 Capsules 220 Stains and Staining Methods 221 Morphological and Physiological Changes due to Changed Environ- ment 222 Culture-media 223 Methods of Work, Apparatus, etc.. . 226 Special means of Differentiating Bacteria 229 Aerobism, Anaerobism 230 Fermentations, Gas-formation, En- zymes, etc 232 Ptomaines, Toxins, Antitoxins, Se- rums, Phagocytosis, etc 235 Attenuation, Virulence 236 Pigments, Green Bacteria 236 Reduction anil Oxidation 239 Nitrifying and Denitrifying Organ- isms, Use of Free Nitrogen 239 Use of Free Carbon Dioxide 241 Luminous Bacteria 241 Hydrogen Sulphide and otherwise unclassified By-products 242 Page. XXIX. Action of Light on Bacteria 243 XXX. Effect of Electricity 244 XXXI. Action on Bacteria of Roentgen Rays, Radium Rays, etc 245 XXXII. Effect of High Pressure on Bacteria. .. 245 XXXIII. Action of Heat and Cold on Bacteria. 246 XXXIV. Thermophilic Bacteria 247 XXXV. Resistance to Dry Air 248 XXXVI. Action of Acids and Alkalies 249 XXXVII. Agglutination and Precipitation 249 XXXVIII. Antiseptics and Germicides 250 XXXIX. Chemotropism, Thermotropism, Geo- tropism, Contact-Irritation, etc 253 XL. Osmotic Pressures 254 XLI. Chemical Analysis of Bacteria 254 XLII. Distribution of Bacteria Geograph- ical and Altitudinal 254 XLII I. Soil-Organisms; Putrefactive Or- ganisms 256 XLIV. Vinegar-bacteria 256 XLV. Silage-bacteria, Fermentation of To- bacco, of Indigo, Retting of Flax, of Sisal Hemp, etc., Softening of Pickles, Sauerkraut, etc 256 XLVI. Bacteria in Water and Ice; Dung- bacteria 258 XLVII. Milk-bacteria; Cheese-bacteria; But- ter-bacteria; Meat-bacteria 259 XLVIII. Bacteria in Bread 260 XLIX. Iron-bacteria 261 L. Sulphur-bacteria 261 LI. Bacteria in Prehistoric Times 262 LII. Preparation of Slides, Cultures, etc., for Museums, etc 262 LHI. Stock-cultures, how best kept; Vital- ity on Media 263 LIV. Color-charts; Nomenclature of Col- ors 263 LV. Photography and Photomicrography. . 263 LVI. Methods and Systems of Classification 264 LVII. Useful Catalogues 265 ILLUSTRATIONS. PLATES. Page. PLATE i. Frontispiece. (1) Ferdinand Cohn, founder of mod- ern systematic bacteriology. De- ceased. (2) Robert Koch, founder of German school of bacteriology, director of the Institute for Infectious Diseases at Berlin. (3) Louis Pasteur, founder of French school of bacteriology. De- ceased. (4) Dr. Roux, one of the leading spir- its of the Pasteur Institute. (5) Em. Duclaux, professor in the University of Paris and director of the Pasteur Institute. De- ceased. 2. Bacterial Olive-knots produced on four plants by needle-pricks 10 3. Cross-section of Petiole of Musk- melon, showing bundles disorgan- ized by Bacillus tracheiphilus 12 4. Datura metclloldes eight days after Inoculation with Bacterium solana- cearum 16 5. Zeiss Horizontal Photomicrographic Outfit 26 6. Arnold Steam Sterilizer, Lauten- schlager Dry Oven, Hot Plate, and Chamberland's Autoclave 48 7. Hydrogen Generator and Wash Bot- tles in use 56 8. Thermostat-room 74 9. Chamberland Autoclave 84 10. Engine for furnishing Vacuum and Compressed Air 94 PLATE n. 12. 13- 14- 15. 16. 17- 18. 19. 20. 21. 22. 23- 24. 25- 26. 27. 28. 29. 30. TEXT FIGURES. Page. FIG. i. Cross-section of Sweet-corn Stem para- sitized by Bacterium Stnvarti 4 2. Cross-section of a Raw Carrot, showing wedging apart of Parenchyma Cells by Bacillus carotovorus 5 3. A Detail from Fig. 2 6 4. Turnip-root, showing Bacterium camfes- trc confined to vicinity of Vessels 7 5. Bacterium campcstre. A small portion of Fig. 4 enlarged IO Page. Culture-room, i. e., place for making Cultures of Bacteria in Still Air loj Movable Hood of Wood and Glass, under which Bacteriological Trans- fers may be made 106 The Reinhold-Giltay Microtome 120 Distilled-water Apparatus 124 Zeiss Stand lla 129 Zeiss Photomicrographic Stand Ic.... 129 Mounted Camera for Enlarging, Re- ducing, and Natural-size Work 134 Lantern-slide Room 144 Black Spot of the Plum 148 Bacterial Disease of Broomcorn 150 Bacterial Black Spot of Walnut 174 Ditto, Late Stage 176 Transmission of Wilt of Cucumber by Insects 178 Brown Rot of Potato. Natural Infec- tion of Tuber, Artificial Infection of Stems 202 Brown Rot of Potato. Shoots wholly destroyed by inoculation 202 Tomato-plant inoculated with Bac.- terium solanacearum 202 Bacterial Wilt-disease of Tobacco 202 Young Pear-shoots blighted by Bacil- lus amylovorus 202 Green Pear-fruits eight days after In- oculation with Bacillus amylovorus. 202 Quince-shoots and Pear-fruits (cross- section) showing Blight due to Bacillus amylovorus 202 Small Green Apples blighted by Ba- cillus amylovorus 202 Page. FIG. 6. Turnip-root, showing Bundle occupied by Bacterium camfestre and the com- mencement of a cavity; a later stage than Fig. 5 1 1 7. Cauliflower-petiole, showing Bundle de- stroyed by Bacterium camfestre 12 8. Melon-wilt due to Bacillus tracheiphilus. 13 9. Cross-section of Bundle of a Cucumber- stem, showing Bacillus tracheiphilus restricted to the Spiral Vessels and Three pitted vessels 15 ILLUSTRATIONS. Page. FIG. 10. Datura metelloides Inoculated by Needle- pricks with Bacterium solanacearum. The same plant as in Plate 4, but six days later 17 11. (a) Capsule of Organism plated from Black Spot of Plum; (b) Viscid Cul- ture-medium from which a was ob- tained 18 12. Yellow Ooze from Black Spot of Plum stained by ordinary method 19 13. Tenuous Threads of Bacillus tracheiphi- lus drawn from a Muskmelon Stem . . 19 14. A detail from Fig. 13, highly magnified. 19 15. Flagella stained from a pure culture of a Bacterium grown in Water contain- ing a few drops of Uschinsky Solution. 21 16. Beyerinck's Drop Bottle 21 17. Double Blow Bulb 22 18. Short Form of Bacterium campestre when crowded 23 19. Long Form of Bacterium campestre when grown on Sugar-agar 23 20. Hanging-drop Culture 24 21. Involution-forms of Bacillus tracheiphi- lus 24 22. Y-shaped Forms from Root-tubercles of Clover 24 23. Zeiss Compensating Ocular, with Screw or Filar Eye-piece Micrometer 25 24. Zeiss Upright Photomicrographic Cam- era 26 25. Hand-lens for examining Bacterial Cul- tures 27 26. Hand-lens for examining Bacterial Cul- tures, showing another form of mount. 27 27. Zeiss Cover-glass Measurer 28 28. Nelson's Photographic Gelatin 30 29. Agar-agar as received from Japan. (Slender "Kanten") 31 30. Another form of Agar-agar made in Japan (Square "Kanten") 32 31,32. Gelidiums furnishing Agar-agar... 33,34 33. Agar-agar Flour as received from Euro- pean Manufacturers 35 34. Schleicher and Schull's Folded Filter Papers 36 35. Thermo-regulator for Blood-serum Oven. 37 36. Iris-rhizome-rot Organism grown on Sterile Raw Carrot 41 37. Tin-box in which Pipettes, Scalpels, etc., may be sterilized 42 38. Fluid Culture showing rise of Viscid Precipitate when twirled rapidly 42 Page. FIG. 39. Platinum-iridium Transfer Wires 43 40. Simple way of filtering with Chamber- land Bougie 44 41. Roux Filter for separating Bacteria from their Products 45 42. Section of the Arnold Steam Sterilizer, showing Principle of Action 46 43. Lautenschlager Centrifuge 47 44. Wire-crate for holding Media to be ster- ilized 48 45. Oven for use in solidifying Blood-serum, etc., at Temperatures below 100 C. .. 49 46. Simple Rack for holding Fermentation tubes 52 47,48,49. Fermentation-tubes in actual use. . 53 50. Ordinary Kipp Gas-generator 54 51. Hempel's Burettes for Gas-analysis 55 52. Hempel's Simple Pipette for Liquid Re- agents 56 53. Small Novy Jar 57 54. Large Novy Jar; the most convenient Form 58 55. Simple Device for growing organisms in Nitrogen ....'. 59 56. Test for Reduction of Nitrates to Ni- trites 63 57. Crystals formed in Nutrient Agar as the Result of Bacterial Growth 66 58. Thick-walled Flask for Filtration or Evaporation in vacua 67 59. Titration-devices 68 60. Sodium-hydrate Bottle 69 61. Effect of Sunlight on Pear-blight Ba- cillus 71 62. Effect of Sunlight on Bean-spot Bacte- rium 71 63. Water-bath for Thermal Death-point Experiments 76 64. Roux Metal-bar Thermo-regulator 77 65. Thermometer for Thermal Death-point Experiments 79 66. Leveling Apparatus 80 67. Dewar Glass for Experiments with Liquid Air 81 68. Petri-dish Poured Plate inoculated with a measured quantity of a Bouillon Culture of Bacillus tracheiphilus 82 69. The same as Fig. 68, but poured after Exposure to Liquid Air 83 70. 'Stomatal Infection by Bacterium pruni in Green Fruits 84 71. Stomatal Infection by Bacterium pruni in Leaf 86 72. Stomatal Infection by Bacterium pruni a. Later Stage in Fruit 88 FIG. 73. Seedling Sweet-corn Plant in Stage when most of Infections occur 89 74. Stomatal Infection of Sweet-corn Leaf by Bacterium Stcwarti 90 75. A Detail from Fig. 74, highly magnified. 91 76. Water-pore Infection by Bacterium cam- pestre 92 77. Bacteria from Fig. 76, enlarged 2,000 93 78. Single Spiral Vessel occupied by Bac- terium campestre 93 79. Water-pore Infection in Cabbage; a later stage than that shown in Fig. 76. 94 80. Angular Leaf-spot of Cotton, Nearly Natural Size 95 81. End of Vacuum-pipe on Laboratory- table 96 82. Portion of Work-table, showing Simple Apparatus for Distilling Water 97 83. Apparatus for rapidly filling Test-tubes with Measured Portions of Fluid Cul- ture-media 98 84. Can for holding Culture-media 99 85. Wrapped Petri Dishes 100 86. Meyer's Hypodermic Syringe 101 87. Sections through Tooth of a Cabbage- leaf, showing Entrance of Bacterium campestre 102 88. Green Cucumbers soft-rotted by Bacillus carotovorus 103 89. Block for holding Test-tube Cultures. .. 104 90. Constant Burner, with Cut-off for re- ducing Size of Flame 105 91. Steel Sewing Needle (Nu. 10) set into Bone-handle and used for Puncture- inoculations 106 92. Compressed-air Tank and Spray-tube... 107 93. Atomizers for use with 92 108 94. Hand-sprayer for Distribution of Bac- teria I0 9 95. Inoculation Cage for Herbaceous Plants, no 96. Labels from Test-tube Cultures m 97. Wooden Labels from Inoculated Plants, in 98. Temperature-record Sheets 112 99. Nitrate-bouillon Records 113 100. Sample from Card-catalogue, Two-thirds Actual size 114 101. Heading of Large Sheet for Volumi- nous Abstracts 1 14 102. Green-cucumber Skin, Contents rotted out by Bacillus aroideae 115 103. Pillsbury Slide-boxes 116 104. Another Form of Pillsbury Slide-box. .. 117 105. Small Paraffin-oven used by writer 118 ILLUSTRATIONS. Page. ... 119 hold- XI Page. FIG. 106. Infiltrated Tissues embedded in Par- affin in a Watch-glass 1 19 107. Infiltrated Material mounted ready to cut 108. Drawer with Compartments for ing embedded material 120 109. Coplin's Staining Jar 121 no. Coplin's Staining Jar, cross-section 121 in. A Series of Coplin's Staining Jars Ready for Use 121 112. A Page from the Paraffin-record-book.. 122 113. A Mounted Slide of Serial Sections 122 114. A, Rodgers knife for serial sections; B, Lentz knife for cutting hard material with slant stroke ; C, Torrey knife for serial sections; D, Torrey knife for free-hand sections, a, b, c, d, end views of A, B, C, D 123 115. Leaf -tooth of Cabbage infected by Bac- terium campestre 124 1 16, 1 17. Details from Fig. 1 15 124, 125 118. Stomatal Infection of Cotton-leaf by Bacterium mahacearum 126 119. The Reinhold-Giltay Microtome Ar- ranged for cutting Celloidin, etc 127 120. Sub-stage Arrangement on Zeiss Stand Ic 130 121. Newer Form of Zeiss-Abbe Camera 131 122. Zeiss Planar Lenses 132 123. Apparatus for Photographing Natural Size 133 124. Swinging Camera for Equal Lighting of Exposed Object 134 125. Petri-dish Poured Plate photographed by transmitted light 135 126. Green Leaf (Delphinium) with Black Spots; photographed on a rapid non- isochromatic plate 138 127. Green Leaf (Delphinium) with Black Spots; photographed on a slow i so- chromatic plate 139 128. The Wager Exposure-scale 141 129. The Collins-Brown Camera, made by Folmer & Schwing 145 130. Cross-level for use with Camera 146 131. Device for cutting out light in Air-shaft. 146 132. Side-view of a Dark-room, convenient for a few persons 147 133. Top-view of a Dark-room, convenient for a few persons 148 134. Side-view of another Small Dark- room . 148 '35- Top-view of a Small Dark-room shown in Fig. 134 149 XII ILLUSTRATIONS. Page. FIG. 136. Case for protecting Squeegee-plates from Dust and Scratches 149 137. Bacterium triloculare, Ehrenberg"s first figure 166 138. Bacterium triloculare, Ehrenberg's sec- ond figure 169 139. Bacterium termo, figured by Cohn 170 140. Dallinger and Drysdale's conception of Bacterium termo 170 141. TVrmo-like Organism obtained by throwing Beans into Water 170 Page. FIG. 142. Iris-rhizome-rot; Crowded Agar-plate after 45 hours at 25 C 179 143. Iris-rhizome-rot ; Thin Sowing on Agar at end of 4 days ; temperature 25 C. . 180 144. Bacillus aroideae grown on Agar-plate at 37 to 38 C 182 145. Bacillus aroideae grown on Agar-plate at 25 C 183 146. Apparatus for Gradual Substitution of Alcohol for Water in Tissues 184 BACTERIA IN RELATION TO PLANT DISEASES. BY ERWIN F. SMITH. BACTERIA IN RELATION TO PLANT DISEASES. BY ERWIN F. SMITH. PART I. AN OUTLINE OF METHODS OF WORK. GENERAL REMARKS. The following outline of methods for the study of bacterial diseases of plants, which are now in use in the Laboratory of Plant Pathology, United States Depart- ment of Agriculture, has gradually assumed its present shape as a result of the writer's field, hot-house, and laboratory experiments during the past thirteen years. In nearly the same shape, so far as arrangement is concerned, but in a less complete form, it was published in the American Naturalist in 1896.* The scheme here presented is entirely practicable and is believed to be not more extended than the exigencies of the case require ; in the interest of better methods of work in plant pathology it is recommended to all who contemplate a special study of bacterial diseases of plants, and also particularly to those who intend to describe and name species of bacteria, whether pathogenic or nonpatliogenic. Those who doubt the necessity for so much work are advised to read procedures recom- mended for the study of bacteria by a committee of the American Public Health Association, and the earlier paper by H. Marshall Ward (Bibliog., III).f It would be still more to the point if they would isolate a dozen bacterial organisms from the soil, air, or water, and undertake faithfully to identify them by means of any of the older descriptive works, e. g., Eisenberg's Diagnostik or Saccardo's Sylloge Fun- gorum, or even by such recent manuals as those of Sternberg, Lehmanii & Neumann, Fliigge, Migula, or Chester (Bibliog., III). Everyone who has carefully inquired into the matter knows that the brief statement of the behavior of an organism on nutrient agar, on gelatin, and on two or three other media, with perhaps a loose statement of its color and size, no longer constitutes a description which describes. Such accounts, of which there are a great many, usually fail to mention just those things which might serve to distinguish the organism from its fellows. If a new species is not to be described so that it can be identified by others, what then is the use of any name or any description ? The name will only serve to encumber future synonymy and to recall the incapacity of its author. *The bacterial diseases of plants: A critical review of the present state of our knowledge, parts i-vi, Am. Nat., August and September, 1896. tFor Bibliography see end of volume. 3 BACTERIA IN RELATION TO PLANT DISEASES. THE DISEASE. The line between disease and health is sometimes a very narrow one, especially when nothing more is involved than some slight change in function. The difference, however, is very striking in many of the diseases here considered. The writer has used the word "disease" in the common acceptation of the term, meaning thereby J/B Fig. I.* any marked deviation from the normal functions or structure of the plant as it now exists, whether wild or greatly modified by cultivation. In a sense, such a change as has taken place in the cauliflower, the normal flower-shoots of which have become *Fic. i. Cross-section of the upper part of a sweet-corn stem parasitized by Bacterium Stcwarti (Erw. Sm.). The location of the bacteria is indicated by black shading. Most of .the affected bun- dles are on the periphery. The (bacteria 'have not escaped into the parenchyma. Jamaica, Long Island, N. Y., July 16, 1902. The section was taken 'several feet from the ground, but the stem in- fection undoubtedly took place #hrough one or more of the flower nodes. Drawn from photomicro- graph of a section stained -with carbol-fuchsin. Exactly similar sections, but with a larger number of infected bundles, have been cut from stems of sweet --corn plants infected by the writer in August, 1902, during the seedling stage shown in fig. 73. THE DISEASE. 5 compacted, aborted, and enlarged into a fleshy edible mass, might well be regarded as a diseased condition, but it is not so regarded for the purposes of this book. On the contrary, a soft rot of the cauliflower head is regarded as a disease. Bacterial diseases of plants usually involve both functional and structural changes. Inasmuch as the word " symptoms " has a subjective as well as an objective connotation in medical terminology, the writer has preferred to substitute the word " signs " for those objective characters which serve to distinguish one plant disease from another. Fig. 2 * The student will, naturally, first turn his attention to a careful study of the disease. Under this head should be considered : (i) Previous literature ; (2) Geographical distribution ; (3) Signs of the disease ; (4) Pathological histology ; (5) Direct-infection experiments. * FIG. 2. Cross-section of a raw carrot, showing wedging apart of parenchyma cells by Bacillus carotovorus Jones; from paraffin-infiltrated material. The carrot was fixed in strong alcohol 72 hours after placing on rts cut surface one loop of a fluid culture. The inoculation was made in Bhe .middle of a cross-section of five whole root, I cm. thick, placed in a sterile Petri dish. The surface of the root was sterilized in mercuric chloride water. This section was made several millimeters l>elow the inoculated surface. A small portion of it at X is 'shown more highly magnified in fig. 3. This section was staine Fig. 11* MORPHOLOGY. SIZE, SHAPE, ETC. The smallest observed bacteria are only a small fraction of a micron in diameter. Migula states that the stained rods of Ps. indigofera (Voges) Mig. from colonies 36 hours old measured only 0.18 by 0.06 micron. Bacillus denitrificans (Amp. & Gar.) Mig. is also a very small rod i.o to 1.5 by o.i to 0.3 micron, according to Migula. Micrococcus progrediens Schroter is said to be only 0.15 micron in diameter. The organism of peri-pneumonia isolated by Nocard & Roux is more minute. It is probable also that still smaller organisms occur, i. -* i? V'-'-'- "- * } ^L Fig. I3.t Fig. 144 *Fic. 12. A portion of the yellow ooze from the black spot of the plum, stained by ordinary methods. X 2,000. tFic. 13. Cobwebby, sticky threads of Bacillus tracheiphilus drawn from the cut end of a muskmelon stem, arranged on a slide and stained with carbol-fuchsin. About three times natural size. Buzzards Bay, Mass., Oct. 8, 1903. Fig. 14 was drawn from the left-hand thread at the point marked X. JFic. 14. Bacillus trachciphilus Erw. Sm. A portion of one of *he threads shown in fig. 13. T*he arrow indicates the direction of the thread, which was extremely tenacious. The distance be- tween the bacterial rods indicates very clearly the extreme viscosity of iBhe unstained substance lying between them and holding them together. X 1,000. 2O BACTERIA IN RELATION TO PLANT DISEASES. cially commended by Dr. Welch ('92, Bibliog., XIII), the capsule is also stained, but remains distinctly paler than the body of the bacterium. They may also be counter- stained, as in Muir's method or Moore's method. Well-defined capsules are shown in fig. 1 1 a. This may be compared with fig. 12, in which the same organism is shown without capsules. Fig. nt> shows the extreme viscidity of a culture due to the formation and deliquescence of capsules. Fig. 13 shows the tenuous threads into which Bacillus tracheiphilus may be drawn as it oozes from the cut stems of cucurbits. Fig. 14 is a detail from the same more highly magnified, the viscid con- necting substance being unstained. FLAGELLA. Ehrenberg was the first to describe flagella on bacteria (Bacterium triloculare, 1838). Nothing more was done until 1872, when Colin discovered them on Spi- rillum vohitans. In 1875 Dallinger & Drysdale saw and figured them on Bacterium tcrmo. In 1875 Warming determined their existence on Vibrio rugula and Spi- rillum tmdula. In 1877 Koch demonstrated their existence on a number of species by the use of stains. In 1878 Dallinger, using unstained material, saw them many times on Bacterium termo and also on Spirillum volutans. After 1879 110 one appears to have disputed their existence. In 1890 Messea proposed to divide the flagellate bacteria into four large groups, monotrichiate, lophotrichiate, amphitri- chiate, and peritrichiate. In 1895 Fischer used the flagella as marks to distinguish subfamilies. In the previous year Migula used their number and mode of attachment as a means of distinguishing genera. The staining of flagella has now become a regular part of laboratory work. Their number and position on the body wall should be determined, if possible, in case of each species studied. This is sometimes quite easy and at other times very difficult. It should also be determined whether the flagella are fugitive or persistent. Flagella may be stained from young agar cultures. Bouillon cultures are to be avoided because of the intense ground stain. Some kinds may be stained readily from cultures grown for some days in a very dilute Uschinsky's solution i to 3 drops in 10 cc. of distilled water (fig. 15). The flagella of some bacteria are stained readily, those of others only with great difficulty. Many sorts seem inclined to throw off their flagella when transferred from agar to water. The cover-glasses must be clean. When cleaned ready for use seize with the forceps and pass them three times through the upper part of the Bunsen flame, with a considerable interval between each flaming, to avoid cracking. Use a minim quantity of the culture stirred in a big drop of water, or even in 2 to 10 cc. of water in a watch glass or test tube. Give the bacteria time to diffuse by waiting half an hour or more. Take the cover between the thumb and finger of the left hand, touch the end centimeter of a platinum needle to the water containing the bacteria, and sweep it deftly across the cover glass. In this way the fluid is spread in a very thin sheet over nearly the whole surface of the cover and is dry almost at once, with the bacteria well separated. If the fluid will not spread, then the cover is not clean and should be discarded. The bacterial sheet may be mordanted and stained at once, or first fixed by gentle heat. To avoid scorching, the cover should be held between thumb and finger when it is passed rapidly through the flame. Beginners usually burn the bacterial layer. STAINING OF FLAGELLA. 21 Smeary dark lines aud other deceptive artefacts must not be mistaken for the flagella. The following methods have been tried by the writer and have given good results, but none can be depended upon always, and much time and patience are sometimes required to get good preparations of a refractory organism : Fischer's modification of Loeffler's stain ; Moore's modifica- tion of Loeffler's stain ; Van Ermengem's nitrate ? \J / \ \. \ of silver method ; L6 wit's S \ copper-sulphate fuchsin Fi - 15 -* mordant, followed by Ehr- lich's anilin-water gentian violet. (For other methods consult " Formulae " and " Bibliography of General Literature," XII.) In connection with flagella-staining a white porcelain tray, such as photogra- phers use, will be found very convenient for washing, and also the double blow-bulb shown in fig. 1 7. This should be attached to a wash-bottle, such as that shown in fig. 16. This will deliver a small stream, veiy good for washing excess of mordant and stain from the covers. To furnish a steady stream the bulb has to be compressed only about once a minute. The flask used for this purpose should hold a liter. SPORES ENDOSPORES, ARTHROSPORES. Do arthrospores really occur? If so, in what respect do they differ from the ordinary vegetative rods? Test spores for resistance to high temperatures in the water bath and to steam heat; study germination in hanging drops. Do the spores require a period of rest or refuse to germinate except in special media? The suspected existence of spores may be definitely settled by seeing the problematic bodies germinate. In the absence of such proof, considerable certainty may be reached by a combination of two methods: (i) the use of watery basic anilin stains, and (2) the use of moist heat. If at room temperatures the glistening bodies refuse to take the simple stains even on long exposure and at the same time are very resistant to steam heat or to hot water, i. e., much more so than the ordi- nary vegetative rods, it may be assumed that they are spores. If, on the contrary, they are destroyed by tem- peratures only slightly above the recorded thermal death- point of the vegetative rods, it must not be assumed that they are spores, no matter how they behave toward Fig. 16.t *Fic. 15. Flagella of yellow organism plated 'rom black spot of plum. Stained from culture grown in 10 cc. distilled water containing a few drops of Usohinsky's solution. X 1,000. tFic. 16. Beyerinck's drop-bottle. The size and number of drops in a given time are regulated by sliding the bent tube through the cork. It is very convenient to have tiiis flask on die microscope table. By a minim infection of the fluid it may also be arranged so that each drop shall deliver a single spore or bacterium for ihanging-drop studies. About two-fifths natural size. 22 BACTERIA IN RELATION TO PLANT DISEASES. stains, unless they can be made to germinate. Many of the older identifications of spores are untrustworthy. Alfred Fischer has shown that many of these determina- tions rested on plasmolysis of the rods, i. e., on misinterpretations. Omelianski reports finding an oval spore which stains readily with ordinary anilin stains. This occurs in a rather large bacillus accompanying his hydrogen cellulose ferment. Dan- napple reports finding spores which are very sensitive to heat ('99, Bibliog., XXXIII). Usually only one endospore occurs in each cell, but Kern ('81, Bibliog., VIII), and Schaudinn ('02, Bibliog., XI) have found bacteria with two in each cell. Excellent directions for the study of spores are given in Part I of Migula's System der Bakterien (see especially the second paragraph on p. 209). CBLL-UNIONS ZOOGI.CE^, CHAINS, FILAMENTS. In some media bacteria are much inclined to separate after division ; in others they remain attached in various ways. The most common method of union is an irregular clumping, which in fluids gives rise to a fine or coarse flocculence. Such unions also occur on solid media and may be designated zoogloese, or pseudo- zoogl&a, if one prefers to retain zooglosae for the more intimately fused and com- pacter gelatinous unions. Sometimes the organisms remain attached end to end. Where the segmentation is distinct, such unions are designated chains. When very long and with obscure segmentation, they may be called filaments. Is there any true branching? What especial conditions of the culture medium favor the formation of zoogloese, of chains, and of filaments? Many bacteria form zooglcese, chains, or very long filaments under certain conditions, while under other Fig. 17* conditions they remain as very short, straight rods. (Compare figs. 18 and 19.) As in case of involution forms unfavorable cultural conditions (thermal, nutrient, etc.) appear to have much to do with their appearance. The growth of bacteria may be studied in hanging drops of bouillon, etc. Hol- low-ground slides (fig. 20) should be used for this purpose, rather than ring-cells, especially with high powers. Hill's hanging-block method is also serviceable ('02, Bibliog., XVII). *Pic. 17. Double blow-bulb for attachment to drop-bottle shown in fig. 16. By use of this de- vice one obtains with a minimum of pumping a constant small stream of water very suitable for washing stained covers, etc. Made by Emil Greiner. It is best used with a larger flask than that shown in fig. 16. Bulbs which have been long in stock should not be purchased, as the rubber de- teriorates rapidly. MORPHOLOGY. INVOLUTION FORMS. Under this name we designate swollen and distorted forms common in old cultures (fig. 2 1). Under what conditions do they occur ? Are they living or dead ? Isolate in hanging drops of bouillon and determine whether they are stages in development or only degenera- tion forms. Are Y-shaped or branched forms such as occur in old cultures of B. tuberculosis Koch, and in the root-tubercles of clover (fig. 22) to be considered as involution forms ? Are such organisms fungi or bacteria ? Branching forms have been detected by many observers. (Consult numerous citations in the Bibliography of General Literature, X). The most recent paper is by Albert Maassen (Arb. a. d. Kais. Gesundh., Bd. XXI, H. 3, Fig. 18* 1904, p. 377, 6 pi.). He found chloride of lithium specially advantageous for provoking these growths, which are re- garded as teratological. He obtained them in 24 hoxirs. GENERAL COMMENT. Great care should be paid to the minute morphology of each organism, not only in the host-plant but also in a variety of cultures, old and young, so that a body of knowledge more exact than we now possess shall be grad- ually accumulated for differential and systematic purposes. Careful drawings and photographs should be made. The Abbe camera is a great help in making drawings (fig. 121). For such study the Zeiss apochromatic lenses and com- pensating oculars can not be recommended too highly, particularly the 16 mm., with the 12 and 18 compensating oculars for studying the margins of colonies, and the 2 mm. 1.30 n. ap., with the 8 and 12 compensating oculars for the more detailed study of the individual rods. The writer has also made much use of the Zeiss 3 mm. 1.40 n. ap. apochro- matic objective. The Zeiss screw, or filar, micrometer com- bined with a No. 12 compensating ocular (fig. 23) will be found very useful. For photographic purposes the projec- tion oculars or the 4 or 6 compensating oculars may be used. Robert Koch was entirely correct in saying : "A general use of photography in microscopic works would certainly have prevented a great number of unripe publications." Fig. 19.t *Fic. 18. Bacterium camfestre. Cover^glass (smear) preparation from the vessels of a cab- bage plant received from Racine, Wis., Sept. 19, 1896. Stained with carbol-fuchsin. Drawn from a photomicrograph. X 1,000 circa. tFic. 19. Bacterium campestre from an old culture on 23 per cent grape-sugar agar, showing long filaments. Cover stained I hour and 20 minu tes in gentian violet ( I part saturated alcoholic solution plus I part water). Many of the rods stained feebly. Tube inoculated June 30, 1898. Cover prepared Aug. 8. Drawn directily from the slide. X 1,000. 24 BACTERIA IN RELATION TO PLANT DISEASES. Good photomicrographs should be seciired if possible. Koch's first photo- micrographs were of various enlargements. He afterwards recommended X 1,000 as the standard magnification, but X 1,500 and X 2,000 are also convenient sizes and occasionally X 500 is better than X 1,000. Most important is it that the exact mag- nification should always be indicated. The Zeiss apochromatic objectives are much better for photographic work than the achromatic ones. For very small magnifica- tions the writer has found the old Zeiss 35 mm. and 70 mm. very useful. For the same purpose the newer Zeiss planars, series la Nos. 15 (fig. 122) are admirable. These have sharp definition and a very flat field, but not much depth of focus. With them objects several centimeters in diameter may be satisfactorily photographed with magnifications from 2 or 3 diameters to 50 or more. The writer obtains as sharp a focus as possible with wide-open diaphragm and then stops down about two-thirds. Fig. 20 * One of the best simple photomicrographic outfits is the Zeiss upright camera (fig. 24). All apparatus is to be rejected which requires the microscope to rest on the same platform as the camera. It should rest on the table independent ot the camera, unless a weak light is used and the exposures r\ are very long, in which case a slight jarring is of no great (1 consequence. Direct sunlight is the best light, but J} \i ^~~ A 8 t ^ ie ^&ht f tne P en s ky ma y be used (with full open " g- 28.* teriological laboratory is so uncer- tain and variable in its composition as gelatin. The gelatin from different factories varies greatly and hardly any two batches from the same factory are alike. One glue chemist has defined gelatin as " 80 per cent glue, 10 per cent dirt, and 10 per cent doubt." It varies greatly in its melting point and power of setting, and in amount of peptones and albumoses it may contain, which is sometimes large. It always con- tains calcium salts and phosphates, which are often antiseptic, and the nature of which varies according as hydrochloric or sulphurous acid has been used in its manufacture. Formaldehyde is sometimes added to it, we are told ; and occasionally agar also, it is *Fic. 28. Nelson's photographic gelatin No. I. Recommended for bacteriological use. VARIABILITY OF GELATIN. 3! said, is added to certain table gelatins to increase their body. Gelatin also contains a variety of decomposition products due to the growth in it of various fungi and bacteria while it is in the vats or in the drying-house. If there is any delay in the drying it is spotted all over with molds and bacteria. It also contains some wax or grease, used to anoint the surface on which it is spread to dry, and this wax or grease is probably also a variable substance. Gelatins also polarize, it is said, in many different ways. An absolutely pure gelatin of uniform character for bacterio- logical purposes is not to be had. That which perhaps comes the nearest to it and which is here recommended is Nelson's gelatin, made in L,ondon and well known to the makers of photographic dry-plates, who use it in large quantities. It comes in two grades, a hard and a soft, and costs about $1.25 per pound. No. i, that which I like best, comes in shreds resembling " excelsior " used for packing (fig. 28). No. 3, which comes in long, broad strips, contains much cell detritus, etc., and filters with difficulty. Other expensive gelatins, said to be of quite uniform quality, are Fig. 29* Lichtdruck gelatin, made by Carl Creutz, Michelstadt, in Hesse, and Geneva Red Cross gelatin made by Winterthur, in Switzerland, under direction of Dr. Eder, of the Imperial Institute of Vienna (Cockayne ). NUTRIENT AGAR. Agar, or agar-agar, as it is usually called, from a Malay word meaning " vege- table," is a manufactured product obtained from various sea-weeds growing in Chinese and Japanese waters. Various species are used as food and the trade is con- siderable. It usually comes into the hands of the bacteriologist as long, slender, yellowish-white strips (fig. 29) or as blocks (fig. 30), or more especially in recent years, in the form of a gray-white fine powder of European manufacture (fig. 33). It is reputed to be the product of species of Gelidium (figs. 31 and 32). *Fic. 29. The kind of agar-agar usually employed in bacteriological work. This is a manu- factured product known to die Japanese as slender " Kanten." The figure represents first quality " Kanten," in unbroken package. (Courtesy of Dr. Hugh M. Smith, Deputy Commissioner of the United States Bureau of Fisheries, who brought the package with him from Japan.) BACTERIA IN RELATION TO PLANT DISEASES. Of the Japanese algse in this group the following, according to Rein (pp. 81-82), deserve special mention : (i.) Chondrus punctatus Sur. (2.) Gigartina tenella Harvey; Jap. Ogo. (3.) G. intermedia Sur. (4.) Gloiopeltis tenax Kg. (Sphaerococcus tenax Ag.) (5.) Gl. capillaris Sur.; Jap. Shiraga-nori. (6.) Gl. coliformis Harv. ; Jap. Kek'Kai. (7) Gl. intricata Sur. ; Jap. Fu-nori. (8.) Gelidium corneum Lamouroux; Jap. Tokoroten-gusa. (9.) G. Amansii Lamour. (10.) G. cartilagincum Gail. (n.) G. rigidum Grev. ; Jap. Tosaka-nori, i. e., cockscomb alga;. (12.) Sphaerococcus confervoidcs A.; Jap. Shiramo. (13.) Gymnogongrus ftabclliformis Harv.; Jap. Home-nori. (14.) G. japonicus, Sur.; Jap. Tsuno-mata. (15.) Kallymenia dentata; Jap. Tosaka-nori. (16.) Porpliyra vulgaris Ag. ; Jap. Asakusa- nori. Fig. 30.* Agar-agar is a neutral or nearly neutral substance which is converted by boil- ing with water into a stiff jelly that hardens in i per cent solution at 39 to 40 C., and is not easily liquefied either by the growth of organismsf or by heat less than that of boiling water. It is a kind of vegetable glue forming a good matrix for various nutrient substances. A chemical analysis by Karten (Descript. Cat. Int. Health, Exhib., London, 1884) gave the following proximate composition : 11.71 per, cent nitrogenous matter (albumen [?]), 62.05 per cent non-nitrogenous matter (evi- dently glue, the pararabin of Reichardt), 3.44 per cent ashes, and 22.80 per cent water. *Fic. 30. Another form of agar-agar known to the Japanese as square " Kanten." The bulk of this goes to Holland, where it is used for clarifying schnapps. Courtesy of Dr. Hugh M. Smith. The actual slize of these sticks is about 10% by 2^2 by 1^4 inches. tMetcalf has described a bacillus which slowly softens k, and the writer has observed similar phenomena. PREPARATION OF NUTRIENT AGAR. 33 For a full account of Japanese methods of making agar-agar consult a paper entitled "The Seaweed Industries of Japan," by Dr. Hugh M. Smith, in the Bulletin of the United States Bureau of Fisheries for 1904. In addition to beef bouillon, or in place of it, various sub- stances, organic and inorganic, may be added to^the agar with advantage. The writer makes much use of litmus-lactose agar, which is made out of ordinary nutrient agar by adding i per cent milk-sugar and enough pure litmus water to give a pur- ple-red color. Glycerin-agar, waltose-agar, etc., may be made up with any amount of the sub- stance desired, generally i or 2 per cent. Formerly it was difficult to filter agar perfectly clear and it was therefore used less than gelatin, but in recent years it has been discovered that this difficulty may be overcome if the agar is first brought into complete solution by prolonged boiling or by a short boiling at a temperature somewhat above 100 C, e.g., uoC. The writer formerly obtained filtered clear agar by soaking the snipped agar in 5 per cent acetic-acid water for some hours, after which a thin cloth was tied over the mouth of the beaker securely, and tap water allowed to run into it for an hour or more /'. e., until all trace of acid was removed. The softened agar was then put into the bouillon, boiled for two hours, and finally filtered through S. & S. filter 6 Fig. 3 1* *Fic. 31. Red sea-weeds from which agar-agar is manufactured, a, Gelidium corncum Lam., one-third natural size; b, Gelidium subcostatum Lam., one-half natural size. From a colored Jap- anese chart showing " The principal aquatic plants of Japan," supposed to be an official publication. Original in the library of the United States Fish Commission. 34 BACTERIA IN RELATION TO PLANT DISEASES. paper,* using a hot-water funnel. Later he followed Schutz's method ('92, Bibliog., XVI), which is a very good one. This consists in cutting the agar into small bits and first heating it very hot in a beaker or enanieled-iron dish in a minimum quantity of water or beef-bouillon over a hot Bunsen flame with constant and rapid stirring and Fig. 32.f *The folded filter papers are the most convenient (fig. 34). These filter papers give the starch reaction (blue) with iodine, and reduce Fehling's solution on being boiled in it. tFiig. 32. -Unnamed species of red sea-weeds (GV/jrfm) furnishing agar-agar. From a Japan- ese chart showing " Tlie principal aquatic plants o f Japan," supposed to be an official publication. One-half natural size. Original in library of United States Fish Commission. PREPARATION OF NUTRIENT AGAR. 35 *X3fe ' ' ' ' ..' . ' * ..._. , *- ~^ ii n^ occasional additions of small quantities of water until it is thoroughly cooked in the form of a thick mush. It is then put into the remainder of the water or bouil- lon and subjected to streaming steam for two hours, after which, if the first heating was sufficient, it niters readily without the use of a hot-water filter, or the necessity of keeping it in the steamer during the filtering. The stirring rod must touch all parts of the bottom of the dish exposed to the flame, every few seconds during the preliminary' heating, otherwise the agar will burn on and be spoiled. On some ^s<~^-^ accounts it is best to begin operations with beakers rather than the enameled iron dishes. In this way all likelihood of using burned agar is avoided, since the moment the agar burns on the beaker cracks and the agar is spilled. For bacte- riological use agar should be clear, not cloudy or filled with unremoved precipitates. The writer now employs an autoclave and uses an agar flour procured from Lautenschlager or Merck (fig-33). If one has an au- toclave the preliminary heating of the agar in an open dish with a minimum quantity of water and all the subsequent stages may be dispensed with and the entire process carried on in the autoclave, unless it is known or suspected that media heated in the autoclave are less well adapted to the growth of par- ticular organisms than those pre- pared at 1 00 C. The amount of agar added to the culture fluid is usually i per cent. On the making of nutrient agar Fig. 33.* consult "Formula:," and the various standard text-books. Is there any difference in the appearance of colonies when grown at 5 to 10, 15 to 20, and 30 to 37 C.? Observe the amount of precipitate that collects in the fluid in the V. For other observations as to growth on this substratum see "Gelatin." Every organism should be studied in numerous Petri-dish poured-plate *Fic. 33. Agar-agar flour as received from European manufacturers, agar flour. Package of Merck's BACTERIA IN RELATION TO PLANT DISEASES. cultures. Too many plate cultures can scarcely be made. Dishes with flat and very thin bottoms (0.3 mm.) are desirable for some purposes, but are difficult to procure. For quantitative work, plates with flat bottoms are necessary, and when photographs are likely to be wanted plates must be selected which do not have rings, wavy places or other flaws in the glass on the bottom. There is room for much improvement in the quality of the Petri dishes now on the market. The stxident is advised to use agar media for all general laboratory work. When he has learned the behavior of an organism on nutrient agar, he may then try gelatin. Do any of the organisms under observation soften or liquefy the medium ? Agar roll cultures may be made in test tubes readily if the amount of fluid agar is reduced to one-half cubic centimeter. When colonies are to be counted, special pains must be taken to dis- tribute the gelatin or agar uniformly over the bottom of the dish. Various persons Pake, Jeffer, Weiss, Mace", et al. have devised ruled plates for counting the number of colonies of bacteria in Petri-dish poured plates. The writer prefers to count by square centimeters or frac- tions thereof. When the plate is sown thin enough, the entire number of colonies should be counted. When it is very dense, the average may be taken of ten square centimeters se- lected with care, provided the bottom is flat, otherwise the whole plate must be counted. If the counting plate is to be placed under the dish, it may be opaque, i. e., a black surface with white lines, not the reverse. If it is to be placed on top of the dish, the latter preferably bottom up, then it should be of glass or some other transparent substance. The spaces may then be ruled on with a diamond, or drawn on in very fine black lines with India ink. The gelatin film of an unexposed, fixed photographic dry-plate is a very good surface for holding the ink. For counting colonies on very densely sown plates, the writer has found convenient a rectangle 20 mm. by 5 mm. divided into tenths. SIUCATK JBW.Y. In recent years, in the hands of Winogradsky and his students, silicate jelly has played an important part in the isolation of various organisms, which do not take Fig. 34* *FiG. 34 Folded fiker papers made by Schleioher & SdiiiU. PREPARATION OF SILICATE JELLY. 37 kindly to culture media containing animal and vegetable products. It is desirable also for exact experiment with other organisms. It may be used in Petri dishes or flasks, or slanted in test tubes. Along with some disadvantages, e. g., tendency to split, it has a number of valuable characteristics, not least among which is the fact that it enables one to offer the organism a solid substratum which is at the same time purely synthetic. It is generally considered to be very difficult to make, but by following the most recent directions of Ome'lian- ski ('99, Bibliog., XXV), and especially certain slight modifications introduced by Moore & Kellerman and by the writer and his assist- ants, it can be prepared without difficulty, and to it may be added any mineral nutrient substances desired. The writer makes it in the following way: To each 100 cc. HC1 (sp. gr. 1.10 Beauine") is added drop by drop 100 cc. sodium silicate (sp. gr. 1.09), the mixture being stirred continually with a glass rod. This is now placed in a collodion sack and dialyzed for some hours in running water. To this is then added in concentrated sterile form whatever synthetic culture medium is desired, after which the jelly is put into Petri dishes or test tubes and sterilized by heating for three hours in the blood-serum oven (fig. 45) on five consecutive days at 90 C., or by one steaming in the autoclave for 15 minutes at 110 C. The thermo-regulator shown in n g- 35 i s useful for maintaining a constant high temperature in the oven. The oven must also contain some water in a capsule or beaker. It is believed that a more detailed account of the manipula- tions connected with the preparation of silicate jelly will be welcome to many. First of all, one must have dialyzing sacks. Collodion sacks are much more convenient than parchment sacks, since they can be prepared at any time, and dialysis takes place through them with great rapidity. They are useful for so many purposes that material for making them should be on hand in every laboratory. The writer follows Kellerman in making his sacks inside of test tubes. These may be large or small according to what the sacks are to be used for. If for dialyzing silicate jelly in some quantity, it is very convenient to make the sacks inside of test tubes 7 inches long and having an internal diameter of i inch. The first thing is to prepare the collodion mixture. This is made by dissolving soluble guncotton, such as is used by photographers, in a mixture of abso- lute alcohol and sulphuric ether. The writer uses equal parts of these two fluids. If too much alcohol is used, the sacks dry slowly, and if too much ether they are said to become brittle. After some Fig. 35.* *FiG. 35. Tollen's thenmo-regulaitor for maintaining blood-serum oven at 80 to 90 C. The stem and bottom of the bulb contain mercury. The remainder of the bulb is filled with glycerin. In the similar thermo-regulator used for the paraffin-ibath chloroform replaces the glycerin. Actual height, 12 inches. Chloroform and glycerin are very useful in such thermo-regulators be- cause their coefficient of expansion is much greater than that of mercury. Toluene may also be used witfi mercury. 38 BACTERIA IN RELATION TO PLANT DISEASES. experimenting it was found that 5 grams of the clean, white guncotton per 100 cc. of the fluid gave a solution very satisfactory to work with. About 24 hours is required to dissolve the guncotton into a homogeneous mixture, of which there should be at least 800 cc. This should be stored in a cork-stoppered bottle of shape convenient to hold in one hand. It is then ready for use. The clean test tube, thoroughly dry on the inside, is now held in one hand in a slanting position, mouth tip, while with the other the collodion is poured slowly and steadily into the tube, while the latter is slowly rotated. In this way air-bubbles are avoided and the entire interior of the tube is moistened. When this has taken place and about an inch of fluid has accumulated in the bottom of the tube, the excess is poured back into the bottle, slowly rotating the slanted tube, as before, so as to cover again the entire interior with as imiform a layer as possible. When the bulk has been poured back, the tube is stood upright, mouth down, to drain on a sheet of clean paper. In two or three minutes it will have drained sufficiently, the excess of accumulations about the mouth being wiped off on the paper now and then. The tube is then seized and rotated in a horizontal position for four or five minutes with the mouth in the draft of an electric fan, or the rotation may be somewhat longer if no air-current is available. A little experience will tell when the sack is dry enough to remove from the tube. The strong smell of ether must have somewhat subsided and the collodion must not feel wet around the mouth of the tube, as will be the case if the layer of collodion is too thick in places. If it is taken out in this condition, the thick, wet places will become clouded. The collodion is now cut free at the lips of the test-tube by means of a pin-point or other sharp instrument and the tube is filled with cool water, taking care to let it also flow between sack and wall of tube if there is any shrinkage. In a minute or two, if the work has been well done, the sack, free from air-bubbles and filled with water, may be readily lifted out of the tube. It is then placed in a jar of water, where it remains until it is ready to receive the sub- stance to be dialyzed. These sacks are quite tough, and there is little danger of tearing them during filling and tying. When the silicate jelly or other substance has been placed in them, the mouth is brought together and tied by means of a small rubber band, the elasticity of which keeps the sacks perfectly tight. Silicate jelly should be dialyzed for at least 1 2 hours, and sometimes for 24 hours, if every trace of salt must be removed. The writer fills the sacks with the silicate jelly in the afternoon and leaves them in running tap water over night. The next morning they are taken out, their contents emptied into a clean beaker, the nutrient salts added, and the fluid immediately pipetted into tubes, flasks, etc., and sterilized by heat. The nutrient substances should be dis- solved in advance, so as not to delay the preparation of the medium. They should be added for this purpose to a minimum quantity of water. Some dissolve slowly, and there is a preferable order of solution, the glycerin being added last in case of Fermi's solution. For the preparation of silicate jelly a Beauine" hydrometer for liquids heavier than water is used. C.P. hydrochloric acid of any specific gravity is diluted with distilled water until it tests 1.10 on the scale of the hydrometer when cooled PREPARATION OF SILICATE JELLY. 39 to 60 F. Clear homogeneous sodium silicate of any specific gravity is then mixed with distilled water until it is of sp. gr. 1.09 Beaum at 60 F. A great deal of water must usually be added to the sodium silicate, and the first dilution is tedious. For example, 100 cc. of a sodium silicate of sp. gr. 1.42 required the addition of 750 cc. of distilled water to give a fluid registering 1.07 Beaume". On adding the fluid containing the nutrient salts, and hardening, sodium silicate of sp. gr. 1.07 Beaume gave a rather too fluid medium, and sodium silicate of much higher sp. gr. than 1.09 Beaumd is apt to set before it has properly dialyzed, or after adding the nutrient salts and before it can be tubed and slanted. Several liters of the diluted acid and sodium silicate may be conveniently made up at one time. When these are ready, equal volumes of the two are mixed. This is done by adding the sodium silicate drop by drop to the acid, rather rapidly, stirring meanwhile with a glass rod. The top part of the apparatus shown in fig. 146 may be used for this purpose. The salty, acid fluid is now ready to be placed in the collodion sacks for dialyzing in running water. It is ready for removal from the water when it is no longer acid to litmus and shows only traces of sodium chloride remaining. An exposure to the running water for 6 hours is scarcely sufficient, unless the sacks are small. For many purposes Fermi's solution is a good one to add to the dialyzed jelly. This is made as follows, for this purpose: Freshly-boiled distilled water, 100; magnesium sulphate, o. 2 ; monopotassium phosphate, i.o; ammonium phosphate, 10.0. Dissolve. Then add glycerin, 45.0. The dialyzed silicate jelly is now poured out of the collodion sacks into a clean beaker and brought to a boil for a minute or two over an open flame (to drive off the absorbed air). It is now cooled down to 50 C. and the Fermi added. If this has been dissolved over night it must also be brought to a boil and cooled, or have the air removed under an air-pump before adding it to the silicate jelly. To 500 cc. of the dialyzed fluid, 90 cc. of the Fermi may be added. This is stirred with a clean glass rod and then quickly pipetted into test tubes. It is now placed in the autoclave without delay in the position desired and heated for 15 minutes at 1 10 C. To avoid tearing the surface of the jelly by steam, the autoclave must be carefully shut steam-tight as soon as the air is driven out, and it must not be opened until the temperature has again fallen to 100 C. It is also necessary to keep the autoclave closed on account of loss of ammonia from the ammonium salt. For this reason it is desirable to dissolve the Fermi in freshly- boiled water and to pump out any absorbed air rather than to boil it out. Other nutrient salts may be added Uschinsky's solution, etc. The writer has had very good success with Fermi for differential purposes. Many organisms grow remarkably well on this substratum, while others do not vegetate, or make only a scanty growth. The observations on this medium are the same as for gelatin or agar. Observe character of growth, staining of substratum (green, pink), etc. SOLID VEGETABLE SUBSTANCES. These should consist of slant cylinders in cotton-plugged test tubes half covered with distilled water and steamed 20 minutes at 100 C. on each of three consecutive 4O BACTERIA IN RELATION TO PLANT DISEASES. days. The addition of considerable water enables one to keep the culture under observation for several months without danger from drying out if the cotton plugs are properly made. Drier culture media may also be used. If one wishes to do so, the potato or other substance may be lifted entirely out of the water by making a constriction in the lower part of the test tube, a la Roux, or by thrusting a wad of absorbent cotton into the bottom of the test tube before the potato is introduced. The writer has not found these methods necessary. In general, I prefer vegetable media which have been sterilized in the steamer rather than in the autoclave. The following are some of the vegetable substances recommended : (1) Potato. (5) Turnip. (9) Onion. (13) Brazilnuts. (2) Sweet potato. (6) Radish. (10) Banana. (14) Apple. (3) Carrot. (7) Salsify. (n) Coconut. (15) Pear or quince. (4) Sugar-beet. (8) Parsnip. (12) Peanuts. (16) Pineapple. These substances may be extended almost indefinitely and are very useful for making preliminary studies, inasmuch as they include many different kinds of chemical substances. The writer has used them for many years. They should be prepared with great cleanliness, especially the roots, so as to avoid including resistant spores. Sterilization is an easy and simple process if these substrata are free from spores when the steaming begins. Roots and tubers should be selected with great care, only those being taken which are sound and free from blemishes. They are now to be washed thoroughly in tap water with scrubbing and then rinsed in distilled water. With clean hands and a clean knife they are then pared, with care to remove all black specks, and thrown into a beaker of distilled, filtered or boiled water. Cylinders of the proper size may now be punched with a clean cork-borer or cut with a clean sharp knife and, after the xipper part has been slanted, are thrown into another beaker of distilled water, from which they are transferred to two others before they are finally put into the tubes. It is not necessary to soak them in water over night or in antiseptic solutions. They will not brown by oxidization if they are kept under water during the early stages of preparation and are steamed as soon as they are placed in the tubes, i. e., exposed to the air. They may be put into the tubes with clean fingers or by means of a pair of clean forceps. On these different media observe the nature, amount, and rapidity of growth (always with due regard to the air-temperature, which should be recorded) . Carefully determine whether there is any retardation of growth at first and, if so, to what it is due, so that more exact studies may be made subsequently in other media. Look for gas-bubbles, formation of acids and alkalies, formation of hydrogen sulphide, of crystals, of stains, of odors, destruction of starch, disappearance of the middle lamella, softening of cellulose, etc. For the first few days all cultures should be examined at least as often as once in 24 hours and, generally speaking, cultures should not be discarded until after the sixth or eighth week. These experiments should be repeated a number of times and the student should avoid drawing a hasty conclusion, since different samples of potatoes, carrots, etc., vary somewhat in composition and will at times give slightly varying results or even resxilts which seem to be contradictory, e.g., a. brown pigment in some instances and not in others. RAW CULTURE MEDIA. The same media, and as many other sorts as are available, should be tested raw in sterile, dry, Petri dishes 10 cm. broad and 2 to 3 cm. deep. For this purpose the vegetables are prepared as follows : First, select sound, clean specimens, especially avoiding those which are cracked open ; next, scrub their surface thoroughly under the tap, and rinse them in distilled water. They are now soaked 5 or 10 minutes, or even 20 minutes, in 1:1000 water solution of mercuric chloride. They are then removed and dried with or without a preliminary rinsing in sterile water. When dry they are put on a sterile paper or plate, are cut into slices about 1.5 to 2 cm. thick with a cold sterile knife, are picked up with sterile forceps, and are put into the Petri dishes in pairs or fours, the cover being immediately replaced. Enough of the mercuric chloride remains on the surface to inhibit the growth of any surface organisms which have not been killed outright, and experience shows that intruders are rarely dragged over the cut surface. The slices may be inoculated at once or after 36 hours incuba- tion in a moist chamber at 30 C., or 48 hours at 25 C. The latter course is preferable. In either case, half of the slices in each dish must be kept uninoculated for I Fig. 36.* comparison (fig. 36). This method is well adapted to the study of various soft-rot organisms such as Bacillus carotovorus, B. aroideiz, B. oleracetz, etc. PI.ANT JUICES (WITH AND WITHOUT THB ADDITION OF WATER). (1) Juice of the host-plant. (4) Prune-juice. (2) Potato-broth. (5) Orange-juice. (a) With sodium hydrate. (6) Coconut-water (unsteamed).t (fc) Without alkali. (7) Yellow corn-meal broth. (3) Cabbage or cauliflower broth. (8) Timothy-hay infusion. *Fic. 36. Iris-rhizome-rot organism grown on raw carrot. The check piece is unchanged, the inoculated piece has browned and softened. Incubated 4 days at about 23 C. fThis is removed directly from the nut to sterile test-tubes by means of sterile pipettes, which are useful in many ways. The pipettes should be dry-heated and kept from contamination in long, narrow, covered tin boxes. These boxes may be cylindrical or rectangular, with an end cover. The upper end of the pipette should be plugged firmly with cotton before sterilization, and this should be pushed in a short distance beyond the end, so that when the finger is placed on the end there will be an air-tight union. Scalpels, etc., should be sterilized in shorter boxes of the same kind (fig. 37). BACTERIA IN RELATION TO PLANT DISEASES. These fluids are only a few of many that may be used. Some of them, e.g., potato-broth, require special care in preparation. My own method of making potato- broth is to pass the clean pared potatoes rapidly through a grating machine and immediately throw the pulp into the re- quired quantity of distilled water (which should be twice the weight of the potato). The beaker is now put into a water-bath and the temperature rapidly raised to 55 C. and kept there with frequent stirring for an hour. The pulp is now filtered from the fluid and the latter is immediately put into the steamer. If the steaming is long delayed the broth will be dark brown (oxidizing action of the potato- enzyme on tannins in the presence of air), and if the temperature rises much above 60 C., before the pulp is removed, some of the starch becomes gelatinous and the fluid will not filter. All media which have boiled away to any Fig- 37.* Fig. 38. t considerable extent must, of course, be made up to the original volume or weight just prior to final sterilization. In these culture-fluids observe the rapidity, density, and persistency of the clouding ; whether the clouding is simple or turbid from the presence of zoogloese ; and finally, whether it is uniform in all parts of the tube. Note the character of the rim and pellicle, if any are formed, and how soon they appear ; also the amount, color, and general appearance of the precipitate. The amount of the precipitate varies greatly with different media. Its quality also varies. Sometimes it consists of loose, easily separable particles ; in other cases it is a viscid mass which rises as a rope-like unit when the tube is twirled (fig. 38). Record the formation of acids, alkalies,! odors, gas-bubbles, stains, crystals. Does the fluid become viscid or ropy ? Some organisms bring about this condition quickly in a variety of media, e. g., Bacterium pericarditidis (Bacillus pyocyaneus pericarditidis), others rarely or never. Precipitates in test-tube cultures vary all the way from a scarcely perceptible trace to masses a centimeter or more in depth. Do not confound chemical precipitates with bacterial growth. Before inoculation always examine media in test-tubes for presence of slight precipitates and for contaminat- ing organisms. In cultures of rapidly growing species, at optimum temperatures, clouding may occur in less than twenty-four hours ; with slow-growing species, and *Fio. 37. Tin box for holding scalpels, forceps, etc., to be sterilized by dry heat. About one- fourth actual size. A similar tin box which is very convenient for holding sterile pipettes measures 2 by 3 by 15 inches. tFic. 38. Twirled culture of the olive-tubercle organism in Uschinsky's solution, showing viscidity of the precipitate in old cultures. ^Bacterial ash is alkaline, and this ash must be carefully washed from the platinum loop in dis- tilled water each time before it is used to transfer drops of the culture-fluid to litmus paper. The wire must, of course, be re-flamed after washing. FILTERS. 43 o 3 Fig. 39.f when the medium has a retarding action, it may not occur until after two or three weeks. Of course, the rapidity of the clouding depends to a considerable extent on the size of the loop and on whether the inoculation was from a young or old, a fluid or a solid culture. Among other tools, the student should be provided with five platinum-iridium wires set into glass handles, three of which are bent at the free end into loops of a definite size, i. potatoes are scrubbed, and the black specks dug out; they a-re then soaked for 45 minutes in I :iooo mercuric-chloride water. Meanwhile the hands are scrubbed clean and given a five minutes washing in the mercuric-chloride water. The tubers are now rinsed in sterile water, pared deeply, grated as for potato-broth, and thrown into beakers containing sev- eral liters of distilled water, where the pulp is worked over with the hands to liberate as much starch as possible. The starchy water is now removed from tine pulp by passing it through several folds of surgeon's gauze, squeezing out of the pulp as much of the fluid as possible. When the starch has settled the brownish fluid and floating fragments are poured off or decanted, and fresh distilled water is added. The smaller fragments of cell-wall, etc., are then removed by forc- ing the starch (stirred up in water) through a moderately fine-meshed towel (not too fine) with gentle hand-rubbing, into another beaker. Most of the medium-sized and finer starch-grains pass through, leaving in the towel the coarser grains and those fragments of cell-wall which passed through the coarser meshes of the surgeon's gauze. The purified starch is now allowed to stand for about a week in the >ice-t>ox in distilled water (3 liters or more per 'beaker or jar). The water is siphoned off twice a day at first, and afterwards once a day, the starch being stirred up thor- oughly every time fresh water is added. Finally the starch is drained very free from water, scooped out with sterile spoons or spatulas, placed in uncovered sterile Petri dishes, and dried in the blood-serum oven at 56 C., the cover being raised an inch (on corks) to let the moisture out. One-half bushel of sound potatoes should yield from 400 to 500 grams of air-dry aseptic starch. Potato starch has been selected because it is easy to prepare, but other starches might yield in- teresting results. Bacteriologists now pay great attention to the fermentation of sugars, but thus far very little consideration has been given to the action of bacteria on starches and celluloses. What- ever starches are used, they should be prepared in the laboratory, under aseptic conditions, so as to exclude spore-bearing organisms. 13. Starch-jelly with addition of various sugars, gums, and alcohols (for study of organisms having little or no action on starch). 14. Tubes of slant nutrient agar ( + 15 of Fuller's scale) with varying amounts of c. p. glycerin, 2 to 10 per cent or more. SYNTHETIC MEDIA. 51 15. Tubes of 10 cc. slant agar with 10, 20, and 30 grams of grape-sugar. 1 6. The same, with the same amounts of cane-sugar. 17. Gelatin with cane-sugar, varying amounts. 18. Gelatin with malic acid. (17 and 18 may be combined.) 19. Gelatin plates with soluble starch and i per cent potassium iodide and with or without i per cent potassium nitrate. Try a mixture of the pear -blight organism and B. coli. Can the colonies be distinguished in this way using the nitrate? 20. Agar plates with various sugars and the addition of calcium carbonate, or zinc carbonate, for detection of acid-forming colonies. ('91, Beyerinck, Bibliog., XX.) 21. Silicate-jelly. See p. 36. Known also as silica-jelly, 22. Nitrate bouillon (+ 15 bouillon with i per cent potassium nitrate). 23. Triple-distilled water and nutrient mineral substances free from nitrogen. The same, with addition of potassium nitrate. The same, with other nitrogen foods, e.g., sodium asparaginate. 24. Bouillon with lead acetate. 25. Bouillon with neutral red. 26. Salt bouillon, i. e., + 15 bouillon with varying amounts of c. p. sodium chloride (i to 5 percent). 27. Standard peptonized bouillon with varying amounts of sodium hydrate (from -f- 40 to 40) for determining the optimum reaction and the tolerated range of acidity and alkalinity. Synthetic media may be varied indefinitely to fit special cases and are often extremely useful as differential tests. They have frequently been condemned because some particular organism has not grown well in them. The very fact of feeble growth or of no growth is, however, a matter of interest, and not infrequently a means of distinguishing organisms which resemble each other in many particulars. The value of such media becomes apparent at once when a number of organisms are compared. Synthetic media afford more exact methods of research than do the common media, and their value must increase rather than diminish as time goes on. (Consult Grimbert in Archives de Parasitologie, T. I, pp. 191-216.) It does not follow, however, that the common media should be at once abandoned. Festina lente is a good rule. The formulae for some synthetic media are given under " Formulae." For others see various text-books and the papers cited in the Bibli- ography under XVI, XVII, XVIII, XXV, etc. RELATION TO FREE OXYGEN. (/) Surface and deep grmvths. Note the behavior of deep stabs in tubes of recently steamed gelatin and agar, or of the colonies in shake-cultures of gelatin and agar which are protected from the free action of air by pouring into the tubes as soon as solidified another tube of gelatin or agar in the surface layers of which, as an additional precaution, some active aerobe may be grown, e. g., Bacillus sub- tilts. Observe also the relative rate of growth of buried and surface colonies in plate cultures, growth under sterile mica plates, etc. Of course, whether an organism will or will not grow under the conditions mentioned depends often to a large extent on the composition of the culture medium. It might be able to respire in the pres- ence of grape-sugar or cane-sugar, but not when milk-sugar or glycerole is substi- 52 BACTERIA IN RELATION TO PLANT DISEASES. tuted. It will not do to conclude that an organism is a strict aerobe until it has been tested anaerobically in the presence of a variety of carbon foods with uni- formly negative results. One who has had some experience may often give a shrewd guess as to behavior in fermentation-tubes by carefully noting the growth of buried and surface colonies in ordinary media. (2) Fermentation-tubes. The fluids may be Uschinsky's solution (without the glycerin unless this is the carbon compound to be tested); peptone water (2 per cent Witte's peptone with 0.5 per cent sodium chloride); and filtered tap water, or sugar-free beef bouillon with addition of i per cent Witte's peptone (preferably for most purposes this latter fluid). The substances to be tested (which should be chemically pure or as nearly so as possible) are grape-sugar, fruit-sugar, cane-sugar, milk-sugar, galactose, maltose, dextrin,* mannit, dulcit, raffinose, glycerin, ethyl alcohol,! methyl alcohol, acetone, ammonium lactate, ammonium tartrate, asparagin, sodium asparaginate, urea, etc. One to 5 per cent of the various sugars, etc., may be used ; 2 per cent is a good quantity. Fig. 464 Observe carefully what substances induce clouding in the closed end and whether any gas is produced. Test from time to time for acids. The relative vigor of growth in the open end should also be noted. Does growth stop in the U with a sharp line of demarcation? Does the addition of calcium carbonate reduce or prevent the formation of gas or favor growth in any way? Is the reaction in the closed end, as the result of growth, different from that in the open end ? Pipette out all the fluid from the open end, determine its reaction to litmus, and then test the reaction of that which remains. How is the difference, if any, accounted for ? If growth finally ensues in the closed end, is there any reason for thinking it due to absorbed air? How can this be determined ? It should be remembered that often, after a time, air is absorbed into the closed end of fermentation-tubes and may lead to confusing results. For this reason, if they have stood on the shelf any length of time after sterilization, they should be re-steamed and the bubble of air tilted out before they are inoculated. They *The dextrin should be freely soluble in cold water and should not give any red reaction with iodine t. e., should be free from amylo-dextrin (erythro-dextrin). Such dextrin is hard to procure. tThis and the next four should be added, after sterilization, by means of sterile pipettes. The ammonium salts may be obtained in a sterile condition without loss of ammonia by dissolving 10 grams in 200 cc. of water and forcing this through a Chamberland filter into a sterile flask, from which the proper quantity may be pipetted into the culture medium after sterilization. \ FIG. 46. 'Wooden carrier for fermentation-tubes, the flanging base being held under the grooves. Muoh reduced. RELATION TO OXYGEN. 53 should be disturbed as little as possible after inoculation, and especially all tiltings or rough jarring should be avoided. They may be carried in a wooden rack (fig. 46). All culture-media, whether inoculated or not, should be protected from light. Figs. 47, 48, 49 show fermentation-tubes in actual use. The pattern of fermentation-tube preferred by the writer is that slight modi- fication of Einhorn's tube designed by Dr. Theobald Smith (see Wilder Quarter Century Book). The tubes may be had from Emil Greiner, New York. Certain Fig. 47* Fig. 48.t Fig. 494 forms of tubes should not be used. One of these, a short, thick tube with a wide U, in use in some laboratories in this country, allows air to pass readily into the closed end and is entirely worthless. A sample tube of this sort was filled with *Fic. 47. Fermentation-tube with Bacillus tracheifhilus, showing absence of gas and uniform clouding in open and closed end in the presence of grape-sugar. The fluid consisted of water, 400; Savory & Moore's peptone, 4; sodium chloride, i; c. p. grape-sugar, 2; saturated solution carbonate of soda (20 C), 20 drops, '. e., enough to render the fluid slightly alkaline to litmus. tFiG 48. Fermentation-tube with Bacillus tracheiphilus, showing inability of organism to grow anaerobically with glycerin as the carbon food. Fluid, distilled water with I per cent Witte's pep- tonum iiccum and I per cent Schering's c. p. glycerin. Copious growth in open end and in outer part of U ; none in the closed end. jFic. 49. Fermentation-tube of cane-sugar peptone water inoculated with a white, gas-forming organism plated from a spot disease of sisal hemp. The total amount of gas produced and its rate of evolution at 20 to 23 C. are indicated by marks on the closed end of the tube. 54 BACTERIA IN RELATION TO PLANT DISEASES. beef-bouillon and steamed every twenty-four hours for seven or eight days, a large bubble being tilted out each time and appearing just as regularly during the next steaming. Naturally, no strict anaerobe would grow in such a tube and every aerobe would appear to be a facultative anaerobe. The neck of the fermentation- tube should be as narrow as consistent with filling and cleaning. All wide-necked tubes should be discarded. The behavior of the closed end with reference to the absorption of air may be tested by adding litmus- water and 5 per cent grape-sugar to tlie bouillon. On steaming, the litmus is reduced. If there is no air in the closed end the litmus remains reduced, while in the open end exposed to the air it soon oxidizes back to its original color. Other things to be observed are : ( j) Growth in hydrogen. (-mixture is advised. The writer also usually wires in the rwaxed top parts. The gas inflow is cut off by twisting the uppermost (horizontal) ground-glass stopper, which must be care- fully vaselined. One-third actual size. ANAEROBIC CULTURES. 59 , crowded a tight-fitting, soft, rubber stopper. This end is finally buried for an inch or so in a small beaker of glycerin and is perfectly air-tight. A rimless test-tube about 5 inches (13 cm.) long and of a diameter such that it will just slip easily up the other arm of the U-tube, is now packed by means of a pencil or glass rod with 8 or 10 grams of pyrogallic acid, covered quickly with 25 cc. of 10 or 15 per cent caustic-potash water, and slipped up the open end of the tube, which is immediately plunged into a dish of mercury and held there (under a shelf ) until enough of the oxygen is absorbed so that it will stay down of its own weight. The exposure should be made at 25 or 30 C., or at least at temperatures considerably above zero, since the absorption of the oxygen is slow in cool air. The tube containing the pyrogallic acid and potash mixture floats on the mercury and rises, of course, in the arm of the U-tube as the oxygen is absorbed and the mercury enters it. This tube must not, therefore, be too long so as to hit against the curves of the U-tube before all of the oxygen has been absorbed ; otherwise the mer- cury will pass up between the two tubes and overflow into the mixture. In other words, several centimeters must be allowed for the rise of the mercury. A few experiments will determine how much of the mixture is necessary for a tube of a given bore and how long it takes to absorb all of the oxygen, f The level of the mercury in the open end with all the oxygen absorbed may be recorded by a scratch on the tube as a rough guide in subsequent work. At least half a dozen of these tubes will be found useful. They may be made in any laboratory or may be procured from dealers in glassware. In the use of carbon dioxide, especially with sensitive organisms, two factors must be considered, (i) the simple exclusion of air, as in case of hydrogen, and (2) the change in the reaction of the medium due to absorption of the gas (forma- tion of carbonic acid). *Fic. 55. A simple device for growing organisms in air deprived of its oxygen. In the left arm are the cultures ; in the right arm is a test-tube containing a mixture of pyrogallol and caustic- potash water. The heaker contains mercury. About one-third actual size. A modification of Ganong's apparatus for study of germinating seeds. fMace states that I gram of the pyrogallol and 10 cc. of the 10 per cent potash-water are suffi- cient for each 100 cc. of air space. Fig. 55.* 6O BACTERIA IN RELATION TO PLANT DISEASES. LUMINOSITY. Numerous saprophytic bacteria are luminous under certain special conditions. Luminosity is also a striking characteristic of at least one bacterial animal disease the white disease, or sluggish disease, of sand fleas ( Talorchestia longicornis and T. megalophthalmid), common on the shores of France and ot Massachusetts at Woods Hole. Decaying potatoes and other vegetables are sometimes luminous. The question of luminosity should therefore be kept in mind by the student of plant diseases, although no luminous species are known to live in plants. Most of these interesting luminous bacteria have been found in salt water or near it, or on the flesh of quadrupeds and fish. Gorham has been able to grow them on strictly synthetic media. The most recent treatise is by Hans Molisch (Leuchtende Pflanzen, Jena, Gustav Fischer, 1904, pp. ix, 169, with 2 plates and 16 text figures). Molisch records 26 species of luminous bacteria. He found that salt-water fish and the flesh of cattle exposed in the markets were very often luminous 48 per cent of 70 samples of the latter and nearly all the former. Of horse flesh 65 per cent and of cattle flesh 89 per cent became luminous on putting it into 3 per cent solution of sodium chloride, allowing a part of it to project into the air. Fresh-water fish are very seldom luminous. Seedlings exposed to Petri-dish poured plates curved heliotropically toward the light, but they did not become green. Other chlorides than that of sodium stimulate growth and light-production, e, g., potassium, magnesium, or calcium chloride. Certain non-chlorides, such as potassium iodide, potassium sulfate, and magnesium sulfate have the same action (3 per cent or less). Potassium nitrate was also active on B. phosphoreum but not on B. phologenus. Manganese sulfate stimulated growth very noticeably but had no corresponding effect on- the luminosity, which was weak. The spectrum of B. phosphoreum differs from that of the West Indian beetle, Pyrophorus noctilucus, and from that of a luminous fungus known as mycelium X. No biological importance is attributed to the luminosity which is ascribed to an hypothetical phologen. It is an oxidization phenomenon which can take place only in the presence of free oxygen. A tem- perature of 30 C. for forty-eight hours is sufficient to kill B. phosphoreum in gelatin cultures. The minimum temperature for this organism is below zero, the optimum is about 16 to 18 C., and the maximum is 28 C. The bacteria are luminous from minus 5 to plus 28 C. Light production is most intense from 5 to 20 C. FERMENTATION PRODUCTS. The old conception of fermentation involves an evolution of gas (fervere, to boil), but the term is now used with a wider meaning. Like many other terms, it is difficult to use it always logically. In general, it means the breaking up of carbon compounds into simpler substances, either by the direct action of the protoplasm of the organism (hypothetical) or by chemical substances (enzyms, diastases) secreted by the protoplasm. Acids and alcohols are produced ; gases may or may not be evolved. Other volatile products are also produced, e. g., esters, but usually only in very small quantities. Certain of the bacterial fermentations are of large com- mercial importance, e. g., the acetic, the lactic. The breaking up of albumen and FERMENTATION PRODUCTS. 6l other complex nitrogen compounds, i. e., putrefaction, is also sometimes called fer- mentation, and at present there is really no very sharp line to be drawn. Consult Green and Duclaux for the English and French views (Bibliog., XX). The student should observe : (1) Gases. Amount, rate of development, kinds (carbon dioxide, oxygen, hydro- gen, nitrogen, marsh gas). (2) Acids. Volatile and non- volatile (lactic, acetic, butyric, etc.). (3) Alcohols (ethyl, methyl, butyl, glycerin, mannit, etc.). (4) Ethers and esters. (5) Aldehyds, sugars, gums. (6) Albumoses, peptones, amido-bodies. The isolation and determination of the amount of these various products belongs to the province of the chemist, but the work should be done in the bacteri- ological laboratory and under the eye of the biologist if all sorts of errors, due to the unsuspected multiplication of intruding organisms, are not to creep in and render the work worthless. Only some crude determinations, as of proportion of the various gases evolved, may be made by the bacteriologist who is not a chemist. The volume of gas evolved from day to day may be measured in fermentation-tubes (fig. 49). Frost has devised a convenient gasometer for roughly estimating it (see his Laboratory Guide, plate I). These may be made in any laboratory out of cardboard. If the gas is carbon dioxide it may be absorbed by shaking with 10 per cent NaOH. To do this, fill the bowl (fig. 49) even full of the strong caustic-soda water, place the thumb or forefinger over the mouth so as not to include any air, invert the tube so that the gas shall flow into the bowl and come into contact with the alkali, and shake vigorously until all of the carbon dioxide is absorbed. Tilt the fluid back into the open end, and remove the finger so as to equalize the pressure. If any gas remains after equalizing the air-pressure, place the finger over the mouth of the tube, tilt the gas into the bowl and apply a lighted match close to the mouth as the finger is removed. If it is hydrogen or marsh gas it will explode in the open end of the tube when the finger is removed and a flame applied. If it is nitrogen it will not support combustion (see Bibliog., XX, especially '90 Smith and '93 Smith). How distinguish marsh gas from hydrogen ? Organisms easily inhibited by their own acid products may be kept alive a much longer time by adding a little calcium carbonate to the bouillon or agar. In simple tests for acids, discard bright blue litmus paper, which is very sensitive to carbonic acid (try carbonated water on it), and use instead a good grade of reddish- violet (neutral) litmus paper. Such paper may be made in the laboratory (the best way) or may be purchased of H. Struers, Copenhagen. ALKALIES (AMMONIA, AMINS, CARBONATES OF THE ALKALI METALS). Determine rapidity of formation. Note that they are often masked by the simultaneous formation of acids. Try the litmus test and Nessler's test Do not put Nessler's solution into the culture fluid, but expose it to steam from the culture. Observe the behavior of the organism when grown in peptone rosolic-acid water 62 BACTERIA IN RELATION TO PLANT DISEASES. with just enough HC1 added to counteract the alkali in the peptone, and in neutral or slightly acid peptone-water or sugar-free bouillon containing acid fuchsin. On titration of acids and alkalies see Sutton (Bibliography of General Literature, IV). REDUCING POWERS. Determine rapidity of reduction of litmus, methylene blue, and indigo carmine in various fluids and solids (with and without grape-sugar). Probably all bacteria can reduce litmus, etc., but as the rapidity of reduction varies greatly in different species and in different media, it is desirable to make comparative tests. Consult a recent paper by Albert Maassen ("Ueber das Reduktionsvermogen der Bakterien, und ueber reduzierende Stoffe in pflanzlichen und tierischen Zellen," Arb. a. d. Kais. Gesuudheitsamte, Bd. XXI, 3 Heft, 1904, pp. 377-384). HYDROGEN SULPHIDE. This gas is the product of a reduction. From what media and under what conditions is hydrogen sulphide given off with browning of lead acetate paper ? This paper is readily prepared by dipping strips of white filter paper into a strong solution of lead acetate in distilled water. It should be kept in a tight tin box or a glass-stoppered bottle. Probably most, if not all, bacteria are able to produce hydrogen sulphide in nutrient media containing readily decomposable sulphur com- pounds. Is an enzyme necessary ? When an organism grays potato cylinders in test-tubes, why is no hydrogen sulphide given off? The student should read papers by Petri and Maassen (Bibliog., XXVIII). MERCAPTAN AND OTHER ODORS. We need an odor chart to go along with our color charts. If we could have a set of standard substances with peculiar smells for comparison with the many odors evolved from bacterial cultures it would certainly be a great convenience. The difficulty at present is that the judgment of people varies greatly, in many instances, as to what the smell should be likened. As it is, the bacteriologist must do the best he can to define these penetrating smells, which are sometimes very characteristic of particular organisms. Some of the fishy odors are due to am ins. Mercaptan is a very vile-smelling sulphur compound. INDOL, PHENOL, LEUCIN, TYROSIN, ETC. The production of indol is best studied in peptonized beef-broth naturally free from sugar or which has been deprived of its muscle sugar by growing in it (for a few hours only) Bacillus coli (Theobald Smith), after which it should be filtered clear. If B. coli or B, cloacae will not produce gas in beef-broth in the closed end of fermentation-tubes, it is free from sugar and suitable for this use. Many organisms give the indol reaction in Uschinsky's solution to which peptone has been added. The writer has never been able to obtain the indol reaction in any culture medium which did not contain peptone (using this word in the commercial sense. ) Cultures which do not show the red reaction with sodium nitrite (0.02 per cent solution) and sulphuric acid at room temperature will frequently do so when put into hot water INDOL, PHENOL, ETC. for five minutes (70 to 80 C.). The browning of media due to excess of sodium nitrite must not be mistaken for this pink or red reaction. Uninoculated tubes should be included in the test, which may be made on the second and tenth day. For methods of determining phenol see Lewandowski in Deutsche Med. Wochenschrift, 1890, p. 1186, and Chester's Manual, p. 33. Schmidt (Bd. II, p. 1008) gives the following as a qualitative reaction for tyrosin : Dissolve by boiling in water and add a solution of mercuric nitrate. The red reaction is sharper if a little fuming nitric acid diluted in water is added. Try also the violet reaction with neutral iron chloride.* Leucin crystallizes in white soft scales. REDUCTION OF NITRATES (AND MORE COMPLEX NITROGEN COMPOUNDS) TO NITRITES, TO AMMONIA, AND TO FREE NITROGEN. For the pathologist the iodine-starch reaction is the most satisfactory test for nitrites, because it is not superlatively sensitive and consequently does not indicate traces of nitrite absorbed from the air. It is made as follows : Twenty-five cubic centimeters of distilled water are added to one-half gram (more or less) of pure potato starch and the fluid boiled. One cubic centimeter or more of this starch-water and i cc. of freshly prepared potassium-iodide water (i : 250) are now put into the culture fluid, to which is then added a few drops of strong sulphuric-acid water (2 : i). If any appreciable quantity of nitrite is present the culture immediately becomes blue-black from the liberation of free iodine, which acts upon the starch. Old potassium iodide water should never be used without first testing carefully, as it usually contains some free iodine. It is always best to first make a trial test without the bacteria. Commercial starch frequently contains prod- ucts of bacterial decomposition and starch prepared aseptically should be substituted. At least one-third of the organisms which have fallen under the writer's observation in recent years give the nitrite reaction when grown in peptonized beef-bouillon containing potassium nitrate. *Mann (p. 323) gives the following as a specific tyrosin reaction: Deniges has recommended the well-known phenol aldehyde reaction for the detection of tyrosin. Nasse, in repeating Deniges' observations, has found the following to be a very delicate test for tyrosin, as neither proteids nor peptones give the color-reaction. Proceed thus: Add a few drops of formol solution to con- centrated sulphuric acid, when, on warming with tyrosin, a brown-red color is obtained, which, on addition of acetic acid, becomes green. fFic. 56. Bacterium syringae (van Hall). Nitrate bouillon cultures 5 days old, to each of which has been added boiled starch water, potassium iodide water, and sulphuric acid. In tube a the potassium nitrate was reduced to the nitrite, and on addition of the reagents free iodine was liberated, and the starch blued. In the other no nitrite had formed, no iodine was liberated, and the starch remained colorless. For discrepancy see text Fig. 56.t 64 BACTERIA IN RELATION TO PLANT DISEASES. Fig. 56 shows how differently quite similar-looking cultures may react when submitted to this test. Both of these organisms were received from van Hall under the name of Pseudomonas syringe, a being van Hall's own isolation and b being supposedly a subculture from Beyerinck's isolation. Neither one would produce any blight in lilac shoots. There is no simple way known to the writer of distinguishing ammonia from the amins, as both react to Nessler's reagent Nitrogen may be distinguished from the other gases of fermentation by the fact that it is not absorbed by sodium or potassium hydroxide and will not burn or support combustion. This gas is produced readily from nitrates by a number of green-fluorescent organisms (dung-destroyers) but not by all of them. FIXATION OF FREE NITROGEN AND THE OXIDATION OF AMMONIA AND AMMONIUM SALTS TO NITRITES AND NITRATES. These processes are probably common enough to organisms of the soil, many of which have not been investigated, but they are not known to be brought about by plant parasites exclusive of the root-tubercle bacilli of the Leguminosae, which some believe to be parasites (see Peirce).* They are believed to be of rare occurrence in bacteria which grow well on ordinary culture media. The nodules on roots of plants will hereafter be considered more fully. The reader should consult a paper by Geo. T. Moore on " Soil Inoculation for Legumes," Bureau of Plant Industry, United States Department of Agriculture, Bull. 71, Jan- uary 23, 1905 ; and one by Maria Dawson, "Further Observations on the Nature and Functions of the Nodules of Leguminous Plants," Philosophical Transactions Royal Society of London, Series B, Vol. CXCIII, pp. 51-67, 1900, with 2 plates. ASSIMILATION OF CARBON DIOXIDE. Some soil organisms are believed to obtain their carbon directly from carbon dioxide, and would thus be exceptions to the law that all non-chlorophyllous plants must obtain their carbon from organic substances. This supposition, while probably true, has not, we believe, been established satisfactorily. Its elucidation offers a most interesting line of research (see Bibliog., XXVI.) PIGMENTS. Bacterial growths are often bright colored, and an examination of the pigments should form part of one's study of an organism. They may be considered as follows : (1) Under what conditions formed ? Can they be eliminated by growing the organisms in the dark or under unfavorable conditions, e.g., near the maximum or minimum temperature ? Bacillus prodigiosus is a favorable organism for experiment. (2) In what soluble (water, hydrogen-peroxide in water, ethyl alcohol, methyl alcohol, glycerin, acetic ether, petroleum ether, sulphuric ether, acetone, chloroform, turpentine, benzine, benzole, xylol, toluol, carbon bisulphide, etc.)? The pigment should be tested in as many solvents as possible. *Peirce, George James. The Root-tubercles of Bur Clover (Medicago denticulata Willd.) and of Some Other Leguminous Plants. Proc. Calif. Acad. Sci., 3d series, Botany, Vol. II, No. 10, San Francisco, Cal., June 21, 1902, pp. 295-328, with i plate. PIGMENTS. 65 (3) How are they acted on by acids, alkalies, and other reagents ? (4) Of what use are they to the organism ? Are they oxidation-products ? Examine spectroscopically, if possible. On the addition of acids or alkalies, a bacterial pigment may remain unchanged, may be changed into some different color, may be destroyed, or may be converted into some colorless compound which will regain its original color on changing back the reaction. The yellow pigment of several species of Bacterium (Pseudomonas) remains unchanged in the presence of acids and alkalies. The blood-red color of Bacillus prodigiosus becomes carmine in the presence of certain acids and yellowish- brown in the presence of certain alkalies. The blue color of Bacterium syncyaneum is said to be produced only in acid milk. The beautiful green fluorescence of Bac- terium pericarditidis (Bacillus pyocyaneus pericarditidis~), and probably of all this group of bacteria, is produced only in alkaline media. According to Jordan two pigments are normally produced by many green-fluorescent organisms. The blue pigment pyocyanin is visible by gaslight and is soluble in chloroform. The green- fluorescent pigment is insoluble in chloroform and yellowish by gaslight. By this latter test the two can be distinguished when mixed. Soluble phosphates and sulphates are necessary for the production of green fluorescence. The ability to produce pyocyanin is easily lost. Its production in the culture-medium, unlike that of the fluorescine, is not dependent on the presence of phosphates or sulfates. Pyocyanin turns red with acids, fluorescine becomes colorless ; both return to their original color on adding alkali sufficient to change the reaction. " Asparagin, ammonium succinate, ammonium lactate, and ammonium citrate all proved suitable for the development of the fluorescent pigment." The yellow and black pigments are the result of oxidations. (See papers by Gessard, Thumm, and Jordan, Bibliog., XXIII). The pigments of bacteria range from one end of the spectrum to the other. Thus we have various shades of black, brown, violet, indigo, blue, green, yellow, orange, and red. Many bacteria produce no pigment, i. e., are white when seen in mass. Others produce several distinct pigments. Many of the plant parasites are yellow, e. g., Bacterium campestre, Bact. phaseoli, Bad. hyacinthi, Bact. Stewarti, Bad. juglandis. Some of these yellow organisms stain the host-plant and certain nutrient substrata a deep brown. Other plant parasites are white but also stain the host and certain substrata brown, e.g, Bacterium solanacearum, Bacillus carotovorus, B. aroidece. Others are pure white and are apparently destitute of any pigment- producing powers, e.g., Bacillus amylovorus, B. tracheiphilus. Very many bacteria when grown on cooked potato produce a gray stain in this substratum, especially in that part freely exposed to the air, i. e., out of the water. Some other color changes in the host should be mentioned. Various brown and red stains visible in certain plants when attacked by bacteria are not attributable directly to the presence of the microorganisms in the tissues. These are oxidation phenomena likely to occur when the plants are wounded or destroyed by any agent whatsoever. A few illustrations will make my meaning clear. When the limbs of pear trees are destroyed by blight the foliage becomes black, but this blackening 66 BACTERIA IN RELATION TO PLANT DISEASES. also occurs frequently when the flowers, green fruits, or foliage are killed by other causes. In the leaves of Amaryllis atamasco the writer obtained red stripes by injecting the yellow Bacterium hyacinthi, but no bacterial disease followed, and the same plant reddens when bruised. Broomcorn shows conspicuous red blotches when attacked by the broomcorn organism, but the parasite itself does not produce a red pigment, while the plant reddens easily as the result of aphis-punctures or wounds of any sort. Sugar-cane attacked by Bacterium vascularum shows a con- spicuous red stain in the bundles, but other causes, such as the gnawings of an insect or the presence of a fungus, may lead to a similar stain, while the bacterium itself does not produce any red pigment. CRYSTALS. Determine the nature of the crystals observed in the various media. Many of these are double ammonium salts; others result from the action of.trypsin on pro- teids. Crystals which are not due to the drying out of the media are common phenomena in old ciiltures of many sorts, especially if the media were not originally saturated with alkali (soda or potash). Fig. 57 shows two types of crystals formed in +15 nutrient agar by two green-fluores- cent organisms received from van Hall as Pseudomonas syringte, and a third type produced by the olive tubercle organism. QUESTION OK EXISTENCE OF ENZYMES. The enzymes of English writers are the diastases of Duclaux. They are chemical substances, the exact composition of which has not been determined. They Fig. 57.* may be regarded as the working tools of protoplasm. The following are some of the best known kinds : (i.) Diastasic (starch-destroying). (5.) Lab or rennet (casein-forming). (2.) Inverting (sugar-splitting). (6.) Lipase (fat-splitting). (3.) Cytohydrolytic (cellulose-dissolving.) (7.) Pectic (pectin-splitting). (4.) Proteolytic (peptonizing). (8.) Oxidases (oxidizing). Trypsin is common. Pepsin is not known to be produced by bacteria and should be searched for. Many bacteria invert cane-sugar, but invertase is believed to be rare. This, however, may be an ill-founded conclusion. The experiments of various animal physiologists have shown that when cane-sugar is injected into the blood-stream it is excreted unchanged, and according to Julius Sachs cane-sugar, inulin, etc., must *FiG. 57. Crystals formed in cultures of Bacterium syringae (van Hall). I. From tube II, Aug. 14 (agar stock 693), from van Hall's II, i.e., his own isolation corresponding to a, fig. 56. 2. From tube I, Aug. 14 (stock 693), from van Hall's I, which is from Beyerinck's old isolation (see b, fig. 56) X 3. Nos. i and 2 drawn Aug. 30, 1902. 3. Crystals formed on slant litmus-lactose agar which was inoculated with the organism causing olive-knot. About one-half inch of slant in middle part of culture i month old, i. e., made January 20, 1904; drawn February 17-19. X 3- Tempera- ture during growth, 20 to 25 C. ENZYMES. 67 first be reduced to glucose (grape-sugar), before they can be used as food by plants. When no invertase has been detected the general hypothesis has been that this inversion was due to the direct action of the protoplasm, but the recent isolation by Buchner and others of an invertase (Zymase) from yeast, in which it was long believed that none existed, once more emphasizes the uncertainty of negative conclusions. Diastase is common. Is there more than one kind, i. e., a sort which can only convert the starch into amylodextrin and another which converts it into maltose and dextrine ? In many cases, when the organism is grown on potato, the con- version is carried only a little way and stops, there being always a copious purple or red-purple reaction with iodine. In other cases, e.g., when Bacterium campestre is grown on potato, the starch conversion is so complete that after a few weeks there is little or no color reaction when the potato-cylinder is mashed up and iodine water added. What makes this difference? A substance capable of dissolving the middle lamella appears to be common to all bacterial plant parasites and a true cytase presumably occurs, but much additional study is necessary. Probably several enzymes are confused under this name, just as several chemically different substances are still called "cellulose." The substance which dissolves the middle lamella in some cases is prob- ably ammonium oxalate. The writer has not been able to dissolve it by means of pure oxalic acid, but that of turnips softens in ammonium oxalate. The lab or rennet ferment is rather common. Its action should not be confused with the curdling of milk due to the formation of acids. Tests may be made in litmus milk. Is there more than one kind of such ferment? Some organisms coagu- late the milk promptly into a solid mass which finally shrinks, extruding whey. Others cause the Fig- 58,* casein to separate out of the fluid very slowly as a multitude of separate particles which only become compacted very slowly. The writer has not met with the oxidizing enzymes, unless the substance in bacterial cultures which causes rapid evolution of oxygen from hydrogen peroxide is such an enzyme, as Dr. Loew maintains (Bibliog., XLV). Many other enzymes undoubtedly occur and play their part. The student should search for emulsin, lipase, lactase, maltase (glucase), etc. All known enzymes when freely exposed to steam heat are destroyed at tempera- tures considerably under 100 C. They are less sensitive to heat than the bacteria themselves, but are destroyed by a few minutes exposure to temperatures 15 to 30 C. (moist heat) above the thermal death-point of the organisms which have produced *Fic. 58. Thick-walled Kitasato flask for filtration or evaporation in vacua, etc. Much re- duced. 68 BACTERIA IN RELATION TO PLANT DISEASES. Fig.59.f them.* Some of them are very sen- sitive to the presence of acids, alka- lies, strong alcohol, or antiseptics, or their action is inhibited by the pres- ence of other enzymes or of products of enzymic fermentation in excess, or by the absence of some combining substance, such as lime or some weak acid. Some do not pass readily through the Chamberland filter or through filter papers. Some are destroyed at lower temperatures after precipitation. Some are not pro- duced except in presence of the sub- stance which they can decompose, but this is not true of all. Usually an organism produces more than one ferment and some bacteria are known to produce five or six. Bac- terium campestre produces at least three and probably four, viz, diasta- sic, cytohydrolytic, proteolytic, and rennet. It also inverts cane-sugar, but it is not yet known whether this change is accomplished by means of an invertase. On enzymes derived from bacterial soft-rot organisms the reader should consult recent papers by Jones (Centralb. f. Bakt, 2 Abt., and Vermont Exp. Sta. Rep.). Levy has published an interesting paper on " Some physical properties of en- zymes" (The Jotir. Infect. Diseases, Vol. II, 1905, pp. 1-48). For concentrating fluids in vacuo at low temperatures (50 to 60 C.) the thick-walled Kitasato flask shown *The same amount of dry heat does not affect them, and Loeffler has recently advised exposure of thoroughly air-dried tissues and cultures to 150 C., dry heat, as an easy way of eliminating the bacteria prior to grinding and extraction of the uninjured enzymes and other soluble products. Non-sporiferous bacteria may be heated at 120 C. for 2 to 3 hours. Tissues and sporiferous bacteria should be heated at 150 C. for one-half hour. (Deutsche Med. Wochenschrift, Dec. 22, 1904.) fFic. 59. ^Burettes used by the writer for titrating culture media. Twentieth-normal sodium hydrate is used to determine the acidity, 'and the medium is finally brought to the desired alkalinity with quadruple-normal sodium hydrate. The fluid is boiled and titrated hot, using phenolphthalein as the indicator. The burettes should be graduated to tenths of a cubic centimeter and should hold 50 cc. Alkali should not be allowed to stand in them. EVAPORATION AT LOW TEMPERATURES. iii fig. 58 is very convenient. The side tube is attached to the suction-pipe of an air-pump and into the neck is thrust a rubber stopper carrying a thermometer and a U-shaped glass tube of small bore, the outer arm (36 inches long) ending in a beaker of mercury. Heat may be applied by means of a water-bath. By substitut- ing a funnel for the thermometer the same device may be used to hasten the filtration of thick liquids, hard-pointed filter papers being employed. SENSITIVENESS TO PLANT ACIDS. The tests should be made with malic, citric, lactic, oxalic, and tartaric acids added to neutral beef-broth, peptone-water, or plant-broths, or to synthetic media (see Am. Nat., 1899, p. 208). It is best to titrate with or solutions, to acidify 2N 4.N with - or - solutions, and to reckon the acidity in cubic centimeters of normal N solution ( ) required per liter of medium. If pre- ferred, it may be calculated on 100 cc. portions and expressed in per cents, but there is no advantage in this, and it has the disadvantage of introducing fractions. SENSITIVENESS TO ALKALIES (POTASSIUM OR SODIUM HYDRATE). Determine in each case the optimum reaction of the medium for growth. For the majority of bacteria this is said to lie between +10 and + 15 of Fuller's scale.f The best neutral litmus paper should be used freely, but acid and alkaline media should be titrated with phenol- N N phthalein and or solutions. In some media 10 20 e.g., gelatin, juices of various plants the end-reaction with phenolphthalein and caustic soda is not very sharp. In these cases the titration should be stopped at the first trace of change of color. If one adds alkali until the fluid is decidedly red, then a distinct statement to that effect should be made, since otherwise no comparisons of any value can be made. All of the writer's + and refer- ences to media are based on a reaction stopped at the first distinct trace of pink color. As much again alkali must sometimes be added to obtain a deep-red color. *Fic. 60. Stock bottle of sodium hydrate solution. The small bottle at the right holds con- centrated potash liquor to remove the carbon dioxide from the air which enters the bottle. About one-fourth actual size. fThe plus and minus on Fuller's scale denotes, respectively, acid and alkaline media. The + ID, for example, means that exactly 10 cubic centimeters of normal alkali must be added to a liter of the culture medium to render it exactly neutral to phenolphthalein, and, correspondingly, 10 means that the fluid is alkaline to phenolphthalein and that 10 cc. of normal acid would need to be added to bring I liter back to the neutral point. The student should not confuse the litmus neutral point and the phenolphthalein neutral point, as they are about 23 apart, e. g., + 10 of Fuller's scale (acid side) is distinctly alkaline to litmus. (Consult '95, Fuller, Bibliog., XVI.) 70 BACTERIA IN RELATION TO PLANT DISEASES. The writer has used the foregoing method of determining the reaction of culture media for several years and has, in general, found it exceedingly exact and valuable, but it does not appear to be well adapted for determining the amount of alkali (ammonia and amins) produced by bacteria in culture media (see Button, Bibliog., IV). The apparatus required to make these titrations is shown in figs. 59 and 60. Some experiments recently made by the writer with Bacillus tracheiphilus in peptonized beef-bouillons of varying degrees of acidity (acid of beef-juice) and alka- linity seem to show that toleration of sodium hydrate can be considerably increased by inoculating each time from alkaline bouillons rather than from acid ones. Taken from + 20 bouillon (descended from + 20 bouillon) this organism would cloud the same bouillon only down to o; taken from o or 5 bouillons (descended from 2.7 bouillon) it would cloud the same bouillon down to 10 and probably farther, but not to 20. Bouillon containing various amounts ofc. p. sodium chloride behaved in the same way. The organism would tolerate the largest amount of salt (1.5 to 2 per cent) when first grown in an alkaline bouillon. When inoculated from a +20 bouillon the organism finally grew in i per cent salt bouillon, but only after a decided retardation, and would not grow at all in + 1 5 peptonized beef-bouillon containing 1.5 per cent sodium chloride. Bacteria vary greatly in their toleration of acids and alkalies, the range of growth being from minus 100 (or more) of Fuller's scale to phis 100 (or more). The limits of growth are not known, but it is probable that the extremes of toleration in particular aberrant species is much greater than that here given, e. g., on the acid side in sulphuric acid and vinegar bacteria, and on the alkaline side in case of those organisms which are able to grow in the lime-vats of tanning establishments and in alkaline springs. Lehmann & Neumann ('96, Bibliog., Ill), state that they have found bacteria that will endure 100 cc. of normal acid per liter of fluid culture media, i. ., equal to about i per cent sulphuric acid. Some species are indifferent to a considerable degree, having a wide range of growth either side of the (phenolphthalein) neutral line ; others prefer alkaline media ; others acid media. Many are extremely sensitive to their own acid products (acetic, lactic, butyric, etc., acids). Not a few are differently affected by different acids and alkalies. Every new organism presents a whole series of special problems. EFFECT OF DESICCATION. Drops of fluid cultures or small masses of gelatin or agar cultures are spread on small ("4 -inch) clean, sterile cover-glasses, in covered sterile Petri dishes, and are set away in the dark, in dry air (a dry room). The test is finally made by seizing one of these covers with a pair of sterile forceps and dropping it into a tube of sterile bouillon or other medium of a stock previously determined to be exactly adapted to the growth of the organism, /. e., one which does not exert upon it any retarding influence. Occasionally a tube will become contaminated, but enough must be inoculated so that this will not affect the final result (20 at one time is not too many). Fluid cultures are preferred. Solid cultures do not give strictly compar- able results. EFFECT OF DESICCATION. Jl Organisms believed to be non-sporiferotts show great differences, some being killed by an exposure of a few minutes or a few hours, while others remain alive for many weeks. For further information see the special chapters on Bacillus trache- iphihis, B. carotovorus, Bact. hyacinthi, etc. Tests may also be made in air dried over sulfuric acid or calcium chloride. Harding & Prucha have shown recently that Bacterium campestre remains alive much longer when dried on cabbage seed than when dried on glass cover-slips. In their experiments this organism was dead on glass at the end of -ten days, but alive on seed at the end of thirteen months. EFFECT OF DIRECT SUNLIGHT. The exposures should be made in a thin stratum of nutrient agar, not sowed too thickly (there may be several hundred colonies on the plate, if properly distrib- uted), in thin-bottomed Petri dishes, to an unclouded sun for 5, 10, 15, 30, 45, and 60 minutes, a portion of the bottom of the plate, which is placed uppermost, being covered by some substance impervious to light, such as several folds of Manila paper Fig. 61* Fig.62.t or of the black paper which comes wrapped around photographic dry plates, covered in turn by white paper. Exposures of several hours are not recommended. If the layer of agar is very deep, or if the sowings are too thick, some organisms will screen others and all will not be killed. Ten cubic centimeters is a proper amount of agar to use for a plate having an area of 60 square centimeters. The latitude, altitude, time of year, time of day, and intensity of the light should also be recorded. In the summer- time it is very important that the exposures should be made on blocks of ice or, *Fic. 61. Gelatin culture of Bacillus amylovorus (Burrill) Trev. in a Petri dish. Exposed in 1896 to direct sunlight for four hours on ice after covering portions of the plate with pasteboard figures. The bacteria grew only under the protected parts. Drawn from a photograph made after five days incubation of the culture at about 24 C. The temperature of the gelatin during exposure was about 25 C. Three-fifths natural size. fFic. 62. Agar culture of Bacterium phascoIiCErw. Sm.) in a Petri dish. Right one-half ex- posed to direct sunlight for thirty minutes, on ice, the other half protected by several folds of Manila paper. Dish then set away in the dark for several days. One-half natural size. The scattering colonies on the right side undoubtedly grew from bacteria which were sheltered from the direct rays of the sun by overlying organisms, '. e., the plate was sown too thickly. 72 BACTERIA IN RELATION TO PLANT DISEASES. better, on larger Petri dishes filled with pounded ice ; otherwise, in case of 30 to 60 minute exposures, the temperature may rise nearly or quite to that of the thermal death-point of the organism, and then we shall have the effect of heat complicating that of light. To avoid errors it is always best to take one-half of each dish as a check (rather than the whole of a separate dish), and the rise of temperature should be carefully recorded. In some tests made by the writer in Washington in May the temperature of the plates exposed in the open air to the sun for"45 minutes (without ice) rose from 25 to 51 C. Figs. 61 and 62 show the effect of sunlight upon thin sowings of Bacillus amylovorus and Bacterium phaseoli in poured-plate (Petri-dish) cultures. VITALITY ON VARIOUS MEDIA. By this I mean the determination of the resistance of organisms to their own decomposition products. This varies greatly. Much may be learned by the study of old cultures. Do not discard test-tube cultures until after many weeks. Examine frequently. Make transfers from tubes which have been inoculated for a year or more. Determine whether this vitality is due to spores or persists in the ordinary vegetative rods. On what kinds of media does a particular organism live longest? Can length of life be increased by occasionally neutralizing decomposition products (acids) with sterile carbonate of lime ? or by occasional additions of food ? Some bacteria are veritable revelers in filth ; others are extremely sensitive ; all are soon under abnormal conditions in our culture-tubes. Another way of keeping bacteria alive for a long time is by reducing their growth to a minimum. Stock-cultures, especially of perishable organisms, should, generally speaking, be kept in the ice-box at temperatures under 15 C. This greatly reduces the always heavy burden of keeping alive cultures of organisms which are not in immediate demand for actual experiment. Some will also remain alive a long time when sealed airtight. Particular organisms may be kept a long time in par- ticular media, e. g., Bacterium vascularum in diluted peptonized cane-juice gelatin, Bact. Stewarti in milk, etc. Some organisms are quite resistant to their own decomposition products, e. g., Bacillus coli, Bact. pericarditidis. In the cool box B. coli will often live a year in agar stab cultures. MIXED CULTURES AND MIXED INFECTIONS. The behavior of mixed cultures and mixed infections may be tested in various fluids, making poured plates from time to time ; in tubes of agar, potato, and other solid media ; in crossed streaks on agar or gelatin plates ; and in the plants themselves. When two bacteria, or a bacterium and a fungus, are sown together in a culture- medium, there may be (i) antagonism, with the crowding out of one species ; (2) a more or less complete indifference, both organisms growing well ; or (3) a distinctly favorable effect, i. e., a marked increase in growth or in pathogenic effect due to the presence of the second organism. The antagonism may result in the prompt destruction of one of the organisms, or only in a retardation or inhibition which finally disappears after the first organism has made its growth and subsided. In some cases the favorable effect of one organism upon another is due to the fact that it prepares food for it out of an unfavorable substratum, e.g., maltose from starch. BEHAVIOR OF MIXED CULTURES. 73 In the plant one organism often paves the way for others which complete the destruction, e. g., Bacterium campestre and Bact. solanacearum are often followed by soft white rots. Some of the latter, however, are able to make their way unaided, a fact observed and known to the writer for a white rot of the cabbage as long ago as 1896. The simplest way of studying the antagonistic action of bacteria is by means of crossed streaks on agar or gelatin plates. These may be made either simulta- neously, or one after the other has begun to develop. The action of the antagonistic organism may also be obtained by letting its products diffuse through a collodion sac into bouillon inoculated with the other organism. In practice, the bottom of a test-tube is removed and a collodion sac is securely fastened in its place. This tube is filled with the usual quantity of bouillon and lowered into a larger receptacle (tube or flask), the collodion part being surrounded by bouillon. The inner and outer receptacles are now plugged with absorbent cotton, and the apparatus is sterilized in the steamer or autoclave. The two tubes are then inoculated simultaneously, or the outer one some hours or days after the inner one. (See an interesting paper on Antagonism, by Frost, in Jour. Infect. Diseases, Vol. I, 1904, pp. 599-640). Frost has also devised two new methods for studying this subject, viz, the divided-plate method and the agar-block method. The first is a modification of the ordinary streak method. It is managed as follows : A Petri dish is divided into two equal parts by means of a glass rod fastened to the bottom with collodion. A tube of melted agar is inoculated with the antagonistic organism and poured into one half of this plate. Into the other half sterile agar is poured. Streaks of the other organism are now made crosswise of the hardened surface. If there is marked antagonism there will be a decided difference in the behavior on the two sides of the plate, i. e., on the sterile agar as compared with the inoculated. To insure a uniform streak the inoculated loop should be swept across one half of the plate, then re- inoculated and swept across the other half of the plate. The method by agar-blocks consists in substituting agar-walls for collodion walls. A sterile 3-cm.-deep Petri dish is poured full of nutrient agar. When it has solidified it is cut into rectangular blocks, i by i by 3 centimeters, using a sterile knife and taking all possible precautions to avoid contamination by air-borne organisms. A platinum needle is now dipped into a culture of the supposed antagonistic organ- ism and thrust into the block lengthwise but not entirely through it. The mouth of the needle-track is sterilized and sealed by touching it for a moment with a red-hot iron. The head of a small wire nail set into a suitable handle will answer the purpose. The block is picked up with sterile forceps and dropped into a tube of sterile bouillon, which then may be inoculated with the other organism. More than one block and tube should be inoculated, and it is best to test the sterility of the outer surface of the agar-block by delaying the inoculation of the bouillon for a day or two after the inoculated agar-block has been dropped into place. Still another method has been described by Frankland and Ward. They use the walls of a Chamberland filter to keep the bacteria separate. Bouillon for the one 74 BACTERIA IN RELATION TO PLANT DISEASES. organism is placed in a flask or large tube. That for the other organism is placed inside a Chamberland filter, which is then sunk into the other receptacle, whereupon it is sterilized and inoculated as in the collodion-sac method. The favorable influence of a second organism may be studied in crossed streaks on sterile raw potato, carrot, turnip, etc.; on starch jelly ; or on agar, gelatin, or silicate jelly with addition of varying amounts of the different plant acids, or plant juices, or other vegetable substances. Frost's divided Petri dish may be used for the jellies. REACTION TO ANTISEPTICS AND GERMICIDES. Antiseptic has been defined recently by Duclaux as follows : Any substance the intervention of which modifies in any form whatsoever the march of the phe- nomena (Bibliog., XX, Fermentation alcoolique, p. 461). I still use the word with its old primary meaning (anti, against, and sepsis, decay). In this sense an antiseptic is any substance which prevents the multi- plication of bacteria in putrescible substances. Large doses of antiseptics often exert a germicidal action, but such action does not necessarily follow. Often when the antiseptic substance is removed or diluted beyond a certain point growth takes place. The first seven substances mentioned below possess very active germicidal powers and are antiseptic in correspondingly small doses ; the remainder are more or less valuable antiseptics, but are not valuable germicides. (i.) Mercuric chloride. (5.) Lysol. ( 9.) Benzoic acid. (2.) Sulphate of copper. (6.) Trikresol. (10.) Salicylic acid. (3.) Formaldehyd (formalin). (7.) Methyl violet (Pyoktanin). (11.) Chloroform. (4.) Phenol (carbolic acid). (8.) Thymol. (12.) Sulphuric ether. This list may be extended indefinitely. The student should consult valuable digests in Sternberg's Text Book of Bacteriology and in Miquel & Cambier's Traite de Bacte'riologie. Some caution must be used in drawing conclusions from experi- ments. Mercuric chloride does not always destroy when the culture medium contains albuminoid substances. Sulphate of copper is more active in water than in bouillon.* Some organisms will grow in a solution saturated with thymol (e.g., in bouillon). Others will grow in the presence of chloroform (5 cc. of chloroform in test-tubes with 10 cc. of milk or beef-bouillon). Ten organisms have been found by the writer which, under the conditions named, grew in the presence of chloroform and two which grew vigorously in the presence of thymol. Russell reports one capable of growing in the presence of sulphuric ether. It is, therefore, not always safe to depend on these substances as antiseptics. Newcombe has made the same observation (Cellulose Enzymes, Annals of Botany, Vol. XIII, 1899, p. 60). In the opinion of the writer the statements of physiologists respecting the existence of enzymes in the tissues and fluids of the higher plants and animals must be taken with much allowance when chloroform, thymol, and similar antiseptics have been *Moore, George T., and Kellerman, Karl F. A Method of Destroying or Preventing the Growth of Algae and Certain Pathogenic Bacteria in Water Supplies. U. S. Department of Agriculture. Bureau of Plant Industry, Bulletin 64, 1904, pp. 44; see also Bull. 76, Bureau of Plant Industry. Certain pathogenic bacteria, such as Vibrio cholerae and Bacillus typhosus, are destroyed within a few hours in water containing traces of copper salts or dissolved particles of metallic copper. PLATE 8. A thermostat-room. In the center of the bidding and lighted by electricity. Ventilated in the isrne way a the photographic dark-roonu, i. e., by an exhaust-Ian run by an electric motor. Three of the thermortaU were made by Bauich 8t Lorob. the fourth (felted) a a Rohrbeck. ENZYMES. 75 depended upon to keep the solutions free from bacteria. This has been the case very frequently, and in several places in Greene's interesting book on Fermentations, published in 1899, it is said or inferred that the addition of chloroform will prevent the growth of bacteria. This might or might not be true ; much would depend on the kind of organisms present. The medium to which chloroform or thymol has been added must be shut in and shaken continuously if the full antiseptic value of these substances is to be obtained. THERMAL RELATIONS. The student should determine (1) Maximum temperature for growth (thermostat). (2) Minimum temperature for growth (ice-box). (3) Optimum temperature for growth (room or thermostat). (4) Thermal death-point (ten minutes exposure in the water-bath, in thin- walled test-tubes of resistant glass having a diameter of 16 to 17 mm., ordinarily in 10 cc. of moderately alkaline peptonized beef-bouillon, viz, + 15 of Fuller's scale). (5) The effect of freezing (exposure to liquid air or to pounded ice mixed with coarse salt). Thermal relations are among the most interesting and should be studied with great care in case of every organism. They offer valuable means of differentiation and also very useful suggestions as to geographical distribution and habitat. Good thermostats are made by various people. Several items of construction are important. The water or oil jacket should be of considerable volume (thickness) so as not to change temperature quickly ; the cover should be thick and of the best non- conducting substances. The opening for the thenno-regulator should be at least i ^ inches in diameter (so as to take a Roux metal-bar thenno-regulator) ; the warm chamber should be of good size ; the space beneath should be high enough between floors to accommodate any pattern of safety burner; and last, but not least, the workmanship should be of the very best quality, so that the apparatus will not leak. Nearly every worker has probably had experience with leaky thermostats at some time in his life and knows what a vexation of spirit they cause, particularly if filled with oil. A very excellent kind of thermostat is the old, large-pattern, felt- covered instrument devised by Dr. Hermann Rohrbeck and figured in the lower right-hand corner of plate 8. This plate shows a thermostat room with four thermo- stats in use. All are provided with Roux metal-bar thermo-regulators and Koch safety burners. One is for quick shifts as needed ; and others are generally kept at 30, 37>4 , and 40 or 43 C. These temperatures, in conjunction with the cool boxes, thermal baths, and various room temperatures, enable one to quickly determine the thermal relations of an organism. The height of the room is 10 feet, its depth 7 feet, and its breadth 5 feet 3 inches. A larger room would be more convenient. Such a room should be located and constructed so as to be as little subject as possible to external changes of temperature. It should be lined with asbestos and sheet iron, and efficient safety burners should be used to the exclusion of all others (see L/autenschlager's catalogue). The improved Koch safety burner is probably the best All burners require frequent inspection. 7 6 BACTERIA IN RELATION TO PLANT DISEASES. The writer has no very satisfactory way of making exposures for determining the minimum temperature for growth. His method is to make such exposures in the bottom of a large, well-filled ice-box, which is opened as little as possible during the progress of the tests, and then only for the briefest periods. The degree of cold Fig. 63* is governed by the amount of ice. A good thermometer is exposed in the midst of a bundle of inoculated tubes, and if the temperature shows any tendency to rise more ice is added. Under the most favorable circumstances the temperature of the *Fic. 63. Modification of the Ostwald water -bath used by the writer for thermal death-point experiments. This' consists of a porcelain-lined pot n inches in diameter at the top. This is filled with water kept in motion by a water-wheel turned by electricity. The heat is applied by means of a Friedburg burner and is controlled by Roux's thermo-regulator. Murrill's gas-pressure regulator is shown at the left. THERMAL RELATIONS. 77 air in the bottom of the chest may be kept fairly constant for some days or weeks, but with marked external fluctuations of temperature trustworthy results can be obtained only by constantly watching the box. What one needs for this work is a good- sized room kept at o C., or a little below, in which thermostats may be installed at GV temperatures a little above freezing, e. g., + 2, +5, +7, etc. It would then be very easy to determine the minimum temperature at which any organism will grow as easy as it is now to determine the maximum. Different levels in the same room may afford constant and useful differences in temperature. The thermal death-point, which is a purely arbitrary standard, depending on the age and kind of culture, its volume, and the length of exposure, as well as the temperature, is when properly determined not least valuable. The writer, following that one of Dr. Sternberg's methods which is easiest to carry out, uses 10 cc. portions of moder- ately alkaline (-f-io or +15) peptonized beef-brothf in test-tubes of uniform diameter (16 to 17 mm.), inoculates from recent bouillon- cultures with care not to touch the sides of the tube above the fluid, thrusts the tubes deep into the hot water, and exposes for ten minutes. All who make this test are urged to use standard alkaline beef- bouilloii (for all organisms growing well in this medium) and to limit the exposure to exactly ten minutes, so that easy comparisons may be made. The five minutes exposure which has been recom- mended by some authors is rather too short, since it only a little more than suffices to warm the fluid up to the required temperature. Inoculation while the tubes are in the bath and after the fluid has been brought to the required temperature is inconvenient and has no special advantage. Fig. 64.* *Fic. 64. Roux's thermo-regulator, made by Maison Wiesnegg (P. Lequeux), Paris. The parts requiring description are as follows: A, bar composed of two metals (which expand and contract un- equally) attached at bottom and free at the top, which moves with increased heat in the direction of the arrow; B, arm on which the upper part of the apparatus moves freely when K is turned; C, stiff spring; D, long rod which controls the gas-inflow, and the spring movement of which is in the direction of the arrow except when controlled by the counter movement of A, due to lessened heat; E, gas-inflow; F, gas-chamber, of glass; G, gas-outflow, to the burner; H, rubber stopper; I, cylinder screwing into L, and provided with capped upright tube filled with vaseline to prevent gas from escaping in the direction of D. The button shown in the gas-chamber at the left is part of D, and the gas enters the chamber between it and the left end of L, the size of the opening, and consequently the amount of gas, varying with the slightest movement of A. Different temperatures are obtained by turning the button K. The constant gas-flow is provided for by a small opening on the lower side of L at its extreme left, in the gas-chamber. About two-fifths actual size. fThe thermal death-point in acid media is considerably higher at least that of several organ- isms which have been tested in the author's laboratory. 78 BACTERIA IN RELATION TO PLANT DISEASES. An excellent water-bath is that known as the Ostwald-Pfeffer. The experi- menter may, however, construct one for himself out of a medium-sized, thick- walled, porcelain-lined iron kettle (fig. 63). This should rest on a ring of heavy strap-iron supported by four stout iron legs. The burner required may be Dr. Friedburg's safety burner (a very inexpensive and good pattern). The thermo- regulator may be a common Reichert if the mercury seal is cleaned from oxide frequently. In such regulators a sharper contact and a longer freedom from obstruc- tion is said to be obtained (Dr. Harris) by putting a drop of olive oil on top of the mercury. A much better instrument is the metal-bar mechanism known as the Roux regulator (fig. 64). This may be procured from the Maison Wiesnegg, in Paris. It should be kept from direct contact with the water and consequent rusting by burying it in a close-fitting glass tube filled with olive oil or glycerin. This tube is then sunk deep into the water and clamped to the wall of the kettle, which should have perpendicular sides. The water is kept in motion by means of a hori- zontal paddle-wheel at the bottom of the kettle. This consists of four light, oblique zinc or copper vanes (nickeled copper is preferable) soldered to a long central rod which fits into a socket, below, and near its upper end passes through a hole or loop in a horizontal metal arm (a foot or less above the kettle), the other end of which is clamped to the upright rod of a solid iron tripod, or fastened to a rod bolted to the table. If compressed air can be had, a stiff cardboard windmill fastened to the upper end of the vertical rod completes the mechanism. The central part of the wind- wheel may be of cork. The vertical rod may be a piece of glass tubing, in which case it is cemented into a socket of the short metal post to which the vanes of the water-wheel are soldered. If a wind-wheel is attached, it is more convenient to have the vertical rod in two parts, fastened by a coupling. The rod, with its water-wheel attachment, may also be turned by some electrical device. The latter is the most convenient method. In fig. 63 the electric motor is not shown. This stands in a small box screwed to the under side of the table at the right. The switch is fastened to the wall above and back of the top of the thermo-regulator. The pulley band is of smooth rounded leather one-eighth inch in diameter. The electric current is passed through an Edison lamp screwed under the table to reduce the velocity of the motion. With the lamp in place and the current cut down to the minimum the number of revolutions per minute is 55, and the temperature of the water is the same in all parts of the bath. The simplest contrivance of all is to make the water-wheel and upright shaft of wood, to be turned by hand. In localities where the gas-pressure is exceedingly variable, Paul Murrill's gas- pressure regulator (at the left in fig. 63) will be found useful. This is made by Eberbach & Co., Ann Arbor, Mich, (see Journal of Applied Microscopy, Vol. I, p. 92, or Centralb. f. Bakt, i Abt, Band XXIII, 1898, p. 1056.) The gas-pressure may be somewhat improved by simply passing the gas through a big bottle (see top of thermostat 311 in plate 8). The Anschiitz normal thermometers, with long stem and scale divided into fifths, are very convenient for determining temperatures (fig. 65). They come in sets of seven, but may also be had separately. The most frequently useful are No. i (scale 15 to +55) and No. 2 (scale + 45 to + 105). THERMAL RELATIONS. 79 They cost 9 marks each when ordered direct from Berlin, and can be had without delay. Good American thermometers are made by Henry Green, New York. With this open bath it is easy to keep the range of temperature down to o.i to 0.3 of a degree, and the writer has frequently exposed tubes for ten minutes without appreciable change in temperature. Temperatures may be read easily to o. i degree by means of a Zeiss aplanat lens magnifying six times (fig. 25), and should be recorded for each half minute during the exposure. Under no circumstances should exposures be made in water which is not agitated. Of course, for accurate reading the eye and the center of the lens must be level with the top of the column of mercury. The lens may be supported at the proper level on a grooved piece of cork. If possible the thermom- eter used should be compared with some standard instrument. If not, it should at least be compared with several other good thermometers in the same laboratory. The test-tubes are supported by perforated corks thrust into holes bored through a rectangular piece of hard, heavy wood. The writer formerly made use only of the first four tests. It seemed hardly worth while to recommend that all bacteria be tested for the killing effect of cold, so long as we had nothing but the inconvenient and more or less inexact methods of salt and pounded ice or of ether and frozen CO* ; but now that liquid air may be obtained at a small price in many of the larger cities, can be shipped long distances, and can be used with so little inconvenience, there is no good reason why the effect of freezing should not be determined in all cases, since in some instances it is likely to prove a valuable means of differentiation. The bacteria may be exposed in 5 cc. portions of distilled water or bouillon in block-tin test-tubes, or preferably in tubes of resistant glass, for standard periods, e. g., one-half hour, i hour, 6 hours, 12 hours, 24 hours, 48 hours, etc. They may also be exposed to alternate freezing and thawing every fifteen minutes or thirty minutes until all are dead. To avoid endospores, the depressing effect of by-products, etc., young cultures should be used, and, of course, all should be of the same age and grown in the same medium, i. e., bouillon cultures 24 hours or 48 hours old. The tests should be quanti- tative rather than qualitative. They may be made as follows : Into 5 cc. of sterile water or standard bouillon a carefully-measured quantity, i. e., one loop, 5 drops, ^ cc., etc., of the culture is placed, stirred very thor- oughly, and allowed some time for diffusion. To avoid zooglcese, which form early in some species, and to reach more uniform measurements, it is recommended to take the loop from a bouillon culture rather than from agar or other solid media. After sufficient time has elapsed for Fig. 65* un if onn diffusion, six Petri-dish poured plates are made from each of the inoculated tubes. The plates should be of the same diameter (area of 60 sq. cm.). The amount of agar used for each plate should be 10 cc., and the amount of infec- *Fic. 65. Anschiitz normal thermometer with degrees divided into fifths (Centigrade scale). For use in thermal death-point tests. About three-fourths actual size. 8o BACTERIA IN RELATION TO PLANT DISEASES. tious material used should be the thinnest obtainable film of fluid across a carefully- measured i mm. loop, so as to avoid crowding the plates. The same loop should be used in all cases, and it should be dipped into and out of the fluid always in the same way. After pouring, set the plates on a perfectly level spot (fig. 66), until the agar has hardened. If the work has been well done, there should develop an approximately uniform number of colonies in each plate. The tubes of inocu- lated water or bouillon are then immediately lowered into the liquid air and exposed to it for the predetermined time, after which six additional Petri-dish poured plates, of the same size and inoculated in the same way, are made from each tube for comparison with those prepared before the exposure. The tubes may be thawed out by exposure to the air for three minutes and then to tap-water for five to seven minutes. The exposures are best made in Dewar glasses (fig. 67). When the exposures are long, a loose tuft of absorbent cotton should be placed in the mouth of the glass, or it should be covered with a hair-cloth cap, to prevent excessive Fig. 66* evaporation. Under these conditions the air remains liquid for a number of days. At first the temperature is about minus 190 C., rising gradually to minus 180 C., since the nitrogen evaporates somewhat faster than the oxygen. The glasses are fragile and should be handled carefully, especially when filled with the air. As long as they contain liquid air it is safer to keep them in their containing-case, packed about with cotton or felt. One should be careful to avoid cracking the inner wall of the glass, as might happen by dropping some hard substance into the receptacle, otherwise an explosion will occur, the space between the two walls of the Dewar glass being a very perfect vacuum. When the exposures are made in block-tin tubes, the culture should be frozen at once on pouring into the tube and the second set of plates should be made as soon as the fluid has thawed, /'. ^., within about ten minutes, for which purpose the culture should be poured out into a glass tube, otherwise complications due to *Fic. 66. Leveling (nivelling) apparatus for use in making poured plates. About one-third actual size. THERMAL RELATIONS. 81 the gennicidal action of the metal might arise. In no case should the cultures be incubated in the tin tubes. When exposures are made in test-tubes of resistant Jena glass, the cultures must be lowered into the liquid air gradually, the fluid being frozen from the bottom upward to avoid cracking the tubes. It requires about four minutes to properly freeze a culture in a glass test-tube. Large volumes of culture media should not be lowered into the liquid air, as it is wasteful, the air boiling away rapidly. The writer began his experiments with block -tin tubes, as shown in fig. 67, but now uses tubes of Jena glass. The latter crack occasionally in spite of care. Fig. 67* For very rapid freezing the amount of fluid in the tube may be reduced to i cc. Liquid air in Dewar glasses, and compressed oxygen, hydrogen, and carbon dioxide (?) in steel cylinders may be had from the Eagle Oxygen Company, Incorporated, 121 West Eighty-ninth Street, New York City. The tanks of compressed gases may be bought or rented. The following sizes may be had : Fifty gallons (280 pounds pressure per square inch) ; 100 gallons (240 pounds pressure) ; 150 gallons (225 pounds pressure) ; and 200 gallons (280 pounds pressure). Cylinders may also be had with the gas under much greater pressure. The cost of the oxygen is 2}4 cents *Fic. 67. Dewar glass for liquid air, and block-tin test-tubes used in first low temperature ex- periments with bacteria. About one-sixth actual size. 82 BACTERIA IN RELATION TO PLANT DISEASES. per gallon. The wrought-steel cylinders cost about $10 each. A good quality of resistant-glass test-tubes may be had from Greiner & Friedrichs, Stiitzerbach, Germany. One sort has a faint-blue longitudinal stripe blown into the glass, another kind has the letter " R " etched on the upper part of each tube. Tubes without any distinguishing mark should not be purchased, as they are likely to become mixed with ordinary non-resistant tubes. The cost of these tubes, duty free, is about $16 per thousand. Good Petri dishes may be obtained from the same firm, and also from E. H. Sargent & Co., Chicago. The temperature demands of bacteria are extremely variable. Whole groups of them are able to live under conditions which would be impossible for the higher Fig. 68* plants and animals. Many of the northern forms, especially those which grow in water, are adapted to low temperatures. The organisms of dung-heaps and thermal springs, and the tropical forms, often grow at high temperatures. For a very few species it has been known that prolonged freezing or repeated freezing and thawing destroys the weaker individuals and finally all. (See Bibliog., XXXIII, especially papers by Sedgwick & Winslow, and by Park ; consult also an earlier paper by Prudden, Bibliog., XL, VI.) For the bacteria as a whole, however, it has been assumed that ordinary freezing or even very intense cold simply inhibits *Fic. 68. Petri-dish poured plate of Bacillus tracheifhilus. The 10 cc. of nutrient agar was inoculated with a carefully measured loop of a fluid culture. The fluid culture was then exposed in block-tin test-tubes to the temperature of liquid air, after which another plate (fig. 69) was made. THERMAL RELATIONS. 83 growth for the time being. Such statements have been based on certain qualitative tests and do not tell the whole truth. In the writer's experiments with liquid air great differences have been detected, the reduction by exposure for one-half hour varying from 15 per cent, or less, to 90 per cent, or more, according to the species tested. Fully 50 per cent of many sorts, grown in bouillon, are destroyed by a single short exposure (see figs. 68 and 69). Query : Is intense cold any more harmful to bacteria than simple freezing? Are young or old cultures most susceptible? Are they killed by the rupture of the cell-wall due to the formation of ice- crystals, or simply by the abstraction of water? Why do some resist several freezings? Can endospores be killed in this way ? Consult '01, d'Arsonval (Bibliog., XXXIII) and Fig. 69* Smith & Swingle, the Effect of Freezing on Bacteria, Proc. Sixth Ann. Meeting Soc. Am. Bacteriologists, December 27, 1904; Science, N. S., Vol. XXI, 1905, pp. 481-483. For opposing views see '02, Macfadyen, Bibliog., XXXIII. Live steam acts upon the growing bacteria very quickly. All bacteria not in spore form, or in some other way protected from the direct action of the heat by what surrounds them, are promptly destroyed by steam heat at 100 C., an exposure of a minute or two being ample, except, possibly, in case of some of the thermo- *FiG. 69. Same as fig. 68, but made after exposure for twenty hours to liquid air. Number of colonies reduced two-thirds. Exposed in test-tubes of Jena-glass for one-half hour, the reduc- tion was nearly as great, i. e., over 50 per cent. In this latter case the agar plates were incubated 7 days at 30 C, before the count was made. 8 4 BACTERIA IN RELATION TO PLANT DISEASES. philic species. Usually even the most resistant spores, if freely exposed, are destroyed by one to two hours exposure to 150 C., of dry heat, or by thirty minutes exposure on each of three consecutive days to streaming steam at 100 C. Some very resistant spores have survived a single steaming or boiling of five or six hours duration (eight hours in one of Tyndall's experiments), and it is not unlikely that some slowly germinating sorts may be able to resist discontinuous steamings for three days. It is possible also that there may be some sorts able to germinate and again assume a resistant spore form in less than twenty-four hours although this is not probable. Some spores are destroyed by a short boiling at 100 C., and all spores are quickly destroyed by steam under pressure, i. e., in an autoclave. A Fig. 70 * temperature of 1 10 C. for ten or fifteen minutes is sufficient. Exposure of media to higher temperatures and for longer periods should be carefully avoided. It must be remembered, however, in using autoclaves, that all of the air must be replaced by steam before the apparatus is closed, otherwise the temperature to which the medium is exposed will not correspond to that indicated by the pressure gage. The -most convenient autoclaves known to the writer are the large sizes of the *Fic. 70. Earliest stage of fruit spot on green plums, due to Bacterium pruni (Erw. Sm.). The bacteria have entered through the stoma. They disappear farther in, and also a few micra to either side of this stoma, as shown by an examination of the serial sections. Material fixed in strong alco- hol, infiltrated with paraffin, and cut on the microtome in series. Section stained with carbol-fuchsin and drawn directly from the microscope with the aid of a camera lucida. PLATE 9. Chamberland autoclave. Heat is applied to the bottom by means of a double ring of Bunaen burners. No wrench is required for fastening on the top. About one-eighth natural size. THERMAL RELATIONS. 85 pattern designed by Chamberland and made by the Maison Wiesnegg (P. Lequeux), Paris, France, the steam being generated by gas (plate 9). The steam gage is at the left ; in the middle is the valve through which the hot air is allowed to escape when the instrument is wanned up ; at the right is the steam safety-valve. The temperature is manipulated by regulating this valve. By leaving the vent open the apparatus may be used as an ordinary steam sterilizer. It may also be used as a distilled-water apparatus by attaching a condenser to the exit pipe of the middle vent, but such water must not be used for culture media. A very good autoclave is also made by the Kny-Scheerer Co., New York. Harding recommends for auto- claves the use of steam from the engine-room boiler. This is convenient, provided one can always have steam ready during the summer months. An autoclave, like a steam boiler, which it is, must be watched carefully if it is not some time to explode from excess of heat or lack of water. Each time before use one should see that the apparatus contains sufficient water. Soils are rather difficult to sterilize. They may be spread in thin layers and dry-heated for several hours at 150 C., or may be heated in the autoclave for an hour under a pressure of two atmospheres, taking care to drive all the air out of the soil before closing the apparatus. It is not likely, however, that soils can be treated in this way without undergoing certain physical and chemical changes. Small pots of soil may be heated in the steamer at 100 C. for two hours on each of five successive days. The reason for preparing all media in the autoclave, or by heating in the steamer at 100 C. on three successive days (the ordinary way), is because we are never certain in what particular case resistant spores may be present. One short steaming is often sufficient to sterilize media prepared in a cleanly way, as every bacteriologist knows who has had much experience, but 'now and then, in spite of all care, resistant spores will find their way into culture media, and for this reason it is best in all cases (especially in teaching students) to adhere to a routine of three steamings. Large masses of fluid (beakers, flasks) require longer steamings than test-tube cultures. The writer gives double time, or triple time. Discontinuous boiling as a means of sterilization was introduced in 1877 by Tyndall, who well says respecting the sterilization of liquids : "Five minutes of discontinuous heating can accomplish more than five hours continuous heating."* Most plant-pathogenic bacteria of temperate and cold regions have a lower optimum and maximum temperature for growth and a lower thermal death-point than species pathogenic to warm-blooded animals. The maximum temperature for growth is usually at or below 36 C. We should not, however, expect this to be true of bacterial plant parasites in tropical and sub-tropical regions, about which, however, little is known beyond the mere fact that such parasites occur. Savastano states that the optimum temperature for the olive-knot organism, which is said to be more prevalent at the southern than at the northern limit of olive-growing, *This method appears to have been known to housewives for a much longer time. In Dr. Sam- uel Johnson's Dictionary (first Am. from eleventh London ed.) I find the following definition: " Biscuit, A kind of hard, dry bread made to be carried to sea. It is baked for long voyages four times." 86 BACTERIA IN RELATION TO PLANT DISEASES. i. e., commonest in southern Italy, Sicily, and Algeria, lies between 32 and 38 C. In my own experiments with this organism, obtained from olive trees in California, I have found its maximum temperature to be above 35 and below 37.5 C. The optimum temperature of Bacterium solanacearum, which is very destructive to potatoes and tomatoes in the southern United States, is probably about 35 C. at least it grew readily and remained alive for a long time in bouillon kept at 37 C. Its maximum temperature is 39 + C. Bacillus carotovorus, one of the best known of the soft-rot organisms, grows well in the thermostat at 33 to 34 C. Its maxi- mum temperature is at 39 C. or slightly below (Jones). Bacillus aroidete, whose temperature relations were recently studied carefully by Townsend, has a maximum Fig. 71 * temperature of 41 C. A temperature of 40 C. retards growth, but does not prevent it. This organism was isolated from calla-lily conns, but is capable of causing a soft rot in potatoes, carrots, turnips, and many other plants (fig. 102). The maxi- mum temperature of Bacillus oleraceae, recently described by Harrison, is said to be about 42 C. This causes a soft rot of cauliflower. The range of temperature suited for the growth of particular bacteria varies greatly. Some species are able to grow through a range of 50 C. Many tolerate a range of only about 30 C. Certain animal-pathogenic forms have through long subjection to a peculiar environment become restricted to a still narrower range. *Fic. 71. Bacterium pruni. Early stage of a leaf-spot in the plum. The small spot was water- soaked in appearance, but it had not yet collapsed. The bacteria, which are most abundant in the mesophyll, undoubtedly entered the leaf through the stomata, three of which are shown in the section. Material treated as in fig. 70. Section drawn with the aid of an Abbe camera. It represents as nearly as possible one plane. THERMAL RELATIONS. 87 Some bacteria grow well only in the cool box, others only in the thermostat at blood-heat or at higher temperatures, temperatures elevated enough to quickly destroy the unprotected protoplasm of the higher plants and animals. Few of the bacteria commonly studied will grow at temperatures much above 40 C., but this by no means expresses the whole truth. The lowest temperature at which growth will take place ranges in different species all the way from o C., and probably a few degrees below (certain salt-water bacteria) to -\- 40, + 50, + 56, and even + 60 C. (certain thermophilic species found in dung-heaps, hay-mows, silos, hot springs, etc.). The highest temperature at which growth will take place ranges from as low as 30 C. (and probably lower*) to as high as 75, or 80 C., or even 89 C., according to Setchell. Higher temper- atures have been recorded, but I have here used only those determined with care in the exact places frequented by the bacteria. This will be better appreciated if it is remembered that a temperature of 60 C. (140 F.) can be endured by the fingers only a few seconds, while 70 C. (the optimum for some of these species) is intolerable to the hand even for the shortest period. It seems incredible, on first thought, it is so opposed to our customary observations, that any organism whatsoever should be able to live at a temperature only 1 1 degrees below the boiling point of water Nevertheless, protoplasm is an extremely adaptable substance, and it is conceivable that some organisms might grow at a temperature considerably higher. The thermal death-point (10 minutes exposure) ranges from 43 C. for Bacillus tracheiphilus, the lowest yet recorded, f to temperatures only a few degrees under the boiling point (100 C.). For many species the thermal death-point lies between 50 and 60 C. Russell & Hastings have recently discovered in milk a Micrococcus whose thermal death-point is 76 C. As the upper and lower thermal boundaries of growth are approached some functions are extinguished in advance of others; e. g., pigment production, patho- genicity, and sporulation disappear considerably in advance of loss of power to reproduce by fission. OTHER HOST PLANTS. Plants of related species, genera, and families should be tested. If the disease appears to be new to literature, it is also especially important to inoculate those plants which have been reported to be subject to bacterial disease and the nature of which disease is still in doubt. Many facts of scientific and economic interest will be brought to light in this way, and now and then the experimenter may be able to clear away some of the fog which, owing to the uncertain and contradictory state- ments of a majority of our plant pathologists, still hangs over the origin and nature of most of these diseases. Some plant pathogenes appear to be quite narrowly restricted. They attack only one host plant, or at most a few hosts belonging to related species or genera. Others, particularly some of the soft-rot bacteria, attack many kinds of plants belong- ing to widely different families. The history of pear-blight, however, shows us that *Since this was written Molisch states (1. c., p. 93) that gelatin cultures of his Bacterium phos- phorcum were dead at the end of 48 hours when exposed to a temperature of 30 C. The maximum temperature of this organism is said to be about 28 C. tVery recently Marsh has found a fish parasite which is said to have a thermal death-point of 42 C. (See VI, Bibliography of General Literature.) 88 BACTERIA IN RELATION TO PLANT DISEASES. the restriction of an organism to a single host-plant may be only an inference based on insufficient observation rather than an actual fact. After a time the apple and quince were added to the pear as host-plants, and now we may add also the plum and the loquat. PATHOGENIC OR NON-PATHOGENIC TO ANIMALS? If the organism will not grow in the thermostat at 37 C, or grows only feebly, as is the case with many plant parasites, it may be assumed to be non-patho- genic to animals with warm blood. Only those organisms which grow readily in the thermostat at 37 C., and which closely resemble animal-pathogenic forms or which are suspected of causing some particular disease of animals, need be tested Fig. 72* by animal experimentation for economic purposes. In general, it is best to leave this part of the work to the animal pathologist, for the same reason that the more abstruse chemical problems are turned over to the chemist. All of the plant-parasitic bacteria, so far as tested, have turned out to be non- pathogenic to warm-blooded animals, but it is not unlikely that some exceptions may be discovered. Another question, of special interest to animal pathologists, arises here, namely, whether forms known to be pathogenic to animals and especially to man are ever *Fic. 72. Bacterium pruni. Vertical section through a green plum fruit (var. Hale) showing bacterial cavities and the escape of the organisms through the ruptured stoma. In this case beyond doubt the central stoma is the one through which the infection originally took place. Drawn from a photomicrograph. The material was fixed in alcohol, infiltrated with paraffin, cut on the micro- tome, and differentially stained. PATHOGENIC OR NON-PATHOGENIC TO ANIMALS? harbored by plants. Of those known to cause animal diseases none have ever been found naturally present in plants, but some of them, such as the typhoid bacillus, the anthrax organism, etc., have been shown to live for a number of days or weeks when injected into various living plants, and in some instances have been found to multiply a little in the vicinity of the wounds. In general, their life is short in such situations, they do not penetrate far into the tissues, and they are manifestly on the defensive. If they can do no better when injected into vegetable tissues in enormous quanti- ties, it seems rather unlikely that under ordinary natural conditions they would find their way into plants so as to make them dangerous for food. In this connection the reader is referred to Volume II, where this subject is discussed more fully. More danger is likely to result from pathogenic organ- isms carried on the surface of plants, especially on salads and fruits which are not cooked. In times of the gen- eral prevalence of typhoid fever, chol- era, or the bubonic plague, the writer for one would certainly prefer to forego salads and to eat only freshly cooked vegetables. The danger from such foods in time of epidemics is very great, especially in localities where ditch-water is frequently sprinkled on the vegetables to freshen them, e. g., in parts of southern Italy. Most saprophytes when injected into living plants behave in the same way as the animal parasites, i. e., they either die at once or maintain a pre- carious existence for some weeks in the vicinity of the wound and then succumb. The writer has made many experiments, with negative results. The most extensive published series of experiments are those of Zinsser (Jahrb. f. wiss. Bot., 1897). To get a particular disease, the parasite must be used and not some other organism. This the writer has observed over and over again. This statement holds good with plants the same as with animals. In case, however, of the less typical plant diseases (soft rots) various members of a group of closely related organisms may produce essentially similar phenomena. This is paralleled, however, in certain of the less typical animal diseases. *Fic. 73. Seedling sweet-corn plant extruding water from its leaf-tips. Most of the infections by Bacterium Stewarti take place during this stage of growth, the bacteria passing down the leaf through its vessels and entering the stem through the lower nodes. Natural size. Fig. 73.* BACTERIA IN RELATION TO PLANT DISEASES. ECONOMIC ASPECTS. The economic aspects may be considered under four heads: (i) Losses; (2) Natural methods of infection ; (3) Conditions favoring the spread of the disease ; (4) Methods of prevention. In the United States Department of Agriculture and in our State Experiment Stations, naturally, much stress is laid on economic considerations, especially on 2, 3, and 4. A knowledge of 2 and 3 will frequently lead to some simple and effective means of prevention. LoaSES. It is desirable that there should be made from time to time a careful estimate of the losses caused by each particular disease, not only as a warning to farmers, fruit-growers, market-gardeners, and florists of the exist- ence of these dangers, but also as an aid to legislatures and governments in deciding how much money may be judiciously appropriated for the scientific investiga- tion of these problems. Pathologists are urged to make and publish such records. It is perhaps unnecessary to add that the determinations should be reasonably accurate, otherwise it were much better not to make any records. Occasionally, when diseases are wide- spread and destructive, so that depreciation of land values and the hostility of a community might result from great publicity, the pathologist may have to con- sider discretion the better part of valor and refrain from publishing, but in this event he should not fail to make full records which may subsequently be pub- lished or at least consulted. What we need and must finally have is a large body of accurate statistics, covering a series of years, many localities, and many diseases. To make these statistics most useful, certain meteorological data should be collected in the same localities. To be of most service this data concerning the weather should be recorded by the pathologist him- self, who will be better able than anyone else to note down just those things likely to influence the host- plants favorably or unfavorably. Some of these things Fig. 74 * *Fic. 74. Bacterium Stewarti (Erw. Sm.) attacking sweet corn (Zea mays). The section was cut from the extreme upper part of a seedling leaf which was fixed in strong alcohol six days after placing the bacteria on its tip. At the time of inoculation water was extruding from the leaf-tip, as shown in fig. 73. This figure represents a longitudinal vertical cut. The dotted and heavily shaded parts show the location of the bacteria which have entered through the ordinary stomata and have not yet penetrated the vascular system, although in places, as at D, they are close to the spiral ves- sels. At A, B, and C are three stomata. The substomatic chamber under A is free. B, with its surroundings, is shown more highly magnified in fig. 75. Drawn with help of the Abbe camera. COLLECTION OF STATISTICS. 9 1 are cloudy weather (especially if prolonged), sunny weather, frequent or excessive fogs or dews, amount of rainfall, and frequency of rainfall, snowfall, hail, excessively hot weather, cold spells and frosts, droughts, daily maximum and minimum tem- perature, prevalence of special diseases correlated with special peculiar conditions, absence of other diseases, etc. NATURAL METHODS OF INFECTION. Under this heading the student should be on the watch for transmission of the disease through fungous or insect injuries, by mollusks, by birds or quadrupeds, and by the hand of man. Man contributes to the spread of diseases in various ways, Fig. 75* e.g., by neglect to remove diseased plants, by use of infected knives and other tools, by the introduction of infected seeds, or manures, or soils, or water, and by subjecting his plants to a variety of depressing and unwholesome conditions. A great variety of parasites find their home in the earth, the top crust of which swarms with bacteria and fungi. Such parasites are frequently introduced from one locality to another in infected soils adhering to wagons and other farm tools, to the feet of men and animals, to the roots of transported plants, etc. The soil is a living thing and it should not be transported even from one field to another on the same *FiG. 75. Bacterium Stewarti filling the substomatic chamber and pushing out into the deeper tissues of a maize leaf. The result of an inoculation made by placing a small quantity of a pure culture on the tip of a sweet-corn leaf in the seedling stage. For orientation see fig. 74. The glo- bose bodies are nuclei, which are not enlarged (?). 92 BACTERIA IN RELATION TO PLANT DISEASES. farm without due consideration of what may happen. Certain bacterial diseases might be distributed very readily in this way and good fields rendered worthless for certain crops. The parasite may gain entrance to the plant through wounds (plates 2 and 4 and fig. 8) or by way of the stomata (figs. 70 to 75), lenticels, water-pores (figs. 76 to 79), and nectaries. In recent years the writer has discovered a number of very characteristic infections by way of the stomata and the water-pores, which are only modified stomata, e. g,, in cabbage, mustard, plum, bean, soy-bean, cotton (fig. 80), Fig. 76.* pelargonium, larkspur, broomcorn, sorghum, maize, cucumber, etc. Pear-blight affords one of the most striking examples of wholesale infection by way of the nec- taries. The wilt of cucurbits affords an equally good example of infection through wounds namely, leaf-injuries due to beetles. *Fic. 76. Bacterium campestre. Section of a cabbage leaf parallel to the surface and near the margin, showing the result of infection through the water-pores. The tissues are browned and de- stroyed. Immediately under the leaf-serrature a cavity has formed and the bacteria have begun to penetrate into deeper parts of the leaf by way of the spiral vessels, not all of which are occupied. This figure is slightly diagrammatic, but only to the extent of omitting the protoplasmic contents of the parenchyma cells and of introducing six occupied spiral vessels which belong to the next section in the series. No spiral vessels are visible in the lower part of the section because the knife passed just below them. Material collected on Long Island, July 16, 1902, and fixed in strong alcohol. The spirals here shown are a little too densely occupied by the bacteria to make a good drawing under the oil-immersion objective, but a little farther in (beyond X) they are less abundant and entirely satisfactory for this purpose. ECONOMIC ASPECTS. 93 CONDITIONS FAVORING THE SPREAD OF THE DISEASE. The conditions favoring the spread of diseases may be wholly telluric, such as high temperature, unusual drought, cold weather, fogs, heavy dews, and excessive or continuous rainfall. These diseases may be favored by lack of natural drainage, or may be brought on by a variety of causes which are largely within the control of the grower, such as selection of improper varieties, i. e., very susceptible ones, overcultivation, storage at too high temperatures (in case of cabbage and root crops), the use of infected soils, or manures, or seeds, or plants, and, especially in hot-houses, by the mismanagement of water and heat, and by the neglect to destroy the first diseased plants that appear and such transmitters of disease as insects and slugs, which frequently abound in hot-houses. Fig. 77.* METHODS OF PREVENTION. In case of certain diseases, copper fungicides have been found useful, e. g., in walnut bacteriosis and some of the leaf spots, but in general we know as yet very little about bactericidal treatments. In the early stages of an outbreak some of these diseases may be controlled by extirpation of the affected parts, or by the removal of whole plants as soon as they show signs. Also, if possible, the common carriers of infection should be eliminated. Finally, one should not forget that the substitution of resistant vari- eties for susceptible varieties is one of the most hopeful methods for disposing of certain of these vexatious diseases. Whenever any- thing specially noteworthy has been discov- ered in the way of treatment it will be mentioned under each particular disease. Fig. 78.f *Fic. 77. Bacterium campestre from the cavity shown in fig. 76, illustrating water-pore infec- tion of the cabbage. X 2,000. tFic. 78. Bacterium campestre occupying a spiral vessel in a cabbage leaf near a group of infected water-pores. The tissues to the right and left of this vessel, and also above and below it (slide 223 33, 18.6 by 9.7), are entirely free from bacteria. The body of the leaf and all its inner tissues up to within a few millimeters of the leaf-tooth, and also the outer surface of the leaf up to the water-pores, are sound. On the contrary, an unbroken bacterial occupation can be traced from this vessel outward to the water-pore region. The bacteria in this vessel are also less abundant than in those nearer to the group of water-pores, '. e., its occupation is of more recent date. Even if there were no other evidence of infection by way of the hydatodes than that afforded by this vessel, the presence of the bacteria in it under the circumstances mentioned points conclusively to marginal (water-pore) infection as their only possible source. The position of this vessel is in a small vein a little below and at the left of X in fig. 76. Its distance from the left margin of the bacterial cavity is one field of the 16 mm. Zeiss objective with the 12 comp. ocular. Its distance from the sound leaf margin is two-thirds the diameter of such a field. A nucleus is shown at n. 94 BACTERIA IN RELATION TO PLANT DISEASES. GENERAL CONSIDERATIONS. LOCATION OF THE LABORATORY. If possible, the laboratory should be in a clean building in the middle of a green lawn. If it must be in a crowded and dirty city it should be on an upper floor, as far removed as possible from the dust of the street and from the tramp of feet. It ought not to be located on streets filled with the dust of heavy traffic. If a ground- floor or basement room in a dirty locality is the only available place, then the air which enters the room should be filtered through absorbent cotton. A south front is desirable for the mounting of a heliostat and for other photographic purposes ; a north light is desirable for microscopic use, if one is to work at the instrument continuously. By arranging one's time according to the position of the sun, the light from east or west windows may be used to advantage five or six hours a day, which is quite long enough to fatigue ordinary eyes. The writer has managed to get along very well without north light for the last ten years. If one decides to use with the microscope only ar- tificial light, such as that of the Welsbach burner, work- rooms for this purpose may be located anywhere. If pos- sible, several rooms should be secured and apportioned to the various kinds of work, e.g., general laboratory rooms, chambers for special workers, sterilization-room, thermo- stat-room, cold-storage and stock-culture rooms, storage rooms for chemicals, small glass-inclosed rooms for transfer of cultures, photographic rooms, dark rooms for developing, etc. The general photographic rooms should have overhead light as well as side light. EQUIPMENT OF THE LABORATORY. Many pieces of apparatus may be procured from time to time, as the exigencies of the work demand or as the funds will permit. Other apparatus must be provided on the start, and some of it when the building is constructed or reconstructed. Fig. 79* *FiC. 79. Small portion of a cabbage leaf from Long Island, New York, showing characteristic water-pore infections due to Bacterium campestre. The blackened veins correspond to the location of the bulk of the bacteria which have gained entrance to the vascular system of the leaf by way of the groups of water-pores situated on the serratures of the leaf, particularly those which are conspic- uously blackened. Those parts of the leaf where only the larger veins are shown were green and normal in appearance. Coll. July 16, 1902. Drawn from a photograph. PLATE 10. fl 1 ore S~ *> s 5 C en 03 T IS- II * r> ? 1 6 EQUIPMENT OF THE LABORATORY. 95 There should be hot-water pipes, cold-water pipes, steam pipes, a steam bath, gas-pipes, compressed-air pipes, exhaust-air pipes (plate 10 and fig. 81), and electrical wires for light and motive force. There should be thermostats, water- baths, cooled rooms, ice-boxes, steamers, dry-ovens, autoclaves, a distilled-water outfit, an alcohol-still (by which waste alcohol may be recovered or absolute alcohol prepared), an ether-still, filters, gas-generators, gas-furnaces, anaerobic apparatus, the very best microscopic outfits including apochromatic lenses, photographic and photomicrographic appliances, liquid-air receptacles, cylinders of compressed carbon dioxide and oxygen, microtomes, paraffin baths, glassware, balances, chemicals, and many minor pieces of apparatus. Fig. 80* *Fic. 80. Angular leaf-spot of cotton in which stomatal infections appear to be the rule. This leaf represents the secondary stage of a natural infection, i. e., the spots have browned and shriveled, and they involve the entire thickness of the leaf. In an earlier stage of the disease the spots are limited to the under side of the leaf (mesophyll), and occur in the form of small water-soaked, uncollapsed areas surrounding stomata, under which nests of bacteria occur. These spots gradually deepen so as to involve the palisade tissue, and then they become visible on the upper surface of the leaf. The spots are not yet shriveled or browned, but if the leaf is held up and viewed by trans- mitted light they appear as translucent areas, while by reflected light they are dull and wet-looking. A little later they present the appearance shown in this figure. The writer has obtained all stages of this disease in Washington by spraying upon the plants young agar cultures of Bacterium malvace- arum suspended in sterile water. 9 6 BACTERIA IN RELATION TO PLANT DISEASES. In general, the working capacity of a laboratory will be greatly increased by giving the director a stipulated sum of money per annum and carte blanche to buy laboratory necessities whenever and wherever and in whatever quantity he sees fit, requiring only that he submit vouchers ; also by the employment of a number of subordinate assistants of special fitness, to whom may be assigned much of the purely mechanical and routine work of the laboratory, such as the proper cleaning of glassware, the making of ordinary culture media, the keeping alive of stock cultures, the preparation of staining media, the embedding, cutting, and staining of microtome sections, the making of photographs, the indexing of literature, etc. No scientific man should be willing to trust any piece of work in his own line to an assistant unless he can do it as well himself, or better, but when it has become to him the merest routine, his time, if worth anything, can be more profitably employed in something else. In most American laboratories which the writer has visited there is a woeful lack of intelligent subordinate assistance, such, for example, as that furnished by the German "Diener" and the Malays of Java. Every assistant can not hope to be- come at once an independent investigator, although, if competent, his work should always be shaped toward this desirable end. A good library should be within easy reach, and as a suggestion to this end a list of useful books and papers is appended under the head of Bibliography of General Literature. A card catalogue of current literature is also very useful and in time becomes invaluable if properly made. Fig. 81.* CARE OF THE LABORATORY. The laboratory should be a clean place. Its walls should be of such material that they can be rinsed or wiped down occasionally. The floors, doors, tables, window-sashes, etc., should be wiped every day, every other day, or at least every third day, with clean cloths wet in distilled water, boiled water, or clean lake or artesian water. The use of river water, swarming as it does very frequently with all sorts of bacteria, is not to be commended for cleaning purposes, and brooms should be taboo. No one should enter the laboratory who has not business there, and order and quiet should prevail. *FiG. 81. End of the vacuum-pipe on laboratory table. The gage serves to show the degree of exhaustion, i. e., whether there is any leak in the piping between the engine-room and the labora- tory. The two rooms should be connected by a speaking-tube. CULTURE MEDIA. PREPARATION AND CARE OF CULTURE MEDIA. 97 Everything should be carefully weighed or measured. Everything should be clean as possible to begin with. By water is usually meant distilled water, and this should be free from copper or other gennicidal metals (see Bolton, Bibliog., XXXVIII). Moore & Kellerman have shown very recently that the Bacillus typhosus is destroyed in distilled water if the merest trace of metallic copper is present. Water swarming with this organism was sterilized simply by standing three hours in a copper vessel. The writer found the count of Bacillus tracheiphilus reduced over 30 per cent by exposure in bouillon in block-tin tubes for twenty-one hours. Exposure for forty-eight hours gave the same result, i. e., 33 per cent destroyed. A simple glass still is shown in fig. 82. As far as possible the chemicals should be Fig. 82* c. p., and in many cases it is necessary to make the test for oneself, no matter what the reputation of the firm or the statement on the label. When possible, broken packages should be avoided. It is therefore best to procure most chemicals in several small packages rather than in one large one. If the preparation of culture media is broken off before its completion, by nightfall or interruptions of any kind, the unsterilized or incompletely sterilized media should be put into the ice-box, especially if it is warm weather. Neglect of this precaution frequently results in the spoiling of the media. In steam sterilization one should begin to count time only after the thermometer registers 100 C., or at least 99 C. Those who live in high *Fic. 82. Portion of a work-table showing method of distilling water for use in making culture media. The flasks should be insoluble glass. The cold hydrant water passes through the condenser in the direction of the arrow. In actual use the upright flask and the flame are sheltered from air- drafts by sheet asbestos. One-ninth actual size. BACTERIA IN RELATION TO PLANT DISEASES. mountain regions must use autoclaves. Agar, potato, etc., in test-tubes, may be steamed twenty minutes on each of three consecutive days. Gelatin, beef-bouillon, and all other fluids likely to be injured by long heating should be steamed only ten or fifteen minutes on each of three consecutive days, if in tubes. The writer frequently steams such media fifteen minutes the first day, ten minutes the second, and five minutes the third. Agar, gelatin, bouillon, etc., stored in flasks in large quantity must be steamed a longer time usually thirty to forty-five minutes on each day. The first steaming, when softened gelatin is added to bouillon, usually requires thirty minutes. To melt flasked agar quickly, shake it into fragments or break it with a sterile glass rod before putting it into the steamer. Oversteaming should be carefully avoided. It softens gelatins or altogether prevents their solidi- fication, and is very apt to cause troublesome pre- cipitates in a variety of media. Precipitates in bouillon often occur if the tubes are not clean, or if the bouillon was not well boiled at first before filtering and placing in tubes. If the beef-broth looks greenish in the beaker or flask, rather than a clear yellow, it may be assumed that it needs more boiling and that if tubed in this condition it will throw down whitish particles on subsequent steaming. The writer prefers to obtain his ordi- nary + bouillons by incomplete neutralization with sodium hydrate rather than by addition of hydrochloric acid after full neutralization. The adding of hydrochloric acid precipitates out certain nutrient substances and also seems to interfere with the growth of some organisms. Distilled water and river water should be sterilized in quantity in the autoclave. For details concern- ing the making of particular media the student should consult the standard text-books, a dozen or more of which should be kept within easy reach in every laboratory. Some formulse are given in the middle part of this volume. The autoclave may be used for the preparation of sterile water and some media, but, in general, I prefer media which has not been heated above 100 C., especially for use with sensitive organisms. Media should be heated in the autoclave only for a brief time and at a minimum pressure, generally not more than ten minutes and at not more than 110 C. Milk, gelatin, and media containing sugars should never be sterilized in the autoclave. Sugars Fig. 83 * *Fic. 83. Apparatus for rapidly filling test-tubes with 10 cc. portions of agar, bouillon, etc. By means of tins device an expert assistant can fill 500 tubes an hour. Made to order by Emil Greiner. Height, 23 inches. The bulb above X is essential. PREPARATION AND CARE OF CULTURE MEDIA. 99 and other substances decompose at these high temperatures and the results obtained by the growth of bacteria in such media are not comparable with those obtained on media sterilized at 100 C. Kitchens has recently shown that detri- mental acids are formed when bouillon containing sugar is autoclaved. Peptone water, agar, and bouillon may be sterilized in the autoclave. For titrating culture media the writer uses the burettes shown in fig. 59. The twentieth-normal alkali is stored as shown in fig. 60. Quadruple-normal sodium hydrate solution is used for neutralization. The phenolphthalein solution is made by adding i gram of N the dry powder to 100 cc. of 50 per cent alcohol, and then enough sodium hydrate to carry it fully into solution, removing the yellow color without making the fluid a very decided pink. Fluid media may be filled into tubes very rapidly by means of the device shown in fig. 83. For storing media sterilized in test-tubes and for hold- ing cultures made on such media the writer has found ordinary quinine cans very use- ful (fig. 84). The proper care of culture media after sterilization involves considerable thought if they are not to be used immediately. Stored media lose water and along with this loss, of course, there are physical changes, so that the results obtained are not always comparable with those obtained from similar media containing the standard volume of water. Various devices have been recommended for pre- venting this loss of water. Rubber caps keep in the moisture, but are apt to favor the development of fungi. Paraf- fined plugs made by removing the cotton plug, dipping the lower end of it quickly into and out of hot sterile paraffin, and replacing it in the mouth of the tube or flask before the melted paraffin has had time to cool, answer the purpose very well, but have the objection that all of the tubes must be placed in turpentine or some other solvent of paraffin before they can be cleaned for a second use. On the whole, the use of moderately tight plugs and the storage of the media in cool or cold air are the best methods of retaining the water content of the medium. Nutrient media should be made in small quan- tities and often, rather than in large quantities and at infrequent intervals. The cotton should be dry-heated in bulk before plugs are made from it *Fic. 84. Ordinary quinine cans with a little cotton in the bottom are very convenient for holding cultures and culture-media in test-tubes. One-third actual size. 100 BACTERIA IN RELATION TO PLANT DISEASES. THE CLEANING AND STERILIZATION OF GLASSWARE AND INSTRUMENTS. New glassware may be boiled in soap-suds, rinsed thoroughly, soaked in the chromic-acid cleaning mixture for some hours, rinsed in hydrant water, soaked in several changes of distilled water, soaked or shaken in alcohol, and finally rinsed in distilled water. Neglect to wash in alcohol will frequently leave behind on the walls of the test-tubes an invisible film which causes vexatious precipitates in beef- bouillon, etc. Discarded tubes, flasks, and dishes containing living organisms must be autoclaved or filled with the chromic-acid cleaning mixture before they are washed. Some responsible person should attend to this. If acid is used'it should be allowed to act for some hours. Petri dishes should fit together well, but not tightly, and should be double- wrapped in clean Manila paper before placing them in the hot-air oven, or else should be inclosed in suitable tin boxes. The writer prefers to wrap them. The paper for this purpose may be 12 by 12 inches. The dish should be placed in the middle. The sides of the paper are folded over it; the corners of the projecting ends are then turned in, leaving V-shaped flaps, which are folded down on to the plate. The second cover- ing is folded at right angles to the first and on the other side of the dish. Dishes treated in this way and ready for steril- ization are shown in fig. 85. Pipettes should be dry-heated in the tin boxes already men- tioned (fig. 37) after having the upper end carefully plugged with cotton, which should not project. Knives, scalpels, scrapers, spatulas, needles, forceps, etc., may be sterilized in the Bunsen flame, or, if needed cold in quantity, may be wrapped in Manila paper or put uncovered into short tin boxes and heated in the dry oven at 140 C. for two hours. Petri dishes, test-tubes, and all other apparatus wrapped in paper and put into the oven for sterilization by dry heat should have air spaces between them, i. e., they should not be crowded together tightly, and the recording thermometer should project well down into their inidst. The investigator should test the behavior of his oven when full and empty. Many cheap ovens give very different temperatures in different parts, especially if filled with apparatus, so that cotton or paper may be scorched in one part and not sterilized in another. The best oven known to the writer is that made by L/autenschlager. The improved form of the L,autenschlager oven shown in plate 6 does not require watching and gives a uniform temperature *Fic. 85. Petri dishes wrapped in two layers of Manila paper and ready to be dry sterilized. They are set on edge in the oven. Fig. 85* STERILIZATION. IOI in all parts. It also furnishes a maximum temperature with a minimum con- sumption of gas, hot air being fed to the flame. The apparatus has an inner, outer, and middle wall. A horizontal iron gas pipe, of the relative size shown in the front of the picture, passes entirely around the apparatus at the bottom between the outer and middle wall. On top in this tube are many small openings through which gas escapes and when lighted forms so many small Bunsen flames. Air is drawn in at first and mixed with the gas in the middle open part of the feed pipe in front. The products of combustion escape through the chimney- on top of the oven. There are pilot lights, so that the apparatus is set going easily. The result of this arrangement is that the middle wall becomes heated very hot, and consequently the air between this wall and the inner wall rises, cool air entering through holes in the bottom to take its place. There is thus created a powerful upward mount of hot air. This enters the oven through several hundred holes in its ceiling, is forced downward and escapes through as many holes in the floor. From this place the hot air is continually crowded sidewise and backward through brass tubes into the furnace chamber where it serves to support the com- bustion. Unless the dry-oven has a very uniform temperature through- out, so that there is no danger of scorching the cotton, plugged test-tubes should be tied together loosely and stood on end, cot- ton uppermost. Petri dishes (wrapped in paper as directed) may be set on edge. If the test-tubes have been properly cleaned, dry -heating is not necessary for such as are to hold steam-heated media, provided the cotton used for the plugs is dry-sterilized in advance. The best surgeon's absorbent cotton Fig. 66 * is not too good for this work. It should be unrolled and put into the dry-oven in a loose armful and heated just below the scorching point for several hours (2 to 3 hours at 145 C. will answer), with occasional unfoldings and turnings so that all parts may be heated uniformly. It is now taken out, re-rolled and put away in clean paper until needed. By this means all fungous spores lodged in it are destroyed and *Fic. 86. Dr. George Meyer's hypodermic syringe, made by Lautenschlager. Desirable on account of perfect workmanship, and because it is easily sterilized without injury. This size holds I cc. By twisting the button of the piston the packing at the other end is tightened or loosened at will. The separate parts are enlarged one-fourth. IO2 BACTERIA IN RELATION TO PLANT DISEASES. an oil is driven off which otherwise would be deposited as a whitish distillate on the inside of the test-tubes near the plugs. Hypodermic syringes may be sterilized by boiling in distilled water if the contaminating organism is non-sporiferous, or by soaking twenty-four hours in 5 per cent carbolic-acid water or lysol water and a subsequent soaking and boiling in pure water. The writer prefers the Meyer syringe, made by Lauten- schlager (fig. 86). Syringes which allow the culture media to ooze out around the piston whenever any strong pressure is exerted are danger- ous and should never be used with infectious material. Those which do not admit light or allow the experi- menter to see how much fluid has been used or whether air is present are unsatisfactory. In case of many plants, needle-pricks are more satis- factory than hypodermic injections (pi. 4 and figs. 8 and 88). Needles are sterilized in the open flame as needed. When conveniences are not at hand, as on long trips in the country, the kitchen-oven may be used for sterilizing glassware, or even an open flame (alcohol lamp), and agar and gelatin for the making of poured plates may be melted by placing the tubes in hot water in a tin cup or tea kettle, but, in general, the writer has not found the rooms of ordinary farm houses very well suited for research work. Usually they are too dusty. Surgeon's gauze is very conve- nient for laboratory use, for coarse filters, wipe-cloths, etc. Fig. 87.* *Fic. 87. Early stage in the infection of a cabbage leaf by Bacterium camfestre; a, epidermal layer on the apical part of the tooth of a leaf, showing one of the four stomata ( X ) full of bacteria. For the condition immediately under X see b, which was drawn from the third section in series, the intermediate one including part of the guard-cells. 'Slide 338, Bi, stained with carbol-fuchsin. Drawn with the Abbe camera, 3 mm. Zeiss apochromatic objective and 12 compensating ocular. Material collected and fixed 8 days after infection, which was accomplished by atomizing upon the plant water containing a pure culture of Bacterium campestrc grown on slant agar. When collected many of the serratures had begun to show traces of the brown stain which invariably appears when this organism grows in cabbage. The plant was inclosed in the cage shown in fig. 95, and was ex- truding fluid from its water-pores when it was sprayed. X 500. HOW TO AVOID CONTAMINATIONS. 103 THE MAKING AND TRANSFERENCE OK PURE CULTURES. In addition to what has been said under Pathogenesis, the following suggestions may be of service to the beginner. For the making of plate cultures and for the transfer of organisms from one culture medium to another, select a still day and, if possible, a day when a gentle rain or snow is falling. This offers ideal conditions, since the earth is wet, the outside air has been washed free from dust, and there is no wind to stir up dust within the laboratory. A strict adherence to this rule is sometimes very inconvenient and it is Fig. 88.* not meant to be iron-clad. It is, however, of immense service in keeping cultures free from contaminations, and those who propose to disregard it should remember that haste in the beginning of an experiment often leads to vexation and delay in the end, especially when the success of the experiment depends absolutely upon the purity of the culture. *Fic. 88. Soft rot of green cucumbers inoculated by needle-punctures from a pure culture of Bacillus carotovorus. The only parts not softened are those through which the infected needle en- tered, i. e., the parts rubbed with mercuric-chloride water. In each a little button of tissue under the disinfected area did not decay. The sound fruit at the right was punctured at the same time, but with a sterile needle. The cucumbers had been removed from the vine, but were not flabby. They were exposed after inoculation to the ordinary air of the laboratory. The photograph was made on the seventh day. About two-fifths natural size. 104 BACTERIA IN RELATION TO PLANT DISEASES. When ready to make the transfers or to pour the plates, close the windows, wipe up the tables, and wet down the floor, window-sashes, etc., with distilled water or boiled water, and reduce the air-currents within the laboratory to a minimum (especially when transfers are to be made in the open room) by keeping the doors shut and restricting the movements of all persons who may be in the room. It is much better to do all of this work in specially constructed small rooms (plate n) than under hoods (plate 12). Hoods are open only in front. They may be made of any convenient size. The one here figured is is 32 by 39 by 20^ inches, outside measurements. When one is far from laboratories small hoods may be extempor- ized out of clean paper, or cultures may be poured and transfers made inside of a clean pail or jar, turned down on its side. Any method, in fact, which restricts the movement of air past open plates and tubes will be found serviceable. The work-shelf of the room shown in plate 1 1 faces a window as wide as the room, and extending from the level of the shelf to the height of the other windows in the room. This window faces south and is only 6 feet from a well-lighted win- dow in the outer wall of the building. The room also receives bright light from the west side. At the front end of the shelf are a Bunsen burner with cut-off flame, a box of safety matches, a box of rubber bands, and two tumblers one for burned matches and one for platinum loops, needles, forceps, etc. Immediately under this part is a narrow drawer for pencils, note paper, knives, etc. At the back end are a few wrapped Petri dishes, a nivella- tion apparatus, a flask of sterile water, and a crate of media. Underneath this part is Fig. 89.* a second shelf 3 inches below the first, where Petri dishes and tubes containing solid media may be put out of the light as fast as inoculated. The size of this room (inside measurement) is 4 by 4 by 10 feet, and it is large enough. No provision is made for ventilation, because air-currents in a culture-room are very objectionable. The windows, walls, and floor are wiped up with distilled water before making transfers. Outside is a bit of the author's private laboratory. At the right is the microtome and behind it on the wall are deep and shallow drawers ; 69 is for bulk paraffin; 70 A, B, C, D, E, are cut into small compartments used for paraffin blocks. The very shallow drawers are for ribbons which can not be mounted the day they are cut ; 72 has a series of shelves opening on the south side and is used to hold photographic printing frames. *FiG. 89. Pine block with inch holes, convenient for holding test-tube cultures during exam- ination, or tubes of media which are to be inoculated. A good size is 95/2 by 3 l / 2 by i^ inches. PLATE 11. The author's culture-room. At the left hand (back) are narrow ihelvn lor culture-media, pipette-boxo, etc. At the right a the work-ihelf , covered with plate gU. PREPARATION OF POURED PLATES. 105 The agar may be poured at 42 C. in case of organisms whose thermal death- point is known to be high (50 C. or above). For all others it must be cooled carefully to 40 C. before inoculating for poured plates. This requires five or six minutes in the water bath at 40 C. Even this temperature is too high for some organisms and then gelatin at 30 C. may be used. When ready to pour, take a clean absorbent cloth and carefully wipe all water from the outside of the tube (the lips of which have been previously flamed gently with a rotation of the tube on its long axis), lift the cover of the dish only as much as is necessary, hold the cover aver the dish (not at one side), pour quickly but gently, and re-cover, tilting the dish about quickly but gently, if the fluid has not already covered the bottom. To en- tirely cover the bottom sometimes requires a smart little jerk, if the agar is not very fluid. The student must learn to work rapidly and dextrously, then there will be no complaint that the agar has solidified before the plates are poured. The plates should be set on a level shelf while the agar or gelatin is hardening, or, if the colonies per square centimeter are to be determined, a nivelling appa- ratus such as that shown in fig. 66 must be used, and the dishes should have flat bottoms. When plates have been inoculated too abundantly to secure subcultures from single colonies, these may some- times be obtained from the traces of agar or gelatin left in the tubes from which the plates were poured. With this end in view, these tubes should be re- plugged and laid away, for a few days, the lips and top of the tube which were wet by the agar or gelatin being first heated hot in the flame, care being exer- cised not to crack the tubes. All tubes containing fluids should be opened and inoculated in a position as nearly horizontal as their contents will permit, and tubes of solid media, such as agar, may be held level or inverted for inoculation. A convenient block for holding test-tube cultures during examination is shown in fig. 89. It is usually best to flame the plugs slightly before their removal, particularly if they have been exposed to the air for some days. As an additional precaution the transfers should be made under a glass hood, or in a special culture-chamber. If sterilized needles, loops, knives, forceps, pipettes, or anything else designed to be used in making the transfers have accidentally touched anything wJwtsoever, they are presumably contaminated and must be rejected or reflamed. Do not handle the lips of test-tubes containing gelatin or agar from which plates are to be poured. Your hands may be con- taminated by resistant spores. Take hold of the tubes lower down. To economize gas and avoid heating the air of the small work-chamber to an uncomfortable degree, small, cut-off, constant-flame burners are very convenient (fig. 90). *Fic. 90. A constant Bunsen burner with cut-off flame. Very useful for the laboratory table and the culture room. About two-fifths actual size. IO6 BACTERIA IN RELATION TO PLANT DISEASES. Plates, tubes, and flasks containing pure cultures or designed for inoculation should never be opened in the general laboratory on a windy day or in air currents. Pour two uninoculated agar or gelatin plates in the proper way. Keep one covered and uncover the other fora few moments in a current of air, i. e., as long as the time required to make a plate culture. Then keep the two plates together and com- pare from time to time. A few experiments of this sort will convince the most skeptical of the necessity of avoiding drafts. The person and clothing of the experimenter should be as clean and free from dust as possible. White duck coats are very convenient. They show at .once when they are soiled and need washing and ironing. Organisms which for some reason may be difficult to obtain in ordinary plate cultures and which differ markedly from their associates in some particular way, e; > by more rapid growth, by indifference to heat, to acids, to thymol, to chloro- form, to absence of air, etc., or which can use, as food, substances which will not support the growth of most bacteria, may sometimes be isolated very readily by providing conditions suited to their growth and unsuited to that of the bacteria with which they are mixed. This is Winogradsky's principle of elective culture. As he defines it, this is a culture " which presents conditions favorable only to a single definite function or, more exactly, to a function as strictly limited as possible." Such Fig. 91.* media or methods are exactly the opposite of universal. Nutrient starch jelly and nutrient silica jelly are good examples or such media. Nutrient fluids rich in acid potassium phosphate or destitute of nitrogen are additional examples. Heat is often an excellent means of separation. Winogradsky separated his Clostridium pasteurianum from all but two of the contaminating species by heating ten minutes at 75 C. (Archives des Sci. Biol., Vol. Ill, p. 310). The isolation of Streptococcus (Leuconostoc) mesenterioides by L,iesenberg & Zopf and of Bacillus hortulanus by Sturgis are other examples of separation by heat. Omelianski's separation of his hydrogen-cellulose ferment from his methane-cellulose ferment by exposure of the recently established methane ferment to 75 C. for fifteen minutes is another good example. THE FINAL DISPOSAL OF INFECTIOUS MATERIAL^ Diseased material should not be left around the laboratory any longer than is necessary. When it has served its immediate purpose that which is not to be pre- served permanently should be thrown into the furnace. Small amounts may be sterilized by putting into beakers or jars and covering with cleaning mixture or equal parts of crude sulphuric acid and water. Crude vegetable and animal sub- *Fic. 91. Instrument for making puncture-inoculations. It consists of a bone handle with a metal-screw socket, into which a sewing needle is thrust. The needle is usually of small size a No. 8 or 10. PLATE 12. Work-table with movable frame of wood and glass. Bacteriological trantfen may be nude under this frame in the open room it windows and dooti are kept doted. DISPOSAL OF INFECTIOUS MATERIAL. 107 stances likely to become moldy must never be stored in refrigerators designed for pure cultures. The open ice-box is the proper place for such substances, and they must not be left there indefinitely. Some people have a mania for collecting every- thing and then keeping it a long time without making any use of it. An ice-box treated in this way soon becomes an intolerable nuisance. Discarded plates, tubes, slides, covers, pipettes, contaminated litmus paper, etc., should be autoclaved, or covered or filled with cleaning mixture, or dropped into it, as the case may be. Deep, narrow glass jars or long, rectangular enameled pans are necessary for the pipettes. Soiled hands may be disinfected with mercuric-chloride water (1:1000), which should always be on hand in the laboratory in quantity prop- erly labeled. Slight wounds should be washed five or ten minutes in this fluid. Surfaces of floors, tables, etc. soiled by spilled bacterial cultures should be covered immediately with mercuric-chloride water (1:1000) and wiped up carefully after ten or fifteen minutes with distilled water. Spilled cultures of molds should be soaked in mercuric chloride ( i : 1000) for at least an hour before wiping up. Neglect of these simple rules means the seeding down of the ice-boxes, culture-chambers, and the general laboratory with all sorts of resistant mold spores and bacteria. An abundance of cheap car- bonate of lime should be kept on hand for the prompt neutralization of spilled acids. A mass of cotton waste is convenient for the prompt mopping up of spilled fluids. All contaminated needles, loops, knives, scissors, forceps, etc., may be sterilized in the open flame. Instruments which are too valuable to be flamed may be sterilized in carbolic acid (5 per cent) or formal- dehyd (5 per cent) or lysol (5 per cent). Never put down a platinum needle or loop which has been used Fig, 92* in making transfers until it has been passed carefully its whole length through the flame. Dissections are best made on trays which can be easily cleaned and sterilized. *Fic. 92. Compressed-air tank and spray-tube. The one here shown, made by Boeckel, Phila- delphia, is nickel-plated and very substantially constructed. It is filled by means of a small pump similar to a bicycle pump. The gage registers up to 100 pounds per square inch, but 40 pounds pressure is ample. The bacterial fluid is placed in atomizers of the form shown in fig. 93. The method of attachment is not satisfactory. This device is very convenient when trees or low plants covering a considerable area are to be inoculated. Height, 29 inches. The same firm has devised a compact traveling outfit, the compressed-air tank being about one-half the size of the one here figured. The whole is packed into a neat portable box, and the only disadvantage is the small size of the air-chamber, which requires more frequent pumpings. Of course the apparatus may be used equally well for the distribution of fluid germicides or insecticides. loS BACTERIA IN RELATION TO PLANT DISEASES. METHODS OF INOCULATION. Inoculations may be by punctures with a delicate needle (fig. 91), by abrasions of the surface, by hypodermic injection, by watering the soil with infective material, by plunging aerial parts into infectious liquids for a longer or shorter time, by simply putting the bacteria into drops of water on parts of the plant and protecting from sunlight and evaporation for some hours, or 011 a larger scale by spraying portions of the surface with very dilute culture fluids or, preferably, with water containing the bacteria (figs. 92, 93, 94), by brushing or rubbing cultures into some part of the surface, by allowing insects, snails, etc., to feed on diseased material and then colo- nizing them on healthy plants. The writer has made good use of this last method in case of three different bacterial diseases. Stomatal infections may be secured by sub- jecting the plants to conditions similar to those occurring in nature on dewy nights or during heavy fogs or prolonged rains, i. e., by placing the potted plants on wet sand, atomizing thoroughly with sterile water and covering with tall, roomy bell-jars. The experiment should be undertaken in a cool rather than a warm house. When the right conditions have been obtained, moisture covers the surface of the plant in tiny drops which do not evaporate. The bell-jar may now be raised and the plant again atomized lightly with steril- ized water containing the bacterium. The best time to do this is late in the afternoon, so as to take advantage of the cooler night temperature. When the bell-jar is returned, which should be immediately after spraying, it should be covered with cloth or paper to protect from the light. Usually bell-jars should be removed at the end of twenty-four hours, but exceptionally they may be left on thirty-six to forty-eight hours, if not exposed to the sun. Inoculation cages are very convenient for small plants (fig. 95). In case of trees, or shrubs, or masses of tall herbs, tight-fitting covers of tent-cloth will be found serviceable for obtaining conditions similar to those prevailing in wet weather. They may be left on i to 3 days, the outside of the tent as well as the plants within being sprayed with water often enough to keep everything moist until infections have been secured. When the nature of the plant will permit it and when only a few inocula- tions are to be made, the surface which is to be punctured should be rubbed thor- oughly for three to five minutes with mercuric-chloride water (1:1000) and then *Fic. 93. Atomizers for use with the air- tank (fig. 92). These are made by the Davidson Rubber Company, Boston, Mass. About one-fourth actual size. The De Vilbiss sprayer, made in Toledo, Ohio, and now used by the writer, has several distinct advantages. It is all metal and can be steril- ized in boiling water without becoming twisted out of shape, it can be attached more easily to large flasks and to the tube leading from the compressed-air tank, and the spray may be directed up, down, or straight ahead without changing nozzles. It requires, however, more force to operate than the Davidson sprayers, and consequently is less convenient when used with a hand-bulb. SURFACE STERILIZATION. IOC) washed with equal care in sterile distilled water. When many inoculations are made with large numbers of check plants and when due care has been taken to work under conditions such that accidental contaminations from the same organ- isms are not to be feared, the writer has not found this precaution necessary. The use of mercuric chloride should be avoided, if possible, especially on leaves, as the writer's experiments have shown that it penetrates into the plant (some plants) for a considerable distance and prevents the action of the bacteria to this extent (fig. 88), if not altogether, as has happened in some cases. THE KEEPING OF RECORDS. If one contemplates doing much work, a careful record of what has been done is as important as the experiment itself, since exact remembrance is certain to pass away with lapse of time. In all his work, the student should accustom himself to make very exact statements, so that others may be able to follow him. For example, he should not describe his organism as "yellow" or "red" without qualifications, since there are many yellows and reds, but should carefully compare it with some standard color-scale (Ridgway's, Saccardo's, Standard Dictionary, etc.), and govern himself accordingly. He should not say, " Organism does not grow at room-temperatures," but rather should state the temperature at which growth does not occur, as 15, 25, or 35 C, any one of which may be "room-tempera- ture," depending on the latitudCj altitude, and time of year. He should not say, "Organism is killed at temperature of 65 C.," without at the same time stating F"ic 94 * the age of the culture, condi- tions of exposure, and time required, which might be ten days or five minutes. Every independent worker will in the end devise a method of note-taking which is more or less characteristic of his personal peculiarities and best adapted to his own particular needs. For all persons there is no one best method. The methods described in the following paragraphs have been settled upon as those most con- venient for the writer, but it does not follow that they are the most economical of time, or the best devisable, or the ones which independent workers should follow. They are here given as hints for beginners and because the method a man employs in his work is always a matter of more or less interest to his fellow-workers. First of all, there should be provided a record book in which the method of preparation of each culture medium is carefully described. This should be a good- *Fic. 94. Hand-sprayer which may be used for distributing bacteria on plants. Some form is usually kept in every pharmacy and sold as a cologne atomizer. no BACTERIA IN RELATION TO PLANT DISEASES. sized book, well bound in leather, so as to stand long and hard usage. The entire quantity of a culture medium is known as a " stock " and receives a special number, which is written, pasted, or stamped on any flask or tube that contains it and which serves to identify it. If a stock is subsequently divided and a portion of it is treated in some different way, e. g., receives more sugar, acid, or alkali, this por- tion receives a new number, or the old number with the addition of a letter of the alphabet. Each stock described in the record book is numbered serially from i, and the book continues in daily use as long as the laboratory, or until it is filled with records and carefulry filed away as "Culture Media, Volume I." The small pocket ledger, No. 492 of A. C. McClurg & Co., Chi- cago, is very convenient for certain kinds of notes, especially those made in the field and those required for the identification of alcoholic specimens and stained slides (fig. 112). All records should be in ink, of a sort which does not fade, and in field work a good fountain pen is invaluable. Pencil records, especially those made with rapid- writing soft pencils, soon become illegible and should not be toler- ated except on paper to be sub- jected to steam heat. Large sheets of well-gummed paper should be procured and the labels cut in the laboratory to the size needed. Labels may be cut rapidly in quantity with the appa- ratus used to trim photographic prints for mounts. When exposed to streaming steam such labels come off easily, and it is best not to paste them on the tubes or flasks Fig. 95 * until after the final steam steriliza- tion. In moist climates, stock quantities of such gummed labels must be kept in air-tight boxes or between sheets of paraffined paper. Test-tubes in crates are kept separate during steaming by writing the number of the stock on a slip of paper and thrusting this into the crate with the test-tubes. The number should be written with a lead pencil. Faber's pencils for writing on glass are useful in case of flasks and *Fic. 95. Small cage of wood and glass in which herbaceous plants may be placed for inocu- lation by spraying. The inside measurements are 12 by 12 by 30 inches. The large door is a great convenience. Hook-fastenings are better than spring catches. RECORDS. Ill fermentation tubes, since records made with these pencils will bear streaming steam. An inexpensive black pencil which writes on clean glass very readily and bears steam well (even better than Faber's) may be made by stirring into melted beeswax enough lamp-black to make a thick-flowing liquid (as thick as will flow). This is poured into molds made by wrapping writ- ing paper, in several turns, around a lead pencil or thick glass rod, tying near one end, removing the rod, squeezing the other end flat, turning over its edge, and fastening this flattened end in a split stick or clamp. The paper should be retained as a cover, the string being removed and the loose edge pasted down. A dozen such pencils may be made at a cost of 10 cents. In the absence of such pencils, flasks and fermentation tubes may be distinguished in the steamer by dropping over the neck different-sized rubber bands or different numbers of the same kind of band, or by writing with a lead pencil the number of the stock on a square of letter paper, cut- ting a hole in its center and slipping this over the neck of the flask or tube. When the steaming is over, the regular labels should be pasted on or the stock number written on with the proper pencil. All plate cultures and all subcultures made on a given day, no matter of what organism, are numbered serially, beginning with i. These are i, 2, 3, etc., of that particular day. Those of any other day are also numbered i, 2, 3, etc. The writer Fig. %.* Fig. 97.* usually numbers his plates I, II, III, etc. Labels may be pasted on the covers of the Petri dishes, or all may be done with the glass pencil. Cultures in tubes subject to frequent handling and likely to be needed for some time should have gummed-paper labels written in ink. The above transcripts from labels on four test-tube cultures *FiG. 96. Labels from test-tube cultures. *FiG. 97. 'Wooden labels from inoculated plants. 112 BACTERIA IN RELATION TO PLANT DISEASES. (fig. 96) sufficiently indicate what is necessary to form a satisfactory record. This could, of course, be considerably abbreviated by a system of symbols or by depend- ing to a larger extent on the "Notes." In case of the inoculations, on the contrary, only as many series are made use of as there are diseases under consideration. Each plant is generally given a single number, no matter in how many places it may be inoculated, the separate inocula- tions being kept distinct, if necessary, by sub-numbers. Each series begins with -$i*J^s.s&*&M $^V/3f2bdC&ty). Ju^fi/s t \ 4, (] 5- IP &< '. //, 74 o 7 ' ^ /^ * ^-^ f Jo 7; ,/ro " ? " II , 3, 3 Fig. 98.* No. i and continues in an unbroken sequence as long as the disease is under con- sideration. The labels written on soft wood, covered for this purpose on one side with white paint, are stuck into the earth or wired to the plant. Transcripts from two such labels are shown in fig. 97. *Fic. 98. Three sheets showing method of keeping maximum and minimum temperature rec- ords. One-half actual size. RECORDS. After trying various methods, the writer has settled down (in the absence of a stenographer) to the following style of pen and ink notes on cultures, inoculated plants, etc., as extremely flexible and convenient. Reams of ordinary typewriter paper are cut crosswise into three equal portions, so as to form slips about 8 by 3^ inches. As many of these as are necessary for the particiilar purpose are fastened together at one corner with B, J, N, C, or Z eyelets and the Triumph punch, sold by The W. Schollhorn Company, New Haven, Conn., or by the neat little saw- r~ Itf, S7W; 72>. Fig. 99 * toothed clamp made by The Middleton P. F. Co., Philadelphia. The first page of the slips is devoted to the name of the organism under examination, the kind of experiment, the date of its beginning, etc. The subsequent sheets are numbered serially and are devoted to particular plants or to particular cultures. If there is an overflow in any particular part of the record, it is very easy to insert additional *Fic. 99. Sheets showing method of keeping nitrate-bouillon records. One-half actual size. 114 BACTERIA IN RELATION TO PLANT DISEASES. slips. The following transcripts from actual records will serve to illustrate the method (figs. 98 and 99). As fast as the notes are completed they are filed away in boxes or large envelopes until the whole subject has been worked over, when they are sorted out according to their various sub-heads, and all the data which they contain is thus easily available. The writer also uses a sten- ographer whenever possible, and the typewritten sheets, after immediate careful scru- tiny for errors of fact, are filed away in stout Manila envel- opes with the name of the parasite written on one corner; o6, fn- Fj 16 by 12 inches is a good size for the envelopes. Card-catalogues should be made on the L. B. index slips, made and sold by the Library Bureau, Boston, Mass. Figure 100 is a sample from the writer's catalogue by authors. A larger size should be selected if it is desired to include abstracts. When long abstracts or considerable extracts are made from literature which has been borrowed, or may not be readily accessible in futitre, heavy sheets (6^$ by 83^ 32. XT sS ; O^^--7-^^^_ (7 ... Fig. 101. t inches) have been used by the writer. These have headlines, as shown in fig. 101, and are preserved by tying into covers made for the purpose. A red line down the left side of the sheet preserves a space for a marginal index. A serious objection to the making of many abstracts is the time involved and the danger of degenerating into a mere student of literature in the effort to make a complete catalogue ; another is the fact that, if made in advance of actual need, or *Fic. 100. Sample from card-catalogue. Two-thirds actual size. fFic. 101. Top of large sheet used for voluminous abstracts. A red line near left-hand mar- gin marks off a space on which summarizing catch-words or phrases are written. Breadth of sheet, 6^ inches. RECORDS. "5 by some one not entirely familiar with the subject, it not infrequently happens that the statements in the paper which have been omitted from the abstract as unim- portant prove in the end to be the essential ones so far as the owner of the abstract is concerned. For this reason, when they are within reach, the writer prefers to consult the original papers and to save for original work the time consumed in making long abstracts. When they are rare, frequently needed, and only to be had by borrowing, the writer has sometimes photographed the more essential parts. In one instance a pamphlet was bor- rowed from Europe for this purpose. For the exact measurement of col- onies, etc., a strip of plate glass 35 cm. long and ruled into 350 mm. spaces may be had from Carl Zeiss, and will be found very convenient (fig. 102). Steel rules of any size and of very excellent workmanship, graduated ac- cording to the English or the metric system in any degree of fineness, may be had from the L. S. Starrett Com- pany, Athol, Mass. Two of these rules much used by the writer are, respec- tively, 12 inches and 30 centimeters long. They are one inch wide and about three sixty-fourths of an inch thick. They are graduated on both sides, the metric rule into centimeters, millimeters, and one-half millimeters, and the English into inches, halves, quarters, eighths, sixteenths, thirty- seconds, and sixty-fourths. Stage micrometers made by Zeiss are recommended for the finer measure- ments. These have i millimeter divided into tenths, twentieths, and one-hundredths very accurately. All the magnifications of microscopic objects figured in this book are recorded in terms of such a micrometer. After the drawing has been made it is customary to substitute for the section-slide this stage microineter and throw the image of some portion of the ruled scale on the paper ~ *FiG. 102. Green cucumber soft-rotted by Bacillus aroideae. Contents emptied out and skin filled with water and so photographed, 3 days from date of inoculation, which was by means of a few needle-pricks. The fruit was kept at about 25 C. The black bands are pencil marks on the millimeter rule placed inside. The numerous small dark spots are denser bits of tissue which did not wash free on rinsing out the sack with water. At the left drops of water may be seen oozing through the skin and falling. Photograph, nearly natural size, by Townsend. Fig. 102* I ID BACTERIA IN RELATION TO PLANT DISEASES. where it is drawn, taking care, of course, in case of high magnification, to start one cross line from the outside and the other from the inside of the image of the lines. This method of recording magnifications is urged on all. It takes but a moment, does away with troublesome computations, and enables anyone at any time to deter- mine just what was the magnification. The magnification is determined, of course, by dividing the apparent size by the actual portion of the scale shown. For Fig. 103* example, if the scale drawn on the paper is 10 mm. long and represents o.oi mm. of the actual micrometer scale, then the magnification is X 1000; if it represents the entire millimeter of the micrometer scale, the magnification is X 10. For fine weighings, Christian Becker's balances are very satisfactory. *Fic. 103. Pillsbury slide-boxes empty and full, made by Bausch & Lomb, Rochester, N. Y. These boxes are simple, inexpensive, and satisfactory, especially for serial sections. COLLECTIONS. 117 THE MAKING OF COLLECTIONS. A good, representative collection of diseased material is a prime necessity in every pathological laboratory. This grows into completeness only with the lapse of much time and the aid of many hands. It should include photographs, drawings, paintings, dried material, representative specimens preserved in strong alcohol, and serial sections properly stained and mounted in Canada balsam or Dammar balsam, which must not be dissolved in chloroform, since this gradually removes the stain. With the accumulation of much material, some sort of classification becomes im- perative. At present the writer keeps the material designed for sections in 95 per cent alcohol, arranged in as many groups as there are parasites involved. Each jar of material finally receives the same number as the paraffin block from which sections are cut. This material must be exam- ined at least once a year to see that the alcohol has not evaporated, es- pecially if corks are used. Only the best velvet corks should be pur- chased, and as an additional precau- tion they should be sealed in with paraffin. The negatives are filed away in similar groups, protected by nega- tive bags. The stained sections, mounted in balsam, are filed away in cheap wooden boxes (Pillsbury boxes), each holding 25 slides (figs. 103, 104). These are very conven- ient, if properly made, but some boxes of this sort lead to much vexa- tion of spirit, the grooves being too narrow to receive any but the thin- Fig. 104.* nest slides. Those sold in recent years by Bausch & L,omb have given no trouble. In the form shown in fig. 104 the cover remains on better and the mounted slides are easier to take out, but in drying the preparations with the cover off, these boxes tip over at the least touch. During this drying, which requires from a few days to several weeks, the slides should, of course, lie flat, not on edge. *Fic. 104. Another style of slide-box. The advantages of this box are that the cover is not likely to fall off and that the slides, in case of full boxes, are withdrawn more easily. The disad- vantages are that it is tipped over very easily when standing on end open, that the cover is readily mistaken for the bottom when it is closed, and that if the cover is put on upside down the writing on the edges is divided. These may also be had from Bausch & Lomb. n8 BACTERIA IN RELATION TO PLANT DISEASES. The writer passes material designed for sections from alcohol through chloroform (or xylol) into paraffin. Chloroform is preferred in case the infiltration is to be completed in vacuo ; otherwise xylol is generally employed. A mixture of xylol and alcohol is first used, then pure xylol, after this xylol with as much paraffin as can be dissolved in it cold. The vial is then placed on top of the paraffin bath and Fig. 105* shaved paraffin added until it will dissolve no more at this temperature ; the material is then placed inside the apparatus in pure melted paraffin, and it is finally mounted from a second dish of pure paraffin. The temperature of the paraffin bath is usually *Fic. 105. A small paraffin oven much used in the writer's laboratory. The capacity of the chamber is 6 by 7 by 5 inches. The thermo-regulator is like that shown in fig. 35, but with chloro- form substituted for glycerin. PARAFFIN-INFILTRATION. kept at 59 C., and the material is subjected to this temperature only long enough to secure proper infiltration. Generally a few hours are sufficient. A small oven used for this purpose is shown in fig. 105. For large laboratories or classes of students the separate-compartment paraffin oven designed by Dr. Lillie is very convenient. Griibler's paraffin is preferred, and for the climate of Washington we use mixtures of three grades of hardness, viz, melting point 52 C., 58 C., and 60 C., increasing or decreasing the amount of the harder sorts according to the time of year. Dirty paraffin should never be used. All the stock paraffin should be carefully protected from dust. The same remark applies still more pertinently to the sections cut on the microtome. They should be made in still air, in a clean room, and should be carefully protected from dust until stained and mounted. The paraffin - infiltration is usually a simple process unless the material contains air. The embedded material is given a serial number which is scratched on the paraffin (fig. 106), until it is fastened to the cutting block, when it is written on the latter (fig. 107). These blocks are kept as shown in fig. 1 08. The sections are fastened to clean slides * by a very thin layer of Mayer's egg albumen fixative (see Lee's Vade Mecum, 5th ed., p. 143), or with pure water, or preferably with 0.5 per cent gelatin water (which will not keep untreated, but may be preserved by adding 3 per cent phenol) ; the paraffin is removed (after cautious melting) by exposure to turpentine or xylol, alcohol is then substituted, and thereafter graded mixtures of alcohol and water down to alcohol containing 50 or 60 per cent of water, followed by the stain. Water is then removed by passing through graded alcohols into absolute alcohol ; xylol or bergamot oil is substituted for the alcohol, and the section is finally mounted in balsam. Coplin's staining jar is preferred (figs. 109, no). A series of staining jars, ready for use, is shown in fig. in. The section properly fast- ened to the slide, and dry, is started in at the left after melting the paraffin with gentle heat, and is taken out at the right ready for mounting in balsam.t In this series of jars the gradations are as follows, beginning *Fic. 106. Infiltrated tissues embedded in paraffin in a watch-glass and now ready to cut out and mount on blocks for the machine. fFic. 107. Infiltrated material embedded in paraffin and mounted on a pine block ready to cut on the microtome. Actual size. J Sections designed for photo-micrographic work must not only be cut in clean air, but mounted in absolutely clean balsam. So much trouble has been experienced in finding such dissolved bal- sam on the market that the writer now makes his own. The dry balsam is heated in an oven until all easily volatile products are driven off and it becomes brittle. It is then dissolved in xylol and filtered under a bell jar to exclude dust. The filtering usually requires several days. I2O BACTERIA IN RELATION TO PLANT DISEASES. I at the left : Xylol, second xylol, xylol one-third absolute alcohol two-thirds, 95 per cent alcohol, 75 per cent alcohol, 55 per cent alcohol, 40 per cent alcohol, carbol- fuchsin, 40 per cent alcohol, second 40 per cent alcohol, 55 per cent alcohol, 65 per cent alcohol, 75 per cent alcohol, 95 per cent alcohol, absolute alcohol, second absolute alcohol, xylol, second xylol. From this last jar the material is mounted in balsam. Turpentine may be substituted for xylol in jars i and 2. After the paraffin is fully removed, the slides are passed rapidly from jar to jar (a minute or two in each being generally sufficient) until the stain is reached. After remain- ing in the stain the proper length of time (usually three to ten minutes, but sometimes much longer) the slides usually are allowed to remain in the 40 per cent alcohols for a number of minutes, with frequent inspection. When they appear to be properly bleached (rather pale) they are passed rapidly through the remaining jars until they reach the xylol, in which they may remain for some time without injury, if they can not be mounted immediately, but they must not be allowed to stand for any great length of time in any of the alcohols. The secret of success lies in obtain- ing just the proper amount of differentiation in the 40 per cent alcohol and in not losing any of this later on. To retain the stain it Fig. 108* is necessary sometimes to omit some of the graded alcohols. The time required for properly staining sections varies from one or two minutes to a half day or more, according to the subject and the stain employed. No general rule applicable to all cases can be given. When the material is selected for embed- ding, its serial number, with a full description, is entered in the record book (fig. 112). *Fic. 108. One of a series of drawers divided into small compartments for holding infiltrated, embedded material, cut and uncut. PLATE 13. n -i o 3 ri- IJ I" ^ I ? f 3. 2. S S I' 6 RECORDS. 121 This book must not be lost or misplaced. The advantage of having the serial number written also on the bottle containing the stock of preserved material is very evident if a thing of this sort ever happens. The serial number is written on one edge of the slide-box, and serves to identify it (fig. 103). Some record besides a mere number should also be placed on the slide-boxes. All the slides within bear this num- ber, e. g., 256, and also a series number of their own, i. e., i to 25. The slide- boxes are then filed away on shelves either serially or in groups, according to the parasite. Slides con- N "** taining particularly good fields are marked X, and when the best fields are finally decided upon their location is recorded as de- termined on the mechanical stage. In case a dozen or more serial sections are included on one slide the the extra good ones are marked X on the first exam- ination, and the others O, as shown in fig. 113. When one of these sections has been drawn or photo- graphed, the X is underscored or inclosed by a circle. This method enables one to keep track of any num- Fig. 109* ber of sections. Free-hand sections may be made with the Torrey razor shown in fig. ii4D. This is altogether the best razor the writer has used. When very dull it may be sharpened on an India oil-stone. These stones are said to be made of a mixture of carborundum and clay, baked at a high temperature. They may be Fig. 1114 had of the Norton Emery Wheel Company, Worcester, Mass., in three grades of fine- ness, the finest being usually coarse enough for the dullest razors. The size needed is 8 by 2 by i inch. The finishing may be done on an Arkansas oil-stone, with a *Fic. 109. Coplin's staining jar. About one-half actual size, f FIG. no. Cross-section of Coplin's staining jar. About actual size. JFic. in. A series of Coplin's jars filled and properly arranged for staining sections fastened to slides. 122 BACTERIA IN RELATION TO PLANT DISEASES. few final touches on a good leather strop. The maintenance of good edges on microtome knives is a matter of great importance and considerable difficulty, and where much material is to be cut it is very economical of time to send away such knives to be put in order by some expert. In recent years the writer has sent all such knives to Charles L/entz & Sons, Philadelphia, with very satisfactory results. Knives suitable for serial sections are shown in fig. 1 14 A and C. In fig. 1146 is shown one of a set of knives not in- clined to spring and well adapted to the cutting of hard material with a long slant stroke. These knives were made to order by L/entz & Sons at a cost of about $6 each. An end-on view of all these knives is shown in fig. 1 14 a, &, c, d.. Many plant tissues, especially ma- ture leaves, are full of very hard cal- cium oxalate crystals, and the difficul- ties of properly cutting such material are very great. The cutting of thin sections of bone would be quite as easy. After even a few sections the edge of the knife looks like a minia- ture saw and the sections themselves are badly torn, partly by the dulled knife and partly by the movement of p. |j2* the crystals themselves. In case of the yellow disease of the hyacinth the writer has never been able to make satisfactory thin sections, many of the soft cells being filled with bundles of very hard raphides which he has not been able to dissolve without serious injury to the tissues. In such cases thick free-hand sections are about all that can be hoped for. Serial sections are cut on the microtome. The one shown in pi. 13 and fig. 119 leaves nothing to be desired in the way of a perfect-working durable instrument. The ribbon-carrier is above the table at the left. The knife is stationary. The block moves up and down, and the razor-carrier *Fic. 112. A page from the paraffin record-book. The numbers on the slide-boxes (fig. 103) correspond to numbers in this book. Two-thirds actual size. fFic. 113. A mounted slide of serial sections, showing manner of labeling. ZlVBdL B/O.CK 5pot DODO of P/U77V. X00 /rx/.r >< xxo DOOD fVne cavity iijpareijc/ujma Fig. I13.f SECTIONS. I2 3 moves forward at each stroke a distance governed by the set-screw of the scale (y* J* to 40 ;"). By substituting a wide knife-carrier, sections several centimeters in diameter may be cut, and by using a slanting knife, as for celloidin, very hard mate- rial may be cut. By loosening a set-screw, the razor as here shown may be turned a few degrees to right or left, and the paraffin block may also be moved through a considerable arc in any direction, it being held securely in any position by pressure of a collar-screw on a ball-and-socket joint. On 72 in plate 13 is an apparatus for tracing the edges of the paraffin blocks. B Fig. 1 14 * Collections of living bacteria are also necessary. Fortunately many may now be obtained, as needed, from Krai, in Prague ; but, unfortunately, they do not always correspond to their name. Others must be kept on hand, and the cultures (of some sorts) must be renewed at frequent intervals. That way which has given the writer *Fic. 114. A. Knife for serial sections, furnished with the Reinhold-'Giltay microtome. This is made by Joseph Rodgers & Son, Sheffield, England. One-half actual size. B. Microtome knife made to order by Charles Lentz & Sons, Philadelphia, and found useful in cutting hard material with long slant strokes. One-half actual size. The broad wedge-shaped blade of this knife is shown in 'ft. C. Knife obtained from J. R. Torrey & Co., Worcester, Mass., and found very useful for making serial sections on the microtome. One-half actual size. D. Torrey razor, recommended for free-hand sections. The very thin blade is flat on one face and hollow-ground on the other, as shown in d. It is made of the very best steel and holds an edge well. One-half actual size. a, b, c, d, end views of the cutting edge of knives shown in A, B, C, D. Actual size. 124 BACTERIA IN RELATION TO PLANT DISEASES. least inconvenience is by storage in cool boxes (refrigerators) at temperatures of 10 to 15 C. By tliis method some organisms can be kept alive on agar a year without transfer, and even sensitive organisms will generally live for some months, especially B Fig. 115* if planted in proper media. The writer has never made any attempt to prepare a collection of dead bacteria on culture media to serve as museum specimens, but it is possible to do so, it is said, with considerable success by following the methods described by Hauser and others (Bibliog., L,II). DISTILLED WATER. All laboratories doing much work should have an abundance of distilled water, and where this is not readily obtainable in sufficient quantity and of good quality, provision should be made for it when the laboratory is constructed or when the necessity for it arises. In the construction of such a still many things must be kept in mind, if it is to work satisfactorily and yield water of the desired purity. J Fig. 116.t *Fic. 115. Cross-section of tooth of cabbage-leaf infected by Bacterium campestre. Plant No. 401 sprayed with water containing an agar-culture. Bacterial occupation limited to points between A and B. At X vessels are occupied. At A and B the bacteria lie in the intercellular spaces and have not yet entered the vessels. For details of A and B, see figs. 116 and 117. This section, which is one of a series, was cut 270 ft below the apex of the leaf-tooth. A few micromillimeters further down (370 >u) all trace of the bacteria disappears. In other words, the bacteria are still confined to the leaf-tooth, and there is no cavity like that shown in fig. 76. When sprayed this leaf was extrud- ing fluid from the water-pores. Actual length of section, slightly under I millimeter. Slide 331 3 Plant sprayed December 9, 1904; slightly blackened leaf-tooth fixed in 95 per cent alcohol on December 17, 1904. Inked from a photomicrograph. fFic. 116. Cross-section of leaf-tooth of cabbage infected by Bacterium catnpestre. A detail from fig. 115 at A. The bacteria have not yet entered the vessels. tThat thing which has given the writer most trouble was an entirely unexpected difficulty, viz, a plague of tiny red house ants. These got into the reservoir in spite of all that could be done to render it tight, and, of course, spoiled the water for all delicate work. PLATE 14. Apparatus for Distilling Water. (I) Steam inflow pips; (2) wasts-iteam pipe ; (3) hydrant- water inflow pip: ; (4) hydrant- water outflow pipe (flush) to sewer ; (5) galvanized -iron bailer ; (6) water gage ; (7) brass top, tinned on the under side ; (6) copper catch basin ; (9) steam safety valve; (10) block-tin steam pipe to condenser; (11) block-tin water pipe from condenser; (12) hydrant- water pipe into condenser tank; (13) hydrant -water pipe from condenser tank; (14) flush pipe for condenser tank ; (1 5) reservoir, capacity 80 gallons ; (16) water gage; (1 7) overflow pipe from reservoir ; (1 8) block- tin pipe leading to various rooms ; (19) iron support. DISTILLED WATER. 125 The following description and figure of a distilled-water apparatus devised by the author for use in the Laboratory of Plant Pathology, United States Department of Agriculture, may be of interest, therefore, to some. The apparatus consists of a galvanized-iron boiler similar to those used in kitchen ranges. It is 1 8 inches in diameter and about 5 feet high. The top is sawed off and to it is bolted a stout iron ring with a flange, on which rests a ^-inch brass cover. In the lower half of this boiler is a coil of 52 feet of inch copper pipe, the upper end bent downward and securely fastened in the bottom of the boiler to a steam pipe (i inch) connected with a i ^4 -inch steam pipe leading to the ordinary steam boiler in the engine room in the basement; the lower end connected with an iron steam pipe (i inch) leading to a steam trap (Mark traps are said to be the best). Around this copper steam pipe, which is of course tin- plated, stands the river water which is to be converted into steam by contact with the hot pipe. This hydrant water is kept always at about the same level (level of fig. 5 in plate 14), by means of a tinned-copper ball float (automatic cut-off) which closes the mouth of the inflow pipe when the water rises be- yond a certain point. The upper part of the cylinder is a steam chamber under very moderate pressure (o to ^ pound, rarely more). The excess of pressure is dissipated either by escape of steam through the safety valve (9), which is not weighted, or through the coil of pipe in the condenser. The steam passes from a securely riveted tin-lined copper catch basin (8) into a ^-inch block-tin pipe (10), which is fastened to a tubular projection from the catch basin by means of a collar screw. The tubular projection from the top of the catch basin is soldered in place and also held by a flange inside the copper top, so that it can not be forced out by any attainable degree of steam pressure. The J^-inch block-tin pipe passes to the room above, where it is coiled for a length of 35 feet inside a tin-lined copper tank resting on the floor. The height of the condensing tank is 18 inches and its diameter is the same. When in operation this tank is full of running water. Theoretically, this condensation tank is large enough, and it is so practically when the hydrant pressure *Fic. 117. Detail from fig. 115 at B, showing an early stage of water-pore infection of cabbage. The bacteria have not yet entered the spiral vessels. The large dark bodies are nuclei. 126 BACTERIA IN RELATION TO PLANT DISEASES. is good, but when it is feeble or when the steam pressure is high the water becomes too hot and steam sometimes escapes into the reservoir. The water therefore must be hurried through the tank by the use of a steam pump, or else less steam must be allowed to enter the copper pipe. If the writer were to build another similar apparatus he would make the condensing tank 2 feet higher and add 10 feet to the length of the coil of tin pipe. The condensing tank is provided at the bottom with a i-inch inflow pipe for the cold water (it should be i^-inch), and at the top with a i "^ -inch outflow pipe (it should be 2-inch), for the exit of the wanned water. There is also a i-inch flush pipe at the bottom for the occasional removal of sediment. The size of the outflow pipe, which must be somewhat larger than the in- flow pipe, prevents any possibility of clogging and overflow. All the metal parts which come into contact with the distilled water are tinned or nickel- plated. Connected with the lower end of the block-tin coil (by tin solder, which must not contain lead or zinc) is a smaller (^-inch) block-tin pipe (i i ), which leads the distilled water into (15) the storage tank (j^-inch pipe would be better, and without any joint). The reservoir in this case is a white- enameled bath-tub, on the top of which is clamped down a cover of thin sheet copper (o'o-inch), the inner face of which has been carefully tinned. Plate glass ground to fit would be better, and the tub itself is likely to be discarded in the near future, i. llAoWTfiOOM , FOR CORRECTLY TIMING PHOTOGRAPHIC EXPOSURES. mw TWO MOM jj I. 16 II 8 56 f US S56 138 3 ? 'f ? 1 ' I |[ || If l| It IT V I II II ^ I ^ | || II | 6 MO 5*. Z-fZ IT I *** *T- s ^ rs SETOSE?; SS^T ^;sr;r^?s zt: JT^L, 16 MO 5< ZT_ _e i n 11 ii --- MEMOM 0. | -' 1 . - 1 L _ 87 6 S43J I 1 1 1 M 1 1 1 1 1 II U 1 1 1 1 1 1 1 1 1 rrrrn Fig. 128* $> over and over until exhausted (browned). The quantity named above will suffice for a dozen 5x7 plates properly exposed. This developer may also be used with Velox paper. In this case it should be diluted with more water, say i ounce of the ortol solution, i ounce of the alkali, 6 ounces of water, and 6 drops of 10 per cent potassium-bromide water. *Fic. 128. Exposure scale set to show proper time for buildings and average near views at 10 a. tn. to 2 p. m. in July, with stop 64 (32 f ) and an intense sun. The various makes of plates are divided into eight classes, and the time is read from the middle scale for intense sun and the most rapid plates. Under above conditions a Seed's 27-X plate, or its equivalent (i), would require one-sixth second. For light of a less degree of brightness E is set on the proper stop, and the time is read from the bottom scale. The latter scale (G) is also used for slow plates. With intense sun, i. e., as set above, a Cramer's isochromatic slow plate, or its equivalent (7), would require 2 seconds. In indoor work, scale K is first set on H, according to the quality of the light and num- ber of windows. Scale L (kind of walls) is then set on the proper stop, and the time is read from the bottom scale, according to the speed of the plate used. In latitudes far to the north of Phila- delphia there must be considerable increase of time, and there must be a corresponding shortening of time in tropical regions or desert regions. Considerable judgment must also be used in making indoor exposures, especially toward sunset and soon after sunrise. Near sunset, exposures have to be increased enormously. About three-fourths actual size. 142 BACTERIA IN RELATION TO PLANT DISEASES. Previous to development the exposed plate should be placed in the tray, flooded with water, and gently rubbed with the balls of the fingers, particularly if the exposures have been made for some time, or in dusty weather, or on plates which have been opened for some time. Many "pin holes" will be avoided by this practice, and frequently one will be astonished at the amount of dust which can be felt as the fingers are passed over the plate. Negatives should be fixed in strong hypo for ten minutes (a little longer exposure will not harm them), hardened in alum-water (saturated) five or ten minutes if the weather is hot, and washed in running water one to two hours. If these rules are followed, negatives which are good on the start will not spoil after- ward. Weak hypo should not be used, neither should the solution be saturated, but only nearly so, i. e., a saturated solution diluted with one-sixth water. This is made up in small quantities in advance. The saturation is accomplished, not by throwing the crystals into a jar containing water, but by putting them into a cloth-sack which is brought into contact only with the top layers of the water. On removal from the washing-box the back and face of the negative should be rubbed over carefully under running tap water with a wad of soft cotton, and set away in a clean place to dry after rinsing in distilled water. If one is in a great hurry to get a print from a wet negative, it may be dried in about ten minutes by soaking for eight minutes in 95 per cent alcohol and then holding it near an electric fan. In developing in deserts or in southern climates, in very hot weather, all the fluids must be iced, including the wash-water, or else the plate must be hardened in 2 per cent formalin water for five minutes before the development begins. Alum- water can not be used for this purpose, since it greatly retards development. It often happens, especially with beginners, that a good negative (one rightly exposed) is spoiled by being left in the developing solution too long or by being taken out too soon. An overdeveloped negative may be reduced after soaking it in water (or preferably before it has dried) by placing it for a few minutes in a tray of clean water, to which has been added a small quantity of hyposulphite of soda and a few drops of a 10 per cent solution of red prussiate of potash (Farmer's reduc- ing solution), which, of course, must be uniformly distributed. Thin negatives, free from hypo, may be intensified, if they are thin simply from underdevelopment, by exposure for from two to five minutes (occasionally a little longer) in a strengthening solution made of Agfa intensifier 20 parts and water 180 parts, or by soaking them in a strong watery solution of mercuric chloride until they are whitened through uniformly on the back, and then blacking them by soaking in ammonia water strong enough to give off disagreeable fumes. If the time of exposure is not nearly correct, another negative should be made. Negatives thin from overexposure do not intensify well ; neither do those which were much underexposed. All negatives should have the subject, date of making, and degree of magnification written on them with a lead pencil as soon as they are dry. The proper place for a record is on the margin of the negative itself rather than in a book or on a bag. which may become misplaced, although it is convenient to have it also on the envelope, or negative-bag. EXPOSURE-METERS. 143 The correct time of exposure for photomicrographs varies so greatly with the size of stop, length of bellows, kind of slide, number of objective, quality of light, rapidity of plate, etc., that no very definite rules can be laid down, the right time in special cases in Washington varying all the way from several minutes to j-i_ of a second. If the bellows-length is doubled, of course the time of exposure must be quadrupled. Low powers, and especially Planars, let through a great flood of light and require correspondingly short exposures. With low powers and sunlight the student might begin on ^ second. With an oil-immersion lens and bright light he might try ^ second or second. If the section is densely stained, much allowance must be made for that. It is well, at least for a time, to keep a record book of subjects and exposures to refresh one's memory. It saves the spoiling of many plates. Such a record should include subject, length of exposure, stop used, objective and eye- piece used, length of bellows, distance of the condensing lens from the Abbe con- denser, time of day, time of year, quality of light, kind of screen, kind of stain and density of section, kind of plate, developer used, time required for development, and quality of negative, viz, overexposed, underexposed, or correctly timed. For outdoor work, and also for natural-size or slightly magnified indoor work, a good exposure scale is sometimes useful. The best ones known to the writer are the Wynne and the Wager. Success with the Wynne depends on one's judgment as to the proper changes in a good sensitive paper ; with the Wager it depends on one's judgment as to the quality of the light in the sky. After a little experience very uniform and excellent results may be obtained with either. Personally, the writer prefers to use the Wager (fig. 128), because it is simpler and takes less time. No scale is always to be depended on, there are so many variations in light and so many unprovided-for contingencies. Experience is after all the best guide, but until one has obtained it, genuine aids are not to be neglected. The beginner should first become familiar with the right exposure for one stop and one kind of plate, e. g., stop f. 16 and Seed's 27, with a given bellows length. Having learned correct exposures under these constant conditions, it will be comparatively easy to change to other makes of plates and to other f. stops. Slow isochromatic plates require 10 to 12 times as long exposure as fast plates. In the matter ot stops the length of exposure is, of course, quadrupled every time the f. stop number is doubled, and quartered every time it is halved, e. g., if stop 16 will give a perfect negative with one second exposure, stop 8 will require one-fourth second and stop 32, four seconds. Under the same conditions, stop 4 will require one-sixteenth second, and stop 64 sixteen seconds, and so on. With the Universal stops (those commonly used on the shutters made in this country and England) the exposure is doubled for the next higher stop and halved for the next lower one, instead of quadrupled or quartered, as in the case of the f. stops. For lantern slides the writer converts a small room into a camera box (plate 1 8). This room has a floor space about 6 by 5 feet. It has a north window and a west window. Each window is provided with a double set of roller curtains, the outer made of yellow cloth, the inner of a very dense black cloth known in the trade as double-faced, opaque, black shade-cloth, which lets scarcely any light through, 144 BACTERIA IN RELATION TO PLANT DISEASES. even when held directly toward the sun. A cross-bar is screwed across the base of the uprights of the window frame, 35 inches from the floor and a few inches above the window sill. To this bar a swing shelf is hinged and drops down out of the way when not in use. This shelf is about 24 inches wide by 30 inches long. When in use it is supported in a horizontal position by a removable leg. On top of this shelf is placed a cracker-box or some similar box, to the sides of which, at the bottom, beveled cleats are nailed, which slide through corresponding cleats screwed to the top of the shelf. This enables one to push the box toward the win- dow or draw it back on a regular track. On the top of this box, at the back end, or farthest point from the north window, the camera is placed facing this window and is screwed fast to the top of the box the same as to a tripod. Sidewise move- ment is provided by extending the screw-hole in the top of the box into a slot 6 or 8 inches long. In sliding the camera sidewise it is of course necessary to keep the ground glass parallel to the negative in the window, and this is done by drawing parallel lines on top of the box about *& inch apart and exactly at right angles to the negative-carrier. In moving the camera sidewise all that then remains is to see that one side of the camera at front and back matches one of these ruled lines. This gives ample sidewise movement, and some up-and-down movement is usually provided in the camera itself. The rest is obtained by moving the negative. The upper half of the north window is covered by the curtains. The lower part is filled with a removable wooden framework, the negative-carrier, so arranged that the negative itself may be moved up and down or sidewise, or twisted around at will. The framework of the negative-carrier is made of inch stuff. When in use it is placed upright about 3 inches in front of the window pane and just behind the cross-bar which keeps it in place. In the middle of this frame is a circular wooden disc (which must turn freely), held in place on the back by a ledge and in front by four buttons, and open in the center. The breadth of this disc is 24 inches, and it should be made of well-seasoned lumber of a sort not inclined to warp. On this disc at either side two broad vertical cleats are fastened. These are grooved on the inner edges next the framework, and under them, close to the circular piece, two wide ^4-inch pieces slide up and down freely, carrying the negative between them. The latter fits into marginal grooves and is held in place by buttons. The marginal grooves extend the whole length of the ^-inch pieces and consequently allow the negative to be moved sidewise to any extent desired, while the up-and- down movement is obtained by sliding the two ^-inch pieces and the negative between them as a unit. Over the first grooved cleats, at right angles, i. e., horizon- tally, two similar cleats are screwed. These also have wide s^-inch pieces moving under their grooved edges. These sliding pieces cover the sides of the negative and shut out the side-light in whatever position the negative may be placed. Behind the negative against the window is pasted (by its corners) a good-sized piece of white tissue-paper, which serves to distribute the light evenly and to cut out images of trees, buildings, etc. When in use the double curtains of the west window are drawn down and the door is shut. The north light which enters the room then comes through the negative placed in front of the camera. The focus is obtained PLATE 18. Small room arranged for making lantern slides and enlargements on bromide paper. The parts of the window shutter are as follows : ( I ) frame work, (2) circular piece giving rotary motion, (3) one ol Iwo stationary piece under which No. 4 slides, (4) negative carrier, ( 5) one of two stationary pieces under which No. 6 slides, (6) side pieces designed to cut out all the side light except that which comes through the negative. PHOTOGRAPHY AND PHOTOMICROGRAPHY. 145 on the ground glass as for any picture, remembering that a wide margin (^ inch or more) must be left for binding strips, and that if the negative has any up and down its image must be placed crosswise on the lantern-slide. The writer focuses as Fig. 129.* *Fic. 129. The modified Collins-Brown camera used with tripod for natural-size pictures. Heavy shadows are dissipated by using the glass plate. The size of the camera box is IO}4 by I2J4 by SJ4 inches, and its weight, including lens and shutter, is about 15 pounds, or with tripod 19 pounds. The camera takes a 6j4 by 8J4 plate. It is solidly constructed, of the very best workman- ship, and the only objection is its weight, which is no disadvantage in laboratory use. It is not recommended for field use. 146 BACTERIA IN RELATION TO PLANT DISEASES. Fig. 130* sharp as possible with stop wide open and then stops down to 16 u. s. before making the picture. There are two other ways of making lantern-slides, i. . ALLEN, ALFRED H. Commercial organic analysis. A treatise on the modes of assaying the various organic chemicals and products employed in the arts, manufactures, medi- cine, etc., with concise methods for the de- tection of impurities, adulterations, etc. 8vo. Vol. i. Alcohols, neutral alcoholic derivatives, etc., ethers, vegetable acids, starch, sugars, etc. 3d ed., 1898, pp. xii, 557. Vol. ir, Part I. Fixed oils and fats, glycerol, explosives, etc. 3d ed., 1899, pp. x, 387. Vol. n, Part II. Hydrocarbons, mineral oils, lubricants, benzenes, naphthalenes and derivatives, creosotes, phe- nols, etc. 3d ed., 1901, pp. viii, 330. Vol. n, Part in. Terpenes, essential oils, resins, cam- phors, etc. 3d ed. preparing. Vol. in, Part I. Tannins, dyes, and coloring matters. 3d ed., enlarged and rewritten. Illustrated. 1900, pp. xvi, 589. Vol. in, Part II. The amines, hydrazines and deriva- tives, pyridine bases, the antipyretics, etc. Vegetable alkaloids, tea, coffee, cocoa, etc. 8 vo. 2d ed?, 1892, pp. viii, 584. Vol. ill, Part ill. Vegetable alkaloids, non-basic vege- table bitter principles. Animal bases, animal acids, cyanogen compounds, etc. 2d ed.,8 vo., 1896, pp. xii, 508. Vol. IV. The proteids and albuminous principles. 2d ed., 1898, pp. xi, 584. ('99). COHN, ALFRED I. Indicators and test papers, their source, preparation, application, and tests for sensitiveness, etc. New York, John Wiley & Sons; London, Chapman & Hall, Ltd., 1899. pp. ix, 249. Very useful. ('99). The Dispensatory of the United States of America. iSth ed., 1899. Philadelphia, J. B. Lippincott Company, pp. XLV, 1,999. ('99). BEHRENS, H. Anlekung zur mikrochemi- schen Analyse, pp. xi, 242, with 96 figures. 2d ed. Hamburg and Leipsic, Voss, 1899. Coo). SCHOENICHEN, WALTER, UND KALBERLAH, ALFRED. B. Eyferth's Einfachste Lebens- formen des Tier- und Pflanzenreiches. Naturgeschichte der mikroskopischen Suss- wasserbewohner. 3 vollstandig neubear- beitete und vermehrte Aufl. Mit iiber 700 Abbildungen auf 16 Tafeln in Lichtdruck nach Zeichnungen von Dr. A. Kalberlah. Braunschweig, Verlag von Benno Goeritz, 1900. pp. viii, 556. ('oo). SUTTON, FRANCIS. A systematic handbook of volumetric analysis. 8th ed., enlarged and improved, 1900. pp. xi, 640, with figures. London, J. and A. Churchill. This book should be in every laboratory. Coo). VAN RIJN, J. J. L. Die Glykoside chemische Monographic der Pflanzenglykoside, nebst systematiscber Darstelhmg der kunstlichen Glykoside. Gebruder Borntraeger, Berlin, 1900. pp. xvi, 511. ('oo). BEHRENS, H. Mikrochemische Technik. Ham- burg u. Leipzig, Leopold Voss, 1900. pp. vin, 68. ('oo). BUECHLER, MAX. Die Diastasen, 1900. Not seen. ('oi). GAGE, S. H. The Microscope. An introduc- tion to microscopic methods and to his- tology. 8th ed., revised, 1901. pp. iv, 299, with over two hundred figures. Comstock Publishing Co., Ithaca, N. Y. Deserves a place in every laboratory. Cor). COBLENTZ, VIRGIL. A manual of volumetric analysis, treating on the subjects of indica- tors, test-papers, alkalimetry, acidimetry, analysis by oxidation and reduction, iodome- try, assay processes for drugs with the titrimetric estimation of alkaloids, estima- tion of phenol, sugar, tables of atomic and molecular weights. P. Blakiston's Son & Co., Philadelphia, 1901. pp. viii, 181. Coi). LEE, ARTHUR BOLLES. The Microtomis.t's Vade-Mecum, a handbook of the methods of microscopic anatomy. 5th rev. ed. Phila- delphia, P. Blakiston's Son & Co., 1901. PP- xiv, 532. Very useful. French ed by I,ee and Henneguy. Paris, Octave Doin, 1902. ('oi). CROSS, C. P., AND BEVAN, E. J. Researches on cellulose, 1895-1900. Longmans, Green & Co., London, New York, and Bombay, 1901. pp. vn, 180. ('oi). HANAUSEK, T. F. Lehrbuch der technischen Mikroskopie. With 256 text figures. Stutt- gart, Ferdinand Enke, 1901. pp. x, 456. ('oi). RAWSON, CHRISTOPHER, GARDINER, WALTER M., AND LAYCOCK, W. F. A dictionary of dyes, mordants, and other compounds used in dyeing and calico printing. London, Chas. Griffin & Co., Ltd.; Philadelphia, J. B. Lippincott Co., 1901. pp. 372. ('02). OMLIANSKI, W. Ueberdie Garungder cellu- lose. Centralb. f. Bakt., 2 Abt., Bd. vin, Nos. 7-13, pp. 193-201, 225-231, 257-263, 289- 294, 321-326, 3S3-36i, 385-391, i text fig. and i plate. Jena, 1902. Omelianski finds two morphologically similar bacteria capable of fermenting pure cellulose (Swedish filter pa- per) in mineral solutions with chalk. Both grow ansero- bically, and bear spores in a swollen terminal part. One called the hydrogen ferment breaks up cellulose with the formation of hydrogen, carbon dioxide, acetic acid, and butyric acid. The other, called the marsh-gas ferment, breaks up cellulose with the formation of marsh.gas, carbon dioxide, acetic acid, and butyric acid. The cultures were made by the selective method, by which means most of the accompanying forms were crowded out. The hydrogen ferment was isolated from the methane ferment by heating the material used for the first transfer (from the methane-yielding ferment) for 15 minutes at 70 , the subsequently inoculated flasks then gave only the hydrogen fermentation. The organism of the latter was isolated pure on potato, but only after many trials and with feeble growth and weak ferment powers. The methane bacterium was not obtained pure in colonies. Neither organism colored blue with iodine. The experiments were begun In 1894 and carried through a long series of years, involving an enormous amount of painstaking labor. ('02). MANN, GUSTAV. Physiological histology. Methods and theory, pp. xv, 488. Oxford, The Clarendon Press, 1902. ('02). HOEBER, RUDOLF. Physikalisohe chemie der Zelle und der Gewebe. Wilhelm Engel- mann, Leipzig, 1902, pp. xii, 344, with 21 fig. ('03). KUESTER, ERNST. Parhologische Pflanzenana- tomie. Verlag von Gustav Fischer, Jena, 1903, PP- vii, 312, with 121 figures. ('03). DAVENPORT, CHARLES BENEDICT. Experimental Morphology. Part n. Effects of Chemical and Physical Agents upon Growth. 8vo. The Macmillan Co., New York. 210 BACTERIA IN RELATION TO PLANT DISEASES. ('03). PEIRCE, GEORGE JAMES. A Text-Book of Plant Physiology. Henry Holt & Co., New York, 1903, pp. vi, 291. ('03). COHN, ALFRED I. Tests and reagents, chem- ical and microscopical, known by their au- thor's names, together with an index of sub- jects, ist ed. 383 pp. New York, John Wiley & Sons; London, Chapman & Hall, Ltd., 1903. ('03). VON FUERTH, OTTO. Vergleichende chemische Physiologic der niederen Tiere. Jena, Gus- tav Fischer, 1903. pp. XIV, 670. ('04). NUTTALI,, GEORGE H. F. Blood immunity and blood relationship; a demonstration of cer- tion blood-relationships amongst animals by means of the precipitin test for blood, pp. xn, 444. Cambridge (England) University Press, 1904. V. Books and Papers of More or Less General Interest. ('28). EHRENBERG, C. G. Symbols physicae, seu icones et descriptiones animalium everte- bratorum seposi.tis insectis quae ex itinere per Africam Borealem et Asiam Occiden- talem Friderici Guilelmi Hemprich et Chris- taani Godofredi Ehrenberg medicinae et chlrurgias doctorum studio novae aut illus- tratas redierunt percensuit et regis iussu et impensis edidit Dr. C. G. Ehrenberg. Decas prima. Berolin-i ex Officina Academica Venditur a mittlero MDCCCXXVIII. ('30). EHRENBERG, C. G. Beitrage zur Kenntnis der Organisation der Infusorien, etc. Printed 13 Aug., 1830, as part of Abhandl. d. K. Akad. d. Wissensch. zu Berlin. Physic. Klasse for the year 1830. As a whole the volume bears date of 1832. ('32). EHRENBERG, C. G. Die geographische Ver- breitung der Infusionsthierchen in Nord- Afrika und West-Asien, etc. Abhandl. d. K. Akad. d. Wissensch. zu Berlin. Physik. Klasse. For the year 1829. Printed 1832. ('32). EHRENBERG, C. G. Ueber die Entwickelung und Lebensdauer der Infusionsthiere, etc. Abhandl. d. K. Akad. d. Wissensch. zu Berlin, 1831. Printed January, 1832. ('38). EHRENBERG, C. G. Die Infusionsthierchen als vollkommene Organismen. Ein Blick in das tiefere organische Leben der Natur, von Christian Gottfried Ehrenberg zu Ber- lin. Nebst einem Atlas von vierundsechszig colorirten Kupfertafeln gezeichnet vom Verfasser. Leipzig, Verlag von Leopold Voss, 1838. pp. 548, folio. ('40). HENLE, J. Pathplogische Untersuchungen. Berlin, August Hirschwald. pp. vi, 274. 1840. ('54). SCHROEDER, H., UND VON DuscH, TH. Ueber Filtration der Luft in Beziehung auf Faul- niss und Gahrung. Annalen der Chemie und Pharmacie, Bd. LXXXIX (Neue Reihe, Bd. xin), Heft i. pp. 232-243, Heidelberg, 1854. Extremely Interesting historically. These authors were the first to show that boiled beef bouillon and sim- ilar easily putrescible fluids could be preserved indefi- nitely if protected from the floating matter of the air by means of cotton plugs. Boiled milk, on the contrary, decayed as certainly under the cotton as when exposed directly to the air, for reasons unknown to them. The exact nature of this floating matter of the air was not determined. That remained for Pasteur to work out some years later by means of many ingenious experi- ments. ('54). COHN, FERDINAND. Untersuchungen iiber die Entwicklungsgeschichte der mikroscopischen Algen und Prize. Der Akademie iibergeben den i Mai, 1853. Nov. Act. Acad. Caes. Leop.-Carol. Naturae Curiosorum. Vol. xxiv, Pars i, 1854. pp. 103-256, 6 plates. Breslau and Bonn. ('59). SCHROEDER, H. Ueber Filtration der Luft in Beziehung auf Faulniss, Gahrung und Krys- tallisation. Annalen der Chemie und Phar- maoie, Bd. cix (Neue Reihe Bd. xxxm), Hit. i, pp. 35-52, 1859. Leipzig und Heidel- berg. ('60). PASTEUR, Louis.. Experiences relatives aux generations dites spontanees. C. R. des se. de 1'Acad. des Sci., Paris, 1860. pp. 303-307. ('62). PASTEUR, Louis. Memoire sur les corpuscles organises qui existent dans I'atmosphere. Examen de la doctrine des generations spon- tanees. Ann. de Chimiie et Physique, 1862. 3 s;erie, T. LXIV, pp. 5-110. ('65). DAVAINE, C. SUT 1'existence et la recherche des bacteridies dans la pustule maligne. C. R. d. se. et mem. de la societe de biol. for 1864, p. 93-94 (comptes rendus .part). Paris, 1865. ('68). ROBERTS, W. CHANDLER. On the occurrence of organic appearances in colloid silica ob- tained by .dialysis. Journ. of the Chem. Soc. of London. New series, vol. vi, 1868, pp. 274-276, 2 figs, of fungi. Roberts appears to have been the first to observe the growth of organisms on silicate jelly. ('72). COHN, F. Untersuchungen fiber Bacterien. Cohn's Beitr. zur Biol. der Pflanzen, Bd. i. 2 Hft. 1872. pp. 127-224. i plate. ('73). LISTER, JOSEPH. A further contribution to the natural history of Bacteria and the germ theory of fermentative changes. Quarterly Journal of Microscopical Science. 1873. Vol. xiii. pp. 380-408. ('73). PASTEUR, Louis. Etudes sur le vin, ses mala- dies, causes qui les provoquent, precedes npuveaux pour le conserver et pour le vieillir. Deuxieme edition revue et aug- mentee. Avec 32 planches imprimees en couleur et 25 gravures dans le texte. Paris, 1873. F. Savy. pp. iv, 344. ('76). PASTEUR, Louis, fitudes sur la biere, ses maladies, causes qui les provoquent, pro- cede pour la rendre inalterable, avec une itheorie nouvelle de la fermentation. 12 plates, 85 text figures. Paris, 1876. Gau- thier-Villars. pp. ym, 387. ('77). VON NAEGELI, C. Die niederen Pilze in ihren Beziehungen zu den Infectionskrankheiten und der Gesundheitspflege. Miinchen. R. Oldenbourg, 1877. pp. xxxn, 285. ('78). LISTER, JOSEPH. On the lactic fermentation and its bearing on pathology. Transactions of the Pathological Society of London. 1878. Vol. xxix, pp. 425-467- ('79). NENCKI, M., u. GIACOSO_, P. Giebt es Bac- terien oder deren Keime in den Organen gesunder lebender Thiere? Journ. f. Prakt. Chem.. 1879. Bd. xx, p. 34-44. ('82). MOTT, F. W., AND HORSLEY, V. H. On the existence of bacteria, or their antecedents, in healthy tissues. Journ. of Physiol., vol. in, 1880-1882. pp. 188-194. BOOKS AND PAPERS OF MORE OR LESS GENERAL INTEREST. 211 ('86). ('82). ENGELMANN, TH. W. Zur Biologic der Schi- zomyceten. Bot. Zeitung, 1882, co\ 321-32* and 337-341- ('84). METSCHNIKOFP, E. Ueber die Beziehung der Phagocyten zu Milzbrandbacillen. Archiv f. patholog. Anat. u. Physiologic u. f. Klin. Med., Bd. LXXIX, 1884. pp 502-526 2 plates. ('86). BOLTON, MEADE. Ueber das Verhalten ver- sclnedener Bacterienarten im Trinkwasser Ztschr. f. Hyg. Bd. i, 1886. pp. 76-114. ABBE, E. Ueber Verbesserungen des Mikro- skops mit Hilfe neuer Arten optischen Glases. Sitzungsber. der medicin.-naturw Gesellschaft zu Jena, 1886. Also a separate 24 pp. 8vo. ('88). SOYKA, J., UND BANDLER, A. Die Entwicke- lung von (pathogenen) Spaltpilzen unter dem wechselseitigen Einfluss ihrer Zerset- zungsprodukte. Fortsohr. d. Med. Bd vi 1888. pp. 769-773. Treats of growth of bacteria in media exhausted for other organisms. ('88). BANTI, GUIDO. Sulla distruzione dei batterii neirorganismo. Arch, per lo sc. med., 1888. Vol. xn, pp. 191-221, with 2 pp. of bibliog- raphy, i plate. ('89). VIGNAL, WILLIAM. Contribution a 1'etude des Bacteriacees. Le mesentericus vulgatus Paris, 1889. Coo). KOCH, R. Ueber bakteriologische Forschung Berlin, 1890, Verlag von August Hirsch- wald. pp. 15. ('93). DIXON, H. H. On the germination of seeds in the absence of bacteria. Soi. Trans. Roy. Dublin Soc., vol. v, series 11, 1893-1896. pp. 1-4. Review in Rev. Sci., 1894, pp. 437-438 (94). KOCHS, W. Giebt es ein Zellleben ohne Mikroorgamsmen? Biol. Centralb., 1894. No. 14, pp. 481-491. Answer, yes. Plants are grown from sterilized seeds and kept fourteen months without contact with bacteria Plums refused to decay when the surface had been ster- ilized. ('94). FRANKLAND. Die Bakteriologie in eiraigen ihrer Beziehungen zur chemischen Wissen- schaft. Centralb. f. Bakt., Bd. xv, 1894, pp. IOI-II2. ('95)- WARD, H. MARSHALL. On the biology of Bacillus ramosus, a schizomycete of the River Thames. Proc. Royal Soc. of Lon- don, vol. LVIII, 1895. 8vo. p. 265. Also a separate. ('95). PFEFFER, W. Ueber Election organischer Nahrstoffe. Pringsheim's Jahrbucher, Bd. xxvin, 1895, pp. 205-268. ('95). ACHARD, CH., ET PHULPIN, E. Contribution a 1'etude de 1'envahissement des organes par les microbes pendant 1'agonie et apres la mort. Arch, de med. exper. Tome vn, 1895, pp. 25-47. ('95). BECO, L. Etude stir la penetration des mi- crobes intestinaux dans la circulation generate pendant la vie. Ann. de 1'Inst. Pasteur, T. ix, 1895, pp. 199-209. C95-'96). NUTTALL, GEORGE H. P., UND THIERFELDER, H. Thierisches Leben ohne Bakterien im Verdauungskanal. Ztschr. f. physiol. Chem. Bd. xxi, 1895, pp. 109-121, und Bd. xxn, Hft. i, 1896, pp. 62-73. ( 96). LUNT, JOSEPH. On Bacillus mesentericus niger (a new potato bacillus). Centralb. f. Bakt. 2. Abt., Bd. u, 1896. pp. 572-573. Motile, liquefies gelatin rapidly, produces endospores copiously, blackens potato, curdles milk with subsequent solution of the curd, converts potato starch into sugar i. e. , there is no iodine reaction after a time, but a copious reduction of Fehling's solution. ('98). SANARELLI, G. Das myxomatogene Virus. Beitrag zum Studiutn der Krankheitserreger ausserhalb .des Sichtbaren. Centralb. f Bakt., xxni Bd., 1898, pp. 865-873. Author thinks it improbable that there are unorganized causes of infection, and that therefore certain infectious diseases must be due to organisms too small to be visible to the human eye, even when helped by the best optical appliances. ('99). OMELIANSKY, V. Sur la culture des mi- crobes nitrificateurs du sol. Arch. d. Sci. biol. St. Petersb. Tome vn. No. 4, 1899, pp. 291-302. ( 99). STURGIS, W. C. A soil bacillus of the type of de Bary's B. megaterium. Phil. Tr. Roy. Soc. of London. Series B, vol. 191, pp. 147-169. pi. 14-16, B. 172. London, 1899. Organism described as Bacillus hortulanus. Coo). HOF, A. C. Untersuchungen iiber die Topik der Alkalivertheilung in pflanzlichen Gewe- ben. Botanisches Centralb., Bd. LXXXIII, No. 9, xxi Jahrg., No. 35, 1900, pp. 273-280. Coo). SMITH, R. GREIG. The (bacterial) clouding of white wine. Proceedings of the Linnean Society of New South Wales, 1900. Part 4, Oct. 31. pp. 650-658. Also a separate. Coo). JORDAN, EDWIN O. Some observations upon the bacterial self-purification of streams. Jour. Exp. Med., vol. v, pp. 271 to 314. Dec., 1900. i plate. Coo). FORD, WM. W. Varieties of colon bacilli isolated from man. Montreal Medical Jour- nal, Nov., 1900. pp. 835-844. Also a sepa- rate, 10 pp. Bibliography of 14 titles. Coo). SMITH, THEOBOLD. Adaptation of pathogenic bacteria to different species of animals. Phila. Med. Journ., May 5, 1900. Vol. v., pp. 1,018-1,022. Coi). FISCHER, ALFRED. Ueber Protoplasmastrulctur. Antwort an O. Buetschli. Archiv f. Ent- wickelungsm. d. Org., 1901, Bd. xin, pp. 1-33. ('02). JOEST, ERNST. Unbekannte Infektionsstoffe. Centralb. f. Bakt., Abt. i, Bd. xxxi, Orig- inale, 1902, pp. 361-384, pp. 410-422. Bibliog. of 58 citations. ('03). FORD, W. W. The classification and distribu- tion of the intestinal bacteria in man. Studies from the Royal Victoria Hospital, Montreal, vol. i, No. 5 (Pathology u), May, 1903, 95 PP- ('03). WINSLOW, C. E. A., AND NIBECKER, C. P. The significance of bacteriological methods in sanitary water analysis. Technology Quar- terly of Mass. Institute of Technology, vol. xvi, No. 3, Sept., 1903, pp. 227-239. Also a separate. ('04). PHILLIPS, ORVILLE P. A comparative study of the cytology and movements of the Cyano- phyceae (Plates xxm-xxv, pp. 237-335. Bibliog. Trans, and Proc. Bot. Soc. Penn- sylvania, Vol. i. No. 3, 1904. Finds short cillia on side walls of Cyanophycese. 212 BACTERIA IN RELATION TO PLANT DISEASES. VI. Important Books and Papers on Special Human and Animal Diseases. ('63). DAVAINE, C. Recherches sur les infusoires du sang dans la maladie connue sous le nom de sang de rate. C. R. des se. de 1'Acad. des sci., 1863, T. LVII, pp. 220-223, 351-353, 386- 387. This and many other papers on anthrax are reprinted in "1'Oeuvre de Davaine," Paris, 1889, i vol. 8vo. ('64). DAVAINE, C. Nouvelles recherches sur la maladie du sang de rate consideree au point de vue de sa nature. C. R. des se. et mem. de la soc. d. biol. for 1863, Paris, 1864, pp. 193-202. ('70). PASTEUR, Louis. Etudes sur la maladie des vers a soie, moyen pratique assure de la combattre et d'en prevenir le retour. Tome I, La pebrine et la flacherie, pp. xu, 322, illustrated, and Tome n, Notes et docu- ments, pp. 327, Paris, 1870. Gauthier- Villars. ('76). KOCH, ROBERT. Aetiologie der Milzbrand- krankheit begrfindet auf die Entwicklungs- geschichte des Bacillus anthracis. Cohn's Beitrage z. Biol. d. Pflanzen. Bd. n, p. 277, Breslau, 1876. ('77). PASTEUR, L. fitiologie des maladies charbon- neuses. Archives Veter. T. n, Paris, 1877, pp. 668-671. ('80). PASTEUR, CHAMBERLAND ET Roux. Sur 1'eti- ologie du charbon. Archives Veter. T. V., Paris, 1880, pp. 548-557. ('80). PASTEUR, Louis. Sur les maladies virulentes, et en particulier fur la maladie appelee vul- gairement cholera des poules. C. R. des se. de 1'Acad. des sci. T. xc, 1880, pp. 239-248. ('81). KOCH, ROBERT. Zur Aetiologie des Milz- brandes. Mitteil. a. d. K. Gesundheitsamte. Bd. i, 1881, pp. 49-79. ('82). LOEFFLER UND SCHUETZ. Vorlaufige Mittheil- ung fiber die Arbeiten des K. Gesundheit- samtes, welche zur Entdeckung des Bacillus der Rotzkrankheit gefiihrt haben. Deutsch. med. Wochenschr. Bd. vm, 1882, pp. 707- 708. On the bacillus of glanders. See Vol. CXV, New Syd- enham Society for an English translation. ('83). EBERTH, C. J. Der Typhusbacillus und die intestinale Infection. Vortrag 226, Volk- mann's Sammel. Klin. Vortrage. Innere Med. No. 77, pp. 2,033-2,050, with 2 figs. Leipzig. Breitkopf und Hartel, 1883. Bibliography of 13 titles. ('83). FEHLEISEN. Die Aetiologie des Erysipels. Verlag von Theodor Fischer. Berlin, 1883, pp. 38. ('84). PASTEUR, CHAMBERLAND ET Roux. Sur la rage. C. R. des se. de 1'Acad. des Sci. T. xcvni, Paris, 1884, pp. 1229-1231. Earlier papers will be found in the same journal, T. xcvni, p. 457 ; T. xcv, p. 1187, and T. xcn, p. 1259. ('84). LOEFFLER, FRIEDRICH. Untersuchungen fiber die Bedeutung der Mikroorganismen fur die Entstehung der Diphtheric beim Men- schen, bei der Taube und beim Kalbe. Mitteil. a. d. K. Gesundheitsamte. Bd. n, 1884, pp. 421-499- ('84). GAFFKY. Zur Aetiologie des Abdominalty- phus. Mittheil. a. d. K. Gesundheitsamte, Bd. n, 1884, pp. 372-420. ('84). KOCH, ROBERT. Die Aetiologie der Tuberku- lose. Mitteil. a. d. K. Gesundheitsamte, Bd. n, 1884, pp. 1-88, with 10 plates. See Vol. CXV, New Sydenham Society for English translation. This paper should he read hy every student who desires to know how a good piece of investigation is conducted. ('84). NICOLAIER, ARTHUR. Ueber infectiosen Teta- nus. Deutsche med. Wochenschr., 1884. Bd. x, pp. 842-844. ('84). KOCH, ROBERT. Vortrag fiber die Cholera. Berliner klin. Wochenschr. Nos. 31 and 32, pp. 477-483 and 493-503. Deutsche med. Wochenschr. Nos. 32 and 320, 1884, pp. 499- 507 and 519-523. Reprinted also in Fortschr. der Medicin, Bd. n, Beilage, Heft 16, 1884, pp. 121-134 and Heft 17, pp. 141-168. Describes the " comma bacillus," now generally recog- nized to be the cause of Asiatic cholera. ('84). KOCH, R. Ueber die Cholerabakterien. Deut. Med. Wochenschr., Bd. x, 1884, pp. 725-728. ('84). CHAUVEAU, A., ET ARLOING, S. Etude ex- perimentale sur la septicemie gangreneuse. Bull, de 1'Acad. de med., Paris, 6 mai, 1884, 2e serie, T. xm, pp. 604-615. ('85). NICOLAIER, ARTHUR. Beitrage zur Aetiologie des Wundstarrkrampfes. Inaugural-Disser- tation, Gottingen, 1885, W. Fr. Kaestner, pp. 31- ('87). NOCARD ET Roux. Sur la culture du bacille de la tuberculose. Annales de 1'Inst. Pas- teur, T. i., 1887, pp. 19-29. ('88-'9o). Roux, E., ET YERSIN, A. Contribution a 1'otude de la diphtheric. Ann. 1'Inst. Pas- teur, ler memoire, Tome n, 1888, pp. 629- 661. 2e memoire, Tome HI, 1889, pp. 273- 288. 3e memoire, Tome iv, 1890, pp. 385-426. ('89). BEHRING. Beitrage zur Aetiologie des Milz- brandes. Ztschr. f. Hyg., Bd. vn, 1889, pp. 171-185. ('89). KITASATO, S. Ueber den Tetanus bacillus. Ztschr. f. Hygiene, Bd. vii, 1889, pp. 225- 234, i plate. ('89). Hog cholera. Its history, nature, and treat- ment, as determined by the inquiries and in- vestigations of the Bureau of Animal Indus- try, U. S. Dept. Agric. Gov't. Pr. Office, Washington, D. C., 1889, pp. 197, pi. 16. Bulletin prepared in great part by Dr. Theobald Smith. ('90). SMITH, THEOBALD. On the influence of slight modifications of culture media on the growth of bacteria as illustrated by the glanders ba- cillus. Journal of Comparative Medicine and Veterinary Archives, vol. xi, pp. 158-161. ('91). KITASATO, S. Experimented Untersuchungen fiber das Tetanusgift. Zeitschr. f. Hyg., Bd. x, 1891, pp. 267-305. ('91). SMITH, THEOBALD. Special report on the cause and prevention of swine plague. Bu- reau of Animal Industry, U. S. Dept. of Agriculture. Bull. 6, 1891, pp. 166, 12 plates. ('91). SMITH, THEOBALD. Zur Kenntniss des hog- cholerabacillus. Centralb. f. Bakt, Bd. ix, pp. 253-257, 307-311, and 339-343. ('92). PFEIFFER, R. Vorlaufige Mittheilungen fiber die Erreger der Influenza. Deut. med. Wochensdir., Bd. xvin, 1892, p. 28. ('93). BEHRING. Die Geschichte der Diphtheric. Leipzig, 1893. Not seen. SPECIAL HUMAN AND ANIMAL DISEASES. 213 ('93). BRIEGER, LUDWIG, AND COHN, GEORG. Unter- suchungen iiber das Tetanusgift. Zeitschr. f. Hyg., Bd. xv, pp. i-io, 1893. As small a quantity of the tetanus poison as 0.000,23 gra_m would be a lethal dose for a man weighing 70 kilos. This is an inference based on experiments with mice. ('93). MOORE, VERANUS A. Observations on the morphology, biology, and pathogenic prop- erties of twenty-eight streptococci found in the investigation of animal diseases. Bu- reau of Animal Industry, U. S. Dept. of Agriculture, Bull. No. 3, 1893, pp. 9-30. ('93). MOORE, V. A. Pathogenic and toxicogenic bacteria in the upper air passages of do- mesticated animals. Bulletin No. 3, Bureau of Animal Industry, U. S. Dept. Agric., 1893, PP- 38-48. ('93). PFEIFFER, R. Die Aebiologie der Influenza. Zeitschr. f. Hyg., Bd. xm, 1893, pp. 357-386, 8 plates. ('94). NOVY, F. G. Ein neuer anaerober Bacillus des malignen Oedems. Zeitschr. f. Hyg., Bd. xvn, 1894, pp. 209-233, 2 heliotype plates from photomicrographs by Dr. Pfeiffer. ('94). KITASATO, S. The bacillus of bubonic plague. The Lancet, London, 1894 (n), pp. 428-430. ('95). SiEBER-ScHOUMOW, MME. N. 6. Contribution a 1'etude des poissons venimeux. Sur le Bacillus pisoicidus agilis, microbe pathogene pour les poissons. Arch. d. sci. biol., Tome in, 1895, St. Petersb., pp. 226-256, i colored plate, 7 figs. Author isolated from dying fish and from the of the reservoir containing the sick fish, its inflow an outflow pipes, an anaerobic, gas forming (COz), motil short organism (Bacillus pisciclduit auilis), pathogeni was killed by a heating (5-10 min.) in river water at 68-70 C. , and agar or gelatin cultures yielded the cholera red reaction with hydrochloric acid. The organism is also toxic to white mice, guinea pigs, rabbits, and dogs, but not to pigeons. ('98). SHIGA, KIYOSHI. Ueber den Erreger der Dysenteric in Japan. Centralb. f. Bakt., i Abt., Bd. xxin, 1898, pp. 599-600. ('98). SMITH, THEOBALD. A comparative study of bovine tubercle bacilli and of human bacilli from sputum. The Jour, of Exper. Med., vol. HI, 1898, pp. 45I-5H. ('98). LOEFFLER. Bericht der Commission zur Er- forschung der Maul- und Klauenseuche bei dem Institut fiir Infektionskrankheiten in Berlin. Erstattet an den Cultusrniniister von dem Vorsitzendn der Commission, Berlin, Aug. 12, 1898. Deutsche mediz. Wochenschr., 1898, No. 35, pp. 562-564. Also a separate. Reprinted in Centralb. f. Bakt., i Abt., Bd. xxiv, 1898, pp. 569-574. Organism passes through a Chamberland filter, and is invisible. ('98). PETRUSCHKY, J. Ueber Massenausscheidung von Typhusbacillen durch den Urin von Typhus-Rekonvalescenten und die epidem- iologische Bedeutung dieser Thatsache. Centr. f. Bakt. i Abt., Bd. xxm, 1898, pp. 577-583. ('98). NOCARD, ED., ET LECLAINCHE, E. Les mala- dies microbiennes des animaux. 3d ed., 1003. Tome i, pp. n, 668 ; Tome, n, pp. 645. Paris, Masson et Cie. ('98) . NOCARD ET Roux. Le microbe de la peri- pneumonie_. Bulletin de la Soc. Central de Med. Veterinaire. Recueil de Med. Veter- inaire Annexe, Paris. Nouvelle sen, T. 16. Mar. 24, 1898, pp. 213-233. See also the Veterinary Journal, London, vol. XLVII, pp. 147-152. Authors describe as the cause of pleuro-pneumonia in cattle an organism of very small size, not visible clearly even after staining. The serum from diseased foci is extremely virulent, but it is impossible to cultivate any- thing from this serum by any of the ordinary methods. Many bacteriologists have tried and failed, including Nocard and Roux. These authors finally succeeded in cultivating it in bouillon in collodion sacks. The bouil- lon was inoculated with a little of the virulent serum and the sacks were then placed in the peritoneum of rabbits, where they were allowed to remain some weeks subject to osmosis. The organism clouds the bouillon slightly, and is visible under high magnifications, in bright light, as innumerable, minute, bright, mobile points. This bouillon is capable of reproducing the dis- ease, but is free from bacteria cultivable on ordinary media. Check sacks incubated in the peritoneum gave no such result, neither did sacks inoculated with heated virus. Collodion sack cultures incubated in the perito- neal cavity of animals were first used (?) by Metchnikoff Roux and Salimbeni in their study of the toxin and anti- toxin of cholera. The authors finally succeeded in cultivating this or- ganism outside of the animal body, by using a special bouillon and a special agar (see Les maladies micro- biennes des animaux, 1903, T. I., p. 450). " Virulent albu- minous liquids, pulmonary serum not diluted, or the Martin serum bouillon filtered through Chamberland or Berkefeld bougies, gives a sterile filtrate. On the con- trary, after dilution of the same liquids in a non-albu- minous medium, the microbe passes through the Berke- feld and the Chamberland bougie. Under these condi- tions the filtration enables one to obtain without diffi- culty a characteristic pure culture, even from impure products." ('98). NOCARD ET Roux. Le microbe de la peri- pneumoniie. Ann. de 1'Inst. Pasteur, 1898, T. XH, pp. 240-262. ('99). GELPKE, THEODOR. Bacterium septatum und dessen Beziehungen zur Gruppe der Diph- therienbacterien(B. diphtheriae [KIebs-L6f- fler], B. pseudodiphtheriticum [Loftier] und B. xerosis). Arb. a. d. Bact. Institut der techn. Hochschule zu Karlsruhe, n Bd., 2 Hefte, 1899, pp. 71-148. 5 plates (40 pho- tomicrographs) and 4 charts. Bibliog. of 45 titles. ('oo). WELCH, WILLIAM H. Morbid conditions caused by the Bacillus aerogenes capsulatus. Phila. Med. Journ., vol. vi, 1900, pp. 202-216. ('oo). FLEXNER, SIMON. On the etiology of tropical dysentery. Phila. Med. Journ., vol. vi, 1900, pp. 414-424- The author calls special attention to Shiga's results. Flexner studied this disease in the Philippines. He says - " That the bacillus is identical with the organism ob- tained by Shiga in the epidemic of dysentery which pre- vailed in Japan, there can be no reasonable doubt. In morphological, cultural, and pathogenic characteristics the two organisms are indistinguishable." ('oo). GORHAM, F. P. The gas-bubble disease of fish and its cause. U. S. Fish Commission, Bull, for 1899, pp. 33-37, 1900, Washington. ('oo). SMITH, R. GREIG. A new bacillus pathogenic to fish. Proceedings, Linnean Soc., New South Wales for 1900. Sydney, 1901, vol. xxv, pp. 122-130. Two heliotype plates. This Is named Bacillus piscidus bipolaris, in allusion to the bipolar germination of its spores. The organism is motile, and liquefies gelatin. 214 BACTERIA IN RELATION TO PLANT DISEASES. Coo). SMITH, R. GREIG. A fish disease from George's River. Proc. Linnean Soc., New South Wales for 1900. Sydney, 1901, vol. xxv, pp. 605-609. ('99-'oo). MOSCHCOWITZ, ALEXIS V. Tetanus. A study of the nature, excitant, lesions, symp- tomatology, and treatment of the disease, with a critical summary of the results of serum therapy. Studies from the Dept. of Pathology of the College of Physicians and Surgeons, Columbia University, New York. Vol. vii, 88 pp. A bibliography of 337 titles. 1899-1900. ('01). SCHULTZ, N. K. De la vitalite du microbe de la peste bubonique dans les cultures. Arch, des Sci. Biol. publiees par 1'Inst. Im- perial de Med. Exper. a St. Petersbourg. T. vin, No. 4, 1901, pp. 373-389. i plate. Organism not sporiferous. Author made various tests from old cultures kept in sealed tubes of bouillon, and concludes that under favorable conditions the pest bacil- lus may preserve its vitality and virulence for four years. In its most modified form, he says, it exists in the state of very small round grains. ('01). TARTAKOWSKY, G., ET DCHOUNKOWSKY. Du microbe de la peripneumonie des boeufs. Arch, des sci. biol. publiees par 1'inst. imper. de med. exper. a St. Petersbourg. T. vin, 1901, pp. 441-460, 6 figs. Confirms the work of Nocard and Roux. ('02) . MARSH, M. C. Bacterium trutta?, a new species of bacterium pathogenic to trout. Science, n. s., vol. xvi, No. 409, pp. 706-707, Oct. 31, 1902. Also a separate. This organism browns nutrient agar. Its thermal death point is said to be 42 C. ('02). SHIGA, K. Weitere Studien iiber den Dysen- teriebacillus. Zeitschr. f. Hyg. Bd. XLI, 1902, pp. 3SS-368. ('02). MOORE, V. A. The pathology and differential diagnosis of infectious diseases of animals. Ithaca, N. Y., 1902. pp. xrv, 380. 73 figs. 8 plates. ('02). KOCH, ROBERT. An address on the transfer- ence of bovine tuberculosis to man. Brit. 'Med. Jour., London, 1902, vol. 2, pp. 1,885- 1,889. ('03). MARSH, M. C. A more complete description of Bacterium truttae. Bull. U. S. Fish Com- mission for 1902. Washington, Govt. Print- ing Office, 1903, pp. 4H-4IS, with two plates. Also a separate. Grows well in media which is neutral or + 5, but there is little or no growth when the acidity is -f- 15. Growth is also inhibited by an alkalinity of 5. Gelatin and blood serum are liquefied. There is no growth on potato unless it is first neutralized ; then there is a scanty white growth. Milk is not coagulated, but it becomes fairly transparent after two weeks. It does not ferment glu- cose, lactose or saccharose. It reduces nitrates to nitrites and to ammonia. It is not clearly mobile It does not pro- duce indol or phenol. The optimum temperature is at or near 20 C . It is actively pathogenic to trout, especially brook trout, in which the disease was first observed. ('03). MONFALLET, D. Bibliographic abregee des infections. Paris et Santiago (Chili), Ch. Goffi, editeur, 1903, pp. 1-65. About 1900 classified references to papers on human and animal diseases of a communicable nature. ('04). REMUNGER, P. Les travaux recent sur la rage. Bull, de 1'Inst. Pasteur, 1904, T. n, pp. 753- 764. " We have shown that if one filters an emulsion of rabies virus through a Berkefeld V bougie and then cen- trifuges the filtrate, the upper layers are deprived of vir- iileuce, but the virulence is kept in the lower layers. Barratt lias obtained the same results with an unfiltered emulsion of a rabid brain." ('04). FLEXNER, SIMON, HOLT, L. EMMETT, and as- sistants. Bacteriological and Clinical Studies of the Diarrheal Diseases of Infancy, with Reference to the Bacillus Dysenteriaa (Shiga). Studies from the Rockefeller In- stitute for Medical Research, New York. Vol. n, 1904, pp. 7-202. VII. Predisposition, Conditions Favoring Infection or Immunity. ('80). CHAUVEAU. See xxn. ('81). LOEFFLER, FRIEDRICH. Zur Immunitatsfrage. Mittih. a. d. K. Gesundheitsamte. Bd. i, iSSi, pp. 134-187. Also a separate, 54 pp. ('87). METCHNIKOFF. See xxi. ('88). NUTTALL. See xxxvm. ('88). NUTTALL. See xxi. ('88). FLUEGGE, C. Studien iiber die Abschwachung virulenter Bakterien und die erworbenc- Immunitat. Zeitsch. f. Hygiene. 1888. Bd. IV, pp. 208-230. ('89). BUCHNER. See xxi. ('89). NISSEN. See xxi. ('90). LEHMANN. See xxi ('91). OGATA. See xxi. ('91). BUCHNER, HANS. Ueber Immunitat, deren natiirliches Vorkommen und kunstliche Erzeugung. Munch. Med. Wochenschr. 1891, 38 Jahrg., pp. 5SI-SS4, 574-579- ('94). EHRLICH AND WASSERMANN. See xxi. ('01). HUEPPE, FERDINAND. Perlsucht und Tuber- culose. Berliner klin. Wochenschrift, 1901, No. 34, pp. 876-878. Also a separate, pp. 10. (*oi). ZABOLOTNY, D. Recherches stir la peste. 2 mem. Experiences d'inoculation, d'im- munisation et de traitemeiit des animaux. Arch, des Sci. Biol. publiees par 1'Inst. Im- perial de Med. Exper. a St. Petersbourg. T. vni, No. 4, looi, pp. 390-427. 2 plates. ('01). METCHNIKOFF, ELIE. L'immunite dans les maladies infectieuses. Paris (Masson), 1901, pp. ix, 600, av. fig. German trans, by Julius Meyer. Jena (Fischer), 1902, pp. xi, 456. Not seen. ('02). AMMON, OTTO. Theoretische Betrachtungen iiber Ansteckung und Disposition. Arch. f. Hyg. Bd. XLII, 1002, pp. 289-305. ('o2-'o4). KOLLE AND WASSERMAN. See in. VIII. Symbiosis, Antagonism. ('81). KERN, EDWARD. Ueber ein neues Milchfcr- ment aus dem Kaukasus. Bull, de la Soc. Imp. d. Nat. de Moscou, Tome LVI, pp. 141- 177, 2d part (No. 3), 2 plates, 1881. Kephir. Dispora caucasica, nov. gen. and n. sp. Plates good. (.'84). KRANNHALS, H. Ueber das Kumys-ahnliche getrank "Kephir" und ueber den "Kephir - pilz. Deutsch. Archiv. f. klin. med. Bd. xxxv, pp. 18-37, i plate. Bibliography of 18 titles. SYMBIOSIS; CARRIERS OF INFECTION; MORPHOLOGY. 215 ('84). STRUVE, UEINRICH. Ueber Kephir. Ber. d. deutsch. chem. Gesellsch. Bd. xvn, 1884, PP- 3M-3i6 and 1,364-1,368. ('87). METCHNIKOFK. See xxi. ('87. GARRE, C. Ueber Antagonisten unter den Bacterien. Correspondenzbl. f. Schweizer. /.oo^ , T Aerzte - J?i hr S- xvn ' l88 7, PP. 385-392. ( 88). NUTTALL. See xxi. ('88). DE FREUDENREICH, E. De 1'anitagonisme des bacteries et de 1'immunite qu'il confere aux milieux de culture. Ann. de 1'Inst. Pasteur, T. n, 1888, pp. 200-206. ('88). DUBOIS. See xxvn. ('88). HKRICOURT. Des associations microbiennes. Rev. de med., T. , 1888, pp. . Not seen. ('88). SIROTININ. Ueber die entwicklungshemmen- den Stoffwechselproduote der Bacterien und die sog. Retentionshypothese. Zeitsch. f. Hyg. Bd. iv, 1888, pp. 262-290. ('89). BEYERINCK, M. W. Sur le kefir. Arch. neer. des sci. ex. et nat, T. xxm, 1889, pp. 428- 444- i fig. ('89). ROGERS, G. H. Quelques effets des associa- tions microbiennes. C. R. hebd. d. se. et mem. de la soc. de. biol., 19 Janvier, 1889, Paris, se. 9, T. i, pp. 35-38. Two bacteria, inoffensive to a given animal, may be- come pathogenic when inoculated together. ('90). ('91). ('93). ('94). ('96?) ('97). Coo). BLAGOVESTCHENSKY, N. Sur 1'antagonisme entre les bacilles du charbon et ceux du pus bleu. Ann. de 1'Institut Pasteur, T. iv, 1890, pp. 689-715. Mix, CHARLES L. On a kephir-like yeast found in the United States. Proc. Amer. Acad. of Arts and Sciences, 1891, n. s. vol. xvm, pp. 102-114. WARD, H. MARSHALL. The ginger-beer plant, and the organisms composing it. A contri- bution to the study of fermentation-yeasts and bacteria. Phil. Trans. Roy. Soc. (B) for 1892. London, 1893, vol. 183, pp. 125 to 197, PI. 6. GALTIER, V. Nouvelle recherches sur 1'influ- ences des associations bacteriennes. Exalta- tion de la virulence de certain microbes. Accroissement de la receptivite. C. R. des se. de 1'Acad. d. sci., T. cxvin, 1894, pp. 1,001-1,004. . VON FREUDENREICH, EDUARD. Bakteriologische Untersuchungen fiber den Kefir. Landw. Jahrbuch d. Schweiz, 1896. Bd. x, pp. 1-20. 2 text figs, and i heliotype plate. VON FREUDENREICH, ED. Bakteriologische Un- tersuchungen fiber den Kefir. Centralb. f. Bakt., 2 Abt. Bd. in, 1897, pp. 47-54, 2 figs. ; 87-95, I35-I4L KRAUSE. See xv. IX. Carriers of Infection. ('88). ALESSI, GUISEPPE. Sulla trasmissibilita dei germi infettivi mediante le deiezioni delle mosche. Arch, per lo sci. med. Vol. xn, Torino, 1888, pp. 279-292. ('91). WAITE, M. B. Results from recent investiga- tions in pear Wight. Proc. Am. Asso. Adv. Sci., 4Oth meeting, Salem, 1892. Mr. Waite obtained his first results in iSyi, and called attention to them at the Washington meeting of the Am. Asso. Adv. Sci. that summer. ( 94). Yersen, La peste bubonique a Hong-Kong. Ann. de 1'Inst. Pasteur. T. vni, 1894, pp. 662-667. The pest is carried by rats. ('95). SMITH, ERWIN F. Bacillus tracheiphilus, etc. Centralb. f. Bakt.,etc. 2 Abt. i Bd., 1895, P- 365. The disease is spread by beetles. ('96). SMITH, ERWIN F. A bacterial disease of the tomato, egg plant, and Irish potato. Wash- ington, 1896, p. 22, and PI. n, fig. 3. Disease communicated by beetles. ('97). MARPMANN. See XLIX. ('97). SMITH, ERWIN F. Pseudomonas campestris (Parnmel). The cause of a brown rot in cruciferous plants. Centralb. f. Bakt. 2 Abt., Bd. HI, pp. 409-410. Disease communicated by slugs and by larva: of the cabbage butterfly. ('98). SIMOND, P. L. La propagation de la peste. Ann. de 1'Inst. Pasteur, T. xn, 1898, pp. 625-687. 5 figs. ('98). NUTTALL, GEORGE H. F. Zur Aufklarung der Rolle, welche stechende Insekten bei der Verbreitung von Infektionskrankheiten spielen. Centralb. f. Bakt., Bd. xxm, i Abt., 1898, pp. 625-635. ('99). NUTTALL, G. H. F. On the role of insects, arachnids, and myriapods as carriers in the spread of bacterial and parasitic diseases of man and animals. Johns Hopkins Hosp. Repts., vol vni, No. 1-2, pp. 1-154. 3 plates. Bibliography of 366 titles. Noticed in Nature, Dec. 14, 1899. Coo). GALLI-VALERIO, BRUNO. Les puces des rats et des souris jouenit-elles un rok important dans la transmission de la peste bubonique a 1'homme? Centralb. f. Bakt., i Abt, Bd. xxvn, 1900, pp. 1-4. 3 figs. ('02). GALLI-VALERIO, BRUNO.. The part played by the fleas of rats and mice in the transmission of bubonic plague. Jour. Trop. Med., Lon- don, vol. v, 1902, pp. 33-36. . BRENNER, W. Die Schwarzfaule des Kohls. Centralb. f. Bakt. 2 Abt. Bd. xn, p. 729. Disease said to be communicated by aphides. X. General Morphology of the Bacteria. Cytology. ('74). BILLROTH, THEODOR. Untersuchungen fiber die Vegetationsformen von Cocco-bacteria sep- tica und den Antheil welchen 'sie an der Entstehung und Verbreitung der accidentel- len Wundkrankheken haben. Berlin, 1874. Verlag von Georg Reimer. Quarto, pp. xrv, 244. 5 plates. ('77). BILLROTH, TH., AND EHRLICH, F. Unter- suchungen fiber Coccobacteria septica. Archiv. f. klinische chir. Bd. xx, pp. 403- 433, I pi., Berlin, 1877. ('78). HALLIER, ERNST. Die Plastiden der niederen Pflanzen, ihre selbststandige Entwickelung, ihr Eindringen in die Gewebe, und ihre verherende Wirkung. Leipzig., 1878, pp. 92, 4 plates. ('82). ZOPF, W. Zur Morphologic der Spaltpflanzen. Spaltpilze und Spaltalgen, Leipsic, 1882, pp. vi, 74. Verlag von Veit and Comp. 7 plates. 2l6 BACTERIA IN RELATION TO PLANT DISEASES. ('86). HUEPPE, FERDINAND. Die Formen der Bak- terien und ihre Beziehungen zu den Gat- tungen und Anten. 1886. pp. vm, 152, 24 figs. Wiesbaden. C. W. Kreidel's Verlag. ('88). POMMER, GUSTAV. Ein Beitrag zur Kenntniss der fa-denbildenden Bakterien. Mitth. hot. Itist. Graz, Jena, 1888, pp. 93-112. I plate. Bacillus brassicse, a sporiferous form, isolated from an imperfectly sterilized decoction of cabbage leaf. ('89). METCHNIKOFF, EL. Contributions a 1'etude du pleomorphisme des baoteries. Ann. de 1'Inst. Pasteur, T. in, 1889, pp. 61-68. i plate. (80). ERNST, PAUL. Ueber Kern-und Sporenbildung in Bakterien. Zeitschr. f. Hyg. Bd. v, 1889, pp. 428-486. 2 plates. ('89). BABES, VICTOR. Ueber isolirt farbbare Antheile von Bakterien. Zeitsohr. f. Hyg. Bd. v, 1889, pp. 173-190. i plate. ('89) WINOGRADSKY, S. Pleomorphisme des bac- teries. Ann. de 1'Inst. Pasteur, T. in, 1889, pp. 249-264. ('89). METCHNIKOFF, EL. Note sur le pleomorphisme des bacteries. Ann. de 1'Inst. Pasteur, T. in, 1889, pp. 265-267. ('90). ALMQUIST, E. Untersuchungen fiber einige Bakteriengattungen mit Mycelien. Zeitsch. f. Hyg. Bd. vin, 1890, Heft 2, pp. 189-197. ('90). BUTSCHLI, O. Ueber den Bau der Bakterien und Verwandter Organismen. Leipzig, C. F. Winter, 1890, pp. 37. i pi. Maintains the existence in the bacteria of a body cor- responding to a cell nucleus. ('91). FAMINTZIN, A. Eine neue Bakterienform, Newskia ramosa. Bulletin de 1'Acad. de St. Petersbourg. Nouvelle serie (n), 1891, T. xxxiv, p. 481. Not seen . ('91). DANGEARD. See xxin. ('91). PROTOPOPOFF. Sur la question de la structure des baoteries. Ann. de 1'Inst, Pasteur, 1891, Tome v, p. 332-336. fgi) ZETTNOW, E. Ueber den Bau der Bakterien. Centralb. f. Bakt. x Bd., 1891, pp. 689-694. i Tafel. ('92) ZUKAL, HUGO. Ueber den Zellinhalt der Schi- zophyten. Ber. d. deutsch. bot. Gesellsch. x, 1892, pp. 5I-S5- ('92) . SJOEBRING, NILS. Ueber Kerne und Theilung- en bei den Bakterien. Centralb. f. Bakt., xi Bd., 1892, pp. 65-68, with i colored Tafel. "In the bacterial body there may be demonstrated con- sequently as in other cells, two components nucleus and cell-body, which, however, can not always be d tinguished." ('92). THAXTER, ROLAND. On the Myxobacteriaceae, a new order of Schizomycetes. Botanical Gazette, vol. xvn, No. 12, pp. 389-406. Pis. xxn-xxv, 1892. ('92). SAUVAGEAU, C., ET RADAIS. Sur les genres Cladothrix, Streptothrix, Actinomyces, et description de deux Streptothrix nouveau. Ann.de 1'Inst. Pasteur. T. vi, 1892, pp. 242- 273. i plate. ('92) . FoERSTER, F. Ueber eine merkwurdige Erschei- nung bei Chromatium Okenii Ehrbg. sp. Centralb. f. Bakt., xi Bd., 1892, pp. 257-264, mit I taf. (colored). Author's figures show curious bands or bridges con- necting the bacterial cells sidewise as well as end to end. ('92). TRAMBUSTI, A., AND GALEOTTI, G. Neuer Beitrag zitrn Studium der inneren Struktur der Bakterien. Centralb. f. Bakt., xi Bd., 1892, pp. 717-722, mit i Taf. (colored). ('92). KLEIN, E. Zur Geschicbte des Pleomorphis- mus des Tuberculoseerregers. Centralb. f. Bakt., xn Bd., 1892, pp. 905-906. ('92). BuETSCHLi, O. Untersuchungen fiber mikro- skopische Schaume und das Protoplasma. 4to, mit 6 lithogr. Taf. u. 23 Fig. im Text, so wie einem Atlas von 19 Mikrophoto- graphieen. Leipzig, 1892. Rev. in Centralb. f. Bakt., Bd. xm, 1893, pp. 436-438. Again maintains the existence in bacteria of a Central- koerper corresponding to a nucleus, and points out that Alfred Fischer's criticism of his former statements has no substantial basis. See especially pp. 75 to 79. ('94). BEYERINCK, M. W. Ueber die Natur der Faden der Papilionaceenknollchen. Centralb. f. Bakt., Bd. xv, 1894, pp. 728-732. ('94). ILKEWICZ, W. Ueber die Kerne der Milz- brandsporen. Centralb. f. Bakt., Bd. xv, 1894, pp. 261-267, mit i Figur. ('95). WARD, H. MARSHALL. The formation of bac- terial colonies. Annals of Botany, vol. ix, 1895, pp. 653-657. ('95). WARD, H. MARSHALL. A false bacterium. An- nals of Botany, vol. ix, 1895, pp. 657-658. ('9S)- WAGER, HAROLD. Preliminary note upon the structure of bacterial cells. Annals of Botany, vol. ix, 1895, pp. 659-661. ('95). COPPEN-JONES, A. Ueber die Morphologic und systematische Stellung des Tuberkelpilzes und liber die Kolbenbildung bei Aktinomy- kose und Tuberkulose. Centralb. f. Bakt., xvn Bd., 1895, pp. 1-16 and 70-76, with i plate. ('95). BRUNS, HAYO. Ein Beitrag zur Pleomorphie der Tuberkelbacillen. Centralb. f. Bakt., xvn Bd., 1895, pp. 817-826, with 8 figs. ('95). LUBINSKI, Ws. Zur Kultivierungsmethode, Biologic und Morphologic der Tuberkel- bacillen. Centralb. f. Bakt., xvm Bd., 1895, pp. 125-128. ('95). BABES, V. Beobachtungen fiber die metachro- matischen Korperchen, Sporenbildung, Ver- zweigung. Kolben-und Kapselbildung path- ogener Bakterien. Zeitschr. f. Hyg. Bd. xx, 1895, pp. 412-437, 2 plates. Fig. 18 e of Table XI is particularly instructive. This shows a true branching in the anthrax organism, but it is confined to the capsule. COPPEN-JONES, A. Ueber die Nomenclatur des sog. " Tuberkelbacillus." Centralb. f. Bakt., i Abt, Bd. xx, 1896, No. 10-11, pp. 393-395. KANTHACK, A. A. Ueber verzweigte Diph- theriebacillen. Centralb. f, Bkt, xx Bd., 1896, pp. 296-297. ('96). ZETTNOW. Bilder von Spirillum undula majus bei freiwilligem Absterben. Centralb. f. Bakt., xix Bd., 1896, pp. 177-180, with i plate, 16 figs. THAXTER, ROLAND. Contributions from the Cryptogamic Laboratory of Harvard Uni- versity, xxxix. Further Observations on the Myxobacteriaceae. Botanical Gazette, vol. xxin. No. 6, 1897, pp. 393-411, 2 plates. ('97) MARPMANN, G. Zur Morphologic und Biologic des Tuberkelbacillus. Centralb. i. Bakt., xxii Bd., 1897, pp. 582-586, with i plate. ('96). ('97). GENERAL MORPHOLOGY OF THE BACTERIA; CYTOLOGY. 2I 7 ('97). JOHAN-OLSEN, OLAV. Zur Pleomorphismus- frage. Centralb. f. Bakt., 2 Abt., Bd. in, 1897, pp. 273-284, 2 plates. Finds branching forms. ('97). KiTT. Die Streptothrixform des Rotlaufbacil- lus. Centralb. f. Bakt., xxn Bd., 1897, pp. 726-732, with 4 figs. ('98). SCHULTZ. See xxxvm. ('98). STOLZ, ALBERT. Ueber besondere Wachstums- formen bei Pneumo- und Streptokokken. Centralb. f. Bakt., xxiv Bd., 1898, pp. 337- 343, with 6 figs. Figures look like involution forms. ('98). ZIEMANN, HANS. Eine Methode der Doppel- farbung bei Flagellaten, Pilzen, Spirillen und Bakterien, sowie bei einigen Amoben. Centralb. f. Bakt., xxiv Bd., 1898, pp. 945- 955, i plate. The author's figures show the body of the spirillum blue enclosing 1-5 carmin colored granules. ('98). RUZICKA, VLAD. Zur Frage von der inneren Struktur der Mikroorganismen. Centralb. f. Bakt., xxin Bd., 1898, pp. 305-307, with I plate. Kinds granules, which take stains, in bacterial body reserves conclusions as to their nature, but thinks they are not pleomorphic or degeneration phenomena. ('98). GRASSBERGER, R. Zur Frage der Scheinfaden- 'bildung in Influenzaculturen. Centralb. f. Bakt., xxin Bd., 1898, pp. 353-364, with I plate and 4 text figs. ('98). BURCHARD, GEORG. Beitrage ztir Morphologic und Entwickelungs-Geschichte der Bacte- rien. Arb. a. d. Bact. Institut der tech. Hochschule zu Karlsruhe, n Bd., i Heft, 1898, pp. 1-64, 2 pi. Twenty new species are described Bacterium pitui- taus, B. perittomaticum, B. flexile, B. turgescens, B. bracliysporum, B. implectans, B petroselini, B. augulans, Bacillus goniosporus, B. pectocutis, B. paucicutis, B. cylindrosporus, B. leptodermis, B. bipolaris, B. loxo- sus, B. myxodens, B. armoraciae, B. idosus, B. loxosporus. B. cursor. ('98). WAGNER, A. Coli- und Typhusbakterien sind einkernige Zellen. Centralb. f. Bakt., xxm Bd., 1898, pp. 433-438, and pp. 489-492, wifch 2 plates and 6 figs. ('98). CRAIG, CHARLES F. The branched form of the bacillus tuberculosis in sputum. The Journ. of Exp. Med., vol. in, 1898, pp. 363-370, i plate. Author thinks it is premature to separate B. tubercu- losis from the bacteria on account of this phenomenon. ('99). SCHULZE, OTTO. Unitersuchungen iiber die Strahlenpilzformen des Tuberculoseerregers. Zeitschr. f. Hyg. Bd. xxxi, 1899, pp. 153- 186, i plate. ('99). GALLI-VALERIO, BRUNO. Contribution a 1'etude de la morphologic du Bacillus mallei. Centralb. f. Bakt., xxvi Bd., 1899, pp. 177- 180, with 5 figs. Author finds branched forms in bouillon and on agar. ('99). BERESTNEW, N. Zur Frage der Klassifikation und systematischen Stellung der Strahlen- pilze. Centralb. f. Bakt., xxvi, 1899, p. 390. ('99). MOELLER, ALFRED. Ein neuer saure- und alko- holfester Bacillus aus der Tuberkelbacillen- gruppe, welcher echte Verzweigungsformen bildet. Centralb. f. Bakt., Bd. xxv, 1899, pp. 369-373, with i plate. ('99). SPIRIG, W. Die Streptothrix (Aotinomyces) Natur des Diphtheriebacillus. Centralb. f. Bakt, xxvi Bd., 1899, pp. 540-541. ('99). LUBARSCH, O. Zur Kenntniss der Strahlen- pilze. Zeitschr. f. Hyg. Bd. xxxi, 1899, pp. 187-220, i plate. ('99). MARX, HUGO. Zur Morphologic des Rotz- bacillus. Centralb. f. Bakt., xxv Bd., 1899, pp. 274-278, with 4 figs., showing branched forms. ('99). MUEHLSCHLEGEL, A. Ein Beitrag zur Mor- phologie und Entwickelungsgeschichte der Bakterien nach Studien an drei Korner- baciHen. Arb. a. d. kaiserl. Gesundheits- amte, Bd. xv, Heft, i, pp. 131-152, 1899. i plate partly colored. Rev. in Centralb. f. Bakt., xxv Bd., 1899, p. 771. 73 titles cited under literature. Coo). FEINBERG. Ueber den Bau der Bakterien. Centralb. f. Bakt., xxvn Bd., 1900, pp. 417- 426, with 5 plates. Author believes he has demonstrated the existence of a nucleus in the bacteria by means of Romanowski's stain- ing method (a mixture of methylene blue and eosin). The plasma stains blue ; the nucleus, which may be small or which may fill nearly the whole bacterial body, stains red or red-brown. Coo). ZETTNOW. Romanowski's Farbung bei Bak- terien. Centralb. f. Bakt., xxvn Bd., 1900, pp. 803-805. Says Dr. Feinberg's papers contribute " nidus Neues " Coo). NAKANISHI, K. Vorlaufige Mitteilungen uber cine neue Farbungsmethode zur Darstellung des feineren Baues der Bakterien. Munch, med. Wochenschr., 1900, No. 6. Rev. in Centralb. f. Bakt., xxvn Bd., 1900, pp. 547- 549- " All bacteria in their young stage, when they have grown under favorable conditions, are one-nucleate short cells." Coo). SKSCHIVAN, T. Zur Morphologic des Pest- bakteriutns. Centralb. f. Bakt., xxvin Bd., 1900, pp. 289-292, with 4 text figs. Finds V-shaped and branched forms. Coo). MARX, HUGO, AND WOITHE, FRIEDRICH. Mor- phologische Untersuchungen zur Biologic der Bakterien. Centralb. f. Bakt., xxvin Bd., 1900, pp. i-ii, 33-39, 65-69, and 97-111, with 3 plates. Coo). GALLI-VALERIO, BRUNO. Seconde contribution a 1'etude de la morphologic du B. mallei. Centralb. f. Bakt., xxvm Bd., 1900, pp. 353- 359, with 26 figs. Coi). MEYER, ARTHUR. Ueber die Verzweigung der Bakterien. Centralb. f. Bakt., i Abt., xxx Bd., 1901, No. 2, pp. 49-60, 2 plates. Coi). ROSENFELD. See xv. Coi). REICHENBACH, H. Ueber Verzweigung bei Spirillen. Centralb. f. Bakt., i Abt., Bd. xxix, 1001, pp. 553-557- i heliotype plate. Many of the spirilla are shown with distinct branches. ('02). BUETSCHLI, OTTO. Bemerkungen fiber Cyano- pbyceen und Bacteriaceen. Archiv. f. Pro- tistenkunde, Bd. i, Heft. i. Jena, Gustav Fischer, 1902. pp. 41-58, i plate. On the nature of the " Centralkoerper." ('02). ERRERA, Lo. Sur une bacterie de grandes dimensions : Spirillum colossus. Recueil de 1'Inst. botanique (Univ.de Bruxelles), T. v, 1902, pp. 347-357- Also a separate. 218 BACTERIA IN RELATION TO PLANT DISEASES. ('02). LOEB, L. M. On branching forms of certain bacteria. Jour, of Med. Research. Vol. vin, 1902 (n. s., vol. in), pp. 415-423. The author finds branching forms in the typhoid ba- cillus. ('02). HEFFERAN, MARY. An unusual bacterial grouping. Centralb. f. Bakt., 2 Abt., vm Bd., 1902, pp. 689-699. Also a separate, (with 5 figures in the text). B. rosaceus metalloides forms rosette-like groupings in liquid media and on some solid media. ('02). MATZUSCHITA, TEISI. Beobachtungen iiber den merkwiirdigen Teilungsprocess bei einem proteusartigen I/uftbacillus. (Vorl. Mit- teilung). Centralb. f. Bakt., Abt. 2, Bd. ix, 1902, pp. 257-260, mil I Taf. ('03). HILL, H. W., AND RICHARDS, B. R. Notes on Morphology, i. "Snapping" as character- istic of the diphtheroid group, n. Branch- ing of the organism of actinomycosis. Am. Pub. Health Ass'n. Proc. 3(rth Ann. meet- ing -at New Orleans, Dec. 9-12, 1902, Co- lumbus, Ohio, 1903. Also a separate, 4 pp., 2 figs. ('03). WOLBACH, S. B., AND ERNST, HAROLD C. Ob- servations on the morphology of Bacillus tuberculosis from human and bovine sources. Jour, of Med. Research, 1903, vol. x, No. 3, PP- 3I3-333- 13 plates. Also a separate. ('04). THAXTER, ROLAND. Contributions from the cryptogamic laboratory of Harvard Univer- sity, LVI, Notes on the Myxobacteriaceae. Bot. Gaz., June, 1904, vol. xxxvii, pp. 405- 416, with 2 plates. Cystobacter becomes Polyangium. ('04). LEPESCHKIN, W. W. Zur Kenntniss der Erb- lichkeit bei den einzelligen Organismen. Die Verzweigung und Mycelbildung bei einer Bakterie (Bacillus Berestnewi n. sp.). Centralblatt f. Bakt., 2 Abt., 1904, Bd. xii, pp. 641-648, and Bd. xni, pp. 13-22, 20 figs. Describes a branched form. XI. Spores. [See also some papers under X, XV, and XXXIV.] ('76). COHN, FERDINAND. Untersuchungen tiber Bac- terien. Beitrage zur Biologie der Pflanzen, Bd. n, Heft 2, 1876, pp. 249-276. ('84). BUCHNER, H. Ueber das Verhalten der Spalt- pilz-Sporen zu den Anilinfarbstoffen. Aertz- liohe Intelligenzbl., Jahrg. xxxi, 1884, pp. 370. ('87). LEHMANN, K. B. Ueber die Sporenbildung bei Milzbrand. Miinch. med. Wochenschr., 1887, pp. 485-488, No. 26. ('88). PRAZMOWSKI, A. Ueber Sporenbildung bei den Bakterien. Verhand. d. k. k. Akad. d. Wissenschaften in Krakau. Math.-naturw. Sektion, Bd. xvm, 1888, p. 35, I Tafel. Author thinks existence of " arthrospores " is not proved. ('88). GLOBIG. See xxxm and xxxiv. ('88). GRUBER. See xxxm. ('88). BUCHNER, H. Ueber die vermeintlichen Sporen der Typhusbacillen. Centralb. f. Bakt., 1888, Bd. iv, pp. 385-390. ('89). KLEIN, LUDWIG. Ueber einen neuen Typus der Sporenbildung bei den endosporen Bac- terien. Ber. d. deutsoh. Botan. Gesellsch., Bd., vn, 1889, pp. (57)-(72). i plate. Five spore-bearing bacilli are described. The spores are green. In one filamentous-septate species the spores generally occur in pairs, with a septum between, i. e. , the spore is situated, if oue may so speak, in the positive pole of one segment, and in the negative pole of its fel- low. ('91). FISCHER. See XL. ('91). MoELLER, H. Ueber eine neue Methode der Sporenfarbung. Centralb. f. Bakt., Bd. x, 1891, pp. 273-277- The author summarizes his method as follows : " The air-dried cover glass preparation is passed through the flame three times and put into absolute alcohol for two minutes ; then two minutes in chloroform ; then washed with water one-half to two mimites It is subsequently plunged into 5 per cent chromic acid and again thor- oughly washed in water, after which carbol-fuchsin is dropped upon it and warmed over the flame to boiling for 60 seconds. The carbol-fuchsin is then poured off, the cover-glass plunged into 5 percent sulphuric acid until it is bleached, and then thoroughly washed in water. The watery solution of methylen blue or malachite green is then allowed to act upon it for 30 seconds, after which it is washed ff. The spores should then be visible as a dark red in a beautiful green or blue bacterial body." ('91). CRAMER, E. Die Ursache der Resistenz der Sporen gegen trockene Hitze. Arch. f. Hygiene, 1891, Bd. xni, pp. 71-112. Resistance is due to extreme drvness of the spores, and to the fact that all their water is hygroscopic water, which evaporates quickly in dry air, leaving presumably pure water-free albumen. ('92). FOTH. Zur Frage der Sporenfarbung. Cen- tralb. f. Bakt., xi Bd., 1892, pp. 272-278. Does not find Moeller's chromic aciil method universally applicable. Author sometimes substitutes chloriodid zinc. He has also found hydrogen peroxide very useful. He also uses aniliu water fuchsin. A pure, colorless, toluidin free anilin may be had from Jon. Wolff, in Bres- lau. In case oi anthrax spores, the proper time of expos- ure is i% minutes with chromic acid ; 2 to 2% minutes with concentrated chlorzinc iodide solution, and 3 min- utes with hydrogen peroxide. Here also the author says he obtained the best results with H2Oa. ('93). FIOCCA, R. Ueber eine neue Methode der Sporenfarbung. Centralb. f. Bakt., xiv, 1893, No. i, pp. 8-9. Cover-glass preparations are plunged 3 to 15 minutes (mostly 3 to 5 minutes) into steaming hot water contain- ing 10 per cent ammonia water, to which has also been added 10 to 20 drapsof some alcoholic anilin solution. Covers thus treated are then plunged for a moment into water containing 20 per cent sulphuric or nitric acid, and are subsequently exposed to a contrast stain. The stains recommended are gentian violet, fuchsin, methylene blue and safranin ; the contrast stains are vesuvin, chry- soidin, methylene blue, malachit green or safrauin. The preparations are said to be clear and satisfactory. ('93). PHYSALIX. See xxxm. ('94). ERNST, PAUL. Farbungsversuche an Sporen mit Hilfe der Maceration. Centralb. f. Bakt., Bd. xvi, 1894, pp. 182- 184. ('95). MIQUEL. P., AND LATTRAYE, E. De la resis- tance des spores des baoteries aux tempera- tures humides egales et superieures a 100 degrees. Ann. de micrographie. Tome vn, 1895, p. no, 158, 205. Rev. in Centralb. f. Bakt., xix Bd., 1896, pp. 360-362. ('95). BUNGE, R. Ueber Sporenbildung bei Bakter- ien. Fortschr. d. Med., Bd. xni, 1895, No. 20 and 21. Rev. in Centralb. f. Bakt., xvm Bd., 1895, p. 718. SPORES ; FLAGELLA. 219 ('96). SCHREIBER, OSWALD. Ueber die physiologis- chen Bedingungen der endogenen Sporen- bildung bei Bacillus anthracis, subtilis, und tumesccns. Centralb. f. Bakt., xx Bd., 1896, 353-374 and 429-437. Forty-five papers cited at close of this article. ('96). BUCHNER, II. Ueber die physiologischen Be- dingungen der Sporenbildung beim Milz- brandbacillus. Centralb. f. Bakt., xx Bd., 1896, pp. 806-807. ("98). CATTESINA, G. Ricerche suH'intima struttura delle spore dei batteri. Separate from Atti d. Soc. veneto-trentina, vol. in, Fasc. 2, Pa- dova, 1898, 10 pages, with I plate. Rev. in Centralb. f. Bakt., xxvi Bd., 1899, pp. 35-36. Some evidence in favor of existence of a nucleus. ('98). MIGULA, W. Der Keimgehalt und die Wider- standsfahigkeit der Bakterien der animalen Lymphe. Arb. a. d. Bact. Institut der tech. llochschule zu Karlsruhe, u Bd., i Heft, 1898, pp. 65-72. ('98). AUJESZKY, ALADAR. Eine einfache Sporen- farbungsmethode. Centralb. f. Bakt., xxm Bd., 1898, pp. 329-33I- The unfixed covers are placed in a boiling hot % per cent solution of HC1 for 3 to 4 minutes, then washed in water, dried, fixed, and stained with hot carbol fuchsin (three times over flame). Covers are then cooled, bleached in 4 to 5 per centsulphuric acid, and couuterstaiued I to i minutes in malachit green or methylene blue. ('99). STEPHANIDIS, PHILOPIMIN. Ueber den Einftuss des NiihrstoffgehaJtes von Nahrboden auf die Raschheit der Sporenbildung und die Zabl und Resistenz der gebildeten Sporen. Arch. f. Hyg., Bd. xxxv, 1899, PP- r - 10 - Review in Centralb. f. Bakt., xxvi Bd., 1899, p. 568. In a poor substratum anthrax spores were formed more rapidly but in less numbers than in a rich medium. To- ward heat the spores from the rich and poor media be- haved alike. ('99). KI,EIN, ALEX. Eiire einfache Methode zur Sporen fartbung. Cenitralb. f. Bakt., xxv Bd., 1899, PP- 376-379- Klein's modification consists in staining the spores be- fore they have dried. In a watch glass he makes a spore emulsion in phys. salt solution. To this is added an equal volume of filtered carbol fuchsin. This is then gently heated over the open flame for six minutes, i. e. , until steam rises. Covers are now prepared and the bac- terial layer fixed by passing twice through the flame. The covers are then passed through I per cent H2SO4 for 1 to 2 seconds, washed in water and counterstained 3 to 4 minutes in alcoholic methylene blue solution diluted with water. ('99). DANNAPPEI,. See xxxm. Coo). SMITH, R. GREIG. The double staining of spores and bacilli. Proceedings of the Lin- nean Society of New South Wales, 1900, Part 3, June 27, pp. 394-397. Also a separate (issued Nov. 22, 1900). ('02). SCHAUDINN, FRITZ. Beitrage zur Kenntnis der Bakterien und verwandter Organismen. i. Bacillus biitchlii, Arch. f. Protistenkunde, Bd. i, 1902, pp. 306-343, i plate. Bibliography of 24 titles. This very large, slow-moving organism was isolated from the intestinal tract of a cockroach, Periplaneta orientalis. The author states that this organism, like Kern's Di- spora caucasica, is constantly disporous. B. buetchlii was selected for study of its inner structure, on account of Its large size. The membrane did not give the cellulose re- action. Seventy-three figures are (*iven ( illustrating inner structure, stages in the formation of the spores (one in each pole ofthe rod), polar germination of the spores, etc. The organism is 24 to 80 ^ long by 3 to 6 n broad, mostly 50 to 60 (i x 4 to 5 (. The bacillus is flagellate, after the manner of B. subtilis. XII. Flagella. ('38). EHRENBERG. See v. ('72). COHN. See v. ('75). DALLINGER, W. H., AND DRYSDALE, J. J. On the existence of flagella in Bacterium termo. The Monthly Microscopical Journal, Sept. I, 1875, PP- 105-108. ('76). WARMING, EUG. Om nogle ved Danmarks Kyster levende Bakterier. Kjobenhavn, 1876. ('77). KOCH. See LV. ('78). DALUNGER, W. H. On the measurement of the diameter of die flagella of Bacterium termo : a contribution to the question of the "Ultimate limit of vision" with our present lenses. Journ. Roy. Micros. Soc., vol. i, 1878, pp. 169-175, 2 plates. From the mean value of 200 measurements (50 with each of 4 high-power objectives) Dallinger concludes that the diameter of the unstained flagellum of B. termo, in round numbers, is one-two hundred and four thousandth (1-204000) of an inch. This is equal to about one-eighth micron. ('79). VAN TIEGHEM, PHILIPPE EDOUARD LON. Sur les pretendus cils des bacteries. Bull, de la Societe Botanique de France, 1879, T. xxvi, PP- 37-45- Van Tieghem maintained that the flagella were moved from within the body of the bacterium, they themselves being inert gelatinous organs, and not vibratile cilia. ('89). LOEFI'LER, F. Eine neue Methode zum Farben der Mikroorganismen, im besonderen ihrer Wimperhaare und Geisseln. Centralb. f. Bakt., vi Bd., 1889, No. 8-9, pp. 209-224, mit 8 Photogrammen. ('89). TRENKMANN. Die Farbung der Geisseln von Spirillen und Bacillen. Centralb. f. Bakt., vi Bd., Oct. 15, 1889, No. 16-17, pp. 433-436. ('90). MESSEA. See LVI. ('90). LOEFFLER, F. Weitere Untersuchungen fiber die Beizung und Farbung der Geisseln bei den Bakterien. Centralb. f. Bakt., Bd. vn, 1890, pp. 625-639. ('90). TRENKMANN. Die Farbung der Geisseln von Spirillen und Bacillen. n. Mitth. Centralb. f. Bakt., 1890, Bd. vm, No. 13, pp. 385-389. Covers on which the bacterial film is dried without heat are put for 6 to 12 hours In water containing 2 per cent tannin and 0.5 to 0.25 of one per cent hydrochloric acid. They are subsequently washed for one hour in iodine water, and then stained % hour in weak gentian violet anilin water, made as follows : Into a test tube holding 25 cc. put a few drops of concentrated alcoholic solution of gentian violet and add 10 cc. of distilled water. Then pour out about one-half of this and fill up with anilin water. The clear stain is said to color the flagella well on a feebly-stained background. ('91). HUMPHREY, J. E. Notes on Technique, n. Rot. Gazette, 1891, pp. 71-73. Cilia of zoospores of alga; and fungi are stained very readily and sharply "in a drop of moderately strong solu- tion (in qo per cent alcohol) of Hanstein's rosanilliu- violet, composed of equal parts of fuchsin and methyl violet," after first fixing them in a couple of drops of i per cent osmic acid solution. 22O BACTERIA IN RELATION TO PLANT DISEASES. ('92). STRAUSS, I. Sur un precede de coloration a 1'etat vivant des cils ou flagella de certiaines bacteries mobiles. C. R. de la Soc. de biologic, 1892, No. 23, pp. S42-S43. Also Bull. Med., 1892, p. 1,003. ('93). SO.AVO. Di un rapido processo per le colora- zione delle ciglia di alcuni microorganism!. Ministera dell'intern. Laboraitori scientifica della direzione di Santa-Roma. Rev. in Centralb. f. Bakt. Bd. xv. p. 507, 1893. ('93). NICOLLE, M., ET MORAX, V. Technique de la coloration des cils, etc. Ann. de 1'Inst. Pas- teur, T. vii, 1893, No. 7, pp. 554-56i. ('93). MOORE, V. A. On the character of flagella on the Bacillus choleraesuis, B. coli communis, and the B.typhi abdominalis. Wilder Quar- ter Century Book, Ithaca, N. Y., 1893, pp. 339-363. ('93). VAN ERMENGEM, E. Nouvelle methode de coloration des cils des bacteries. Trav. du Lab. d. Hygiene et de Bact. de 1'Univ. de Gand, T. I., 1. 3, 1893- Original not seen. A very good method. Reviewed in Zeitschr. f. Wissensch. Mikr. Bd. xi. 1894, pp. 98-99, and in Ann. de Micrographie, T. V., 1893, pp. 394-395- ('94). HESSERT, W. Geisselfarbung ohne Beize. Centralb. f. Bakt., Bd. xvi, 1894, No. 8-9, PP. 346-347- See also A simple stain for ciliated bacteria. Chicago Med. Recorder, 1894, pp. 240-242. ('94). BUNGE, R. Ueber Geisselfarbung von Bak- terien. Fortschr. d. Medizin, Bd. xn, 1894, No. 12, pp. 462-464. ('94). BUNGE, R. Zur Kenntniss der Geisseltragen- den Bakterien. Ibid., No. 17, pp. 653-670. ('94). BUNGE, R. Weitere Mittheilungen iiber Geis- selfarbung. Fortschr. d. Mediz., Bd. xn, 1894, pp. 929-935- ('95). MOORE, VERANUS A. On ithe nature of the flagella and their value in the systematic classification of the bacteria. Jour. Am. Pub. Health Asso., Oct., 1895, Ann. vol. xx, pp. 432-444, 3 plates. ('95). FERRIER. Considerations generates sur le pleomorphisme des cils viforatiles de quelques bacteries mobiles. Archives de medicine experimentale et d'anatomie pathologique. Paris, Serie I. T. vn, 1895, pp. 58-75- I plate. ('96). LOEWIT, N. Zur Morphologie der Bakterien. Centralb. f. Bakt., xix Bd., 1896, pp. 673- 686, with i plate. ('96). KANTHACK, A. A., AND CONNELL, T. W. The flagella of the tetanus bacillus, and other contributions to the morphology of the tetanus bacillus. Jour. Path, and Bact., iv, 1896-97, pp. 452-459- ('98). BOWHIIJ,, TH. Eine neue Methode der Bak- teriien-Geisselfarbung bei Gebrauch einer Oroeinbedze. Hyg. Rundschau, 1898, No. I. Rev. in Centralb. f. Bakt., xxm Bd., 1898, pp. 667-668. ('98). STEPHENS, J. W. Van Ermengem's method of staining flagella; a modification. The Lan- cet, 1898, Oct. i. Rev. in Centralb. f. Bakt., xxv Bd., 1899, p. 392. Substitutes for nitrate of silver a 2 percent largin solu- tion. The flagella; are said to be cleaner and more dis- tinct. ('99). WELCKE, E. Eine neue Methode der Geissel- farbung. Arch. f. klin. Chir., Bd. ux, 1899, Heft, i, pp. 129-143. Rev. in Centralb. f. Bakt., Bd. xxvi, 1899, pp. 520-521. A silver process of about the same complexity as that of van Ertnengem. ('99). ZETTNOW. Ueber Geisselfanbung bei Bakter- ien. Zeitschr. f. Hyg., Bd. xxx. i Heft., March, 1899, pp. 95-106. Discusses van Ermengem's silver method, and gives a gold method which is said to be better. ('99). MORTON, N. Flagella staining. Trans. Jenner Inst, London, vol. i. 2 series, pp. 242-243, 1899. Tap water is recommended for dilutions. A 24-hour agar surface growth furnishes the bacteria. They are allowed to diffuse in a little of this sterilized water in a watch glass. A drop or two of this is then placed on the clean slide or cover and spread as widely as possible with- out use of the needle. The excess is absorbed by blotting paper. The dried film is not fixed by heat. The stain consists of taunic acid i gram, potash alum i gram, dis- tilled water 40 cc. To this is added 0.5 gram of night blue dissolved in 20 cc. of absolute alcohol. The copious pre- cipitate which results is carefully removed by filtration. The fluid is then ready for use. Stain 2 minutes. The flagella are blue, the body of the organism is not stained. Longer exposures cause precipitates. Couuterstaiu for the body by exposure for i or 2 minutes to anilin-water gentian violet. " I consider that the application of heat and the spreading of the film with the needle are very fatal to good results in flagella staining." ('oo). HINTERBERGER, A. Eine Modifikation des Geisselfarbungsverfahrens nach van Ermen- gem. Centralb. f. Bakt., Bd. xxvn, No. 16-17, I 9. PP- 597-605, I plate and I fig. ('oi). WH.UAMS, HUGH. Flagella stain. See Path- ological technique, by Mallory and Wright. Second Ed., 1901. pp. 104-106. W. B. Saunders & Co., Philadelphia and London. ('02). MEYER, ARTHUR. Kurze Mitteilung iiber die Begeisselung der Bakterien. Centralb. f. Bakt., Abt. i, Bd. xxxi, Originate, 1902, PP- 737-739- XIII. Capsules. ('78). CIENKOWSKI. Untersuohung iiber die Gallert- bildungen des Zuckerriibensaftes. Resume allemand du memoire . uniu*. was originally applied to Gigartina (Eucheuma) isifor- mis, G. spinosa, and G. tenax, which is collected near Singapore, for example, in great masses, and shipped to China. The Chinese use them not only for food but make of them Hal-Thao, a transparent glue, with which they stiffen *ilk and other stuffs, and also fill up the interstices of coarse clothes for the manufacture of lanterns. ('89). PETRI, R. J. Nachtrag zu "Ueber den Gehalt der Nalirgelatdne an Salpetersaure." Cen- tralb. f. Bakt., v Bd., 1889, pp. 679-680. ('90). KUEHNE, W. Kieselsaure als Nahrboden fiir Organismen. Zeitsohr. f. Biol. Neae Folge, Bd. ix, Der ganzen Reihe, Bd. xxvn, 1890, pp. 172-179. ('90). TISCHUTKIN, N. Eine vereinfachte Methode der Bereitung von Fleisch-Pepton-Agar. Wratsch., 1890, No. 8, pp. 177-178. Reviewed in Centralb. f. Bakt., Bd. ix, p. 208, 1891. The crude agar-agar is first exposed for 15 minutes in acetic-acid water (5 glacial acid, 100 water). ('91). SCHULTZ, N. K. Zur Frage von der Bereitung einiger Nahrsubstrate. Centralb. f. Bakt., x Bd., 1891, pp. 52-64. ('91). SLESKIN, P. Die Kieselsaure Gallerte als Nahrsubstrat. Centralb. f. Bakt., Bd. x, 1891, pp. 209-213. ('91). MARPMANN. Mittheilungen aus der Praxis. i, Ersatz fiir Agar; 2, Ersatz fiir Gelatine. Centralb. f. Bakt., x Bd., 1891, pp. 122-124. ('92). PETRI, R., UND MAASZEN, ALBERT. Ueber die Bereitung von Nahrbouillon fiir bakteriolo- gische Zwecke. Arbeiten aus d. kaiserl. Gesundheitsamte, Bd. vm, No. 2, 1892, pp. 3II-3I4. ('92). LOEW, O. Ueber einen Bacillus, welcher Ameisensaure und Formaldehyd assimiliren kann. Centralb. f. Bakt., xn Bd., 1892, pp. 462-465. ('92). DE LAGERHEIM, G. Macaroni als fester Nahr- boden. Centralb. f. Bakt., xi Bd., 1892, pp. 147-148- Author states that cultures of chromogenic bacteria on macaroni stand out from the background very distinctly and are very instructive. The whitest macaroni should be selected. It is cut into pieces 4.5 cm. long. These are put into test tubes and covered i cm. over with water and cooked for 15 minutes. The water is then carefully poured off and the media sterilized in streaming steam in the usual way. ('92). SEILER, F. Influence de la composition de la gelatine nutritive sur le developpement des colonies microbiennes. Schweizerische Wochenschr. f. Chemie u. Pharm., 1892, pp. 261-263. ('92). SCHUTZ, J. Q. A rapid method of making nutrient agar. Bull. Johns Hopkins Hos- pital,vol. in, July-Augusit, 1892, p. 92. A useful and easy method. ('93). USCHINSKY. Ueber eine eiweissfreie Nahr- losung fur pathogene Bakterien, nebst einigen Bemerkungen fiber Tetanusgift. 'Centralb. f. Bakt., Bd. xiv, 1893, No. 10, pp. 316-319- ('93). NASTIUKOFF. Ueber Nahrboden aus Eigelb fur Bakterienkulturen. Wratsch., 1893, No. 33 and 34. Rev. in Centralb. f. Bakt., xvn Bd., 1895, pp. 492-493- ('93). HESSE, W. Ueber den Einfluss der Alkalescenz des Nahrbodens auf das Wachsthum der Bakterien. Zeitschr. f. Hygiene, Bd. xv, 1893, pp. 183-191- 3 plates. ('94). FRAENKEL, C. Beitrage zur Kenntniss des Bakterienwachsthums auf eiweissfreien Nahrlosungen. Hyg. Rumlschau, Jahrg. iv, 1894, pp. 769-776. CULTURE-MEDIA. 225 ('94). WESCNER, F. Die Bereitung eines festen un- durchsiditigcn Nahrbodens fur Bakterien aus Hiihnerciorn. Centralb. f. allg. Path, u. path. Anat., Bd. v, 1894, pp. 57-59. Eggs are shaken until the yolks and whites are thor- oughly mixed. They are then boiled hard. The shell is now removed, the egg cut into suitable pieces, put into test tubes and treated exactly like potato cylinders. This media is said to give very characteristic growths with many bacteria. ('95). TuRRO, R. Ueber Stroptokokkenziichtung auf sauren Na'hrboden. Centralb. f. Bakt, Bd. xvir, i Abt., 1895, pp. 865-874. ('95). SEDGWICK AND PRESCOTT. See XLVI. ('95). ELSNER. Untersuchungen iibar eleotives Wachsthum der Bacterium coli-Arten und des Typhusbacillus und dessen diagnostiche Verwerthbarkeit. Zeitschr. f. Hyg., Bd. xxi, 1895, pp. 25-31. ("95). SMITH, THEOBALD. Ueber die Bedeutung des Zuckers in Kulturmedien fur Bakterien. Centralb. f. Bakt., xvin Bd., 1895, pp. 1-9. ('95). BLEISCH, MAX. See xvn. ('95). HAEGLER, CARL S. Zur Agarbereitung. Cen- tralb. f. Bakt., xvn Bd., 189=:, pp. 558-560, with 2 figs. Advises centrifuging to clear the agar. ('95). DEYCKE, G. Die Benutzung von Alkalial- buminaten zur Herstellung von Nahrboden. Centralb. f. Bakt., xvn Bd., 1895, pp. 241- 24S. C9S)- FULLER, GEO. W. On the proper reaction of nutrient media for bacterial cultivation. Jour. Am. Pub. Health Asso., Oct., 1895, vol xx. A very useful paper. It is recommended for general reading. ('95). HEIM, L. Zur Bereitungsweise von Nahr- mitteln. Centralb. f. Bakt., xvn Bd., 1895, pp. 190-195, i phot. ('95). ZUPNIK, LEO. Zur Agarbereitung. Centralb. f. Bakt., xvin Bd., 1895, p. 202. Review in Bot Centralb., Bd. LXV, 1896, p. 52. Author obtains clear agar by filtering it through a thin layer of absorbent cotton, placed funnel-form in the hot water filter, wet with hot distilled water and pressed in place with the fingers. The bouillon must be clear to start with. The flgar powder is then added and cooked i hour in streaming steam. ('95). MAASSEN, ALBERT. Die organischen Sauren als Nahrstoffe und ihre Zersetzbarkeit durch die Bakterien. Arb. a. d. kaiserl. Gesundheitsamte, Bd. xu, Zweites Heft, 1895, PP- 340-411- ('96). CAPALDI, ACHILLE. Zur Verwendung des Eidotters als Nahrbodenzusatz. Cenitralb. f. Bakt., xx Bd., 1896, pp. 800-803. ('97). HESSE. See XLVI. ('97). MARPMANN. See XLIX. ('97). FORSTER, J. Niihrgelatine mit hohem Schmelz- punkte. Centralb. f. Bakt., xxn Bd., 1897, pp. 341-343. By careful minimizing of heat the author obtains sterile nutrient gelatin melting at 290-30 C. ('97). STODDART, F. WALLIS. New method of sepa- rating the typhoid bacillus from the bacillus coli communis, with notes on some tests for the typhoid bacillus in pure cultures. Uni- versity College, Bristol. The Jour, of Path- ology and Bacteriology, iv, 1896-97, p. 429. ('97). BOKORNY, TH. Grenze der wirksamen Ver- diiniiung von Nahrstoffen bei Algen und Pilzen. Bio. Centralb. June 15, 1897, pp. 417-426. Author states that 0.002 per cent peptone serves no longer as a nutrient for bacteria, but o.oio per cent does. In mineral solutions, monopotassium phosphate, mag- nesium sulphate, and calcium nitrate, a dilution to o.ooi per cent still nourishes algae, but not bacteria ; with 0.005 per cent solutions, bacteria appeared. ('97). LONDON, E. S. Schnelte und leichte Methode zur Bereitung des Nahragars. Centralb. f. Bakt., xxi Bd., 1897, pp. 686-687. ('97). Hiss, PHILIP HANSON. On a method of isolat- ing and identifying bacillus ityphosus, based on a study of bacillus typhosus and members of the colon group in semi-solid culture media. Jour. Exp. Med., vol. u, 1897, pp. 677-700. ('98). SMITH, ERWIN F. Potato as a culture-medium with some notes on a synthesized substitute. Proc. Am. Asso. Adv. Sci., Vol. XLVII, 1898, p. 411. Also a separate. Centralb. f. Bakt., 2 Abt., Bd. v, p. 102. ('98). BOKORNY, TH. Sources of Carbon for Bac- teria. See table by Bokorny in his Lehrbuch der Pflanzenphysiologie, pp. 56-59. Paul Parey, Berlin, 1898. ('98). GIESENHAGEN, K. Eine Vorrichtung zum Filtrieren von Nahragar. Centralb. f. Bakt., xxiv Bd., 1898, pp. 501-502. ('99). YOKOTE, T. Ueber die Darstellung von Nahragar. Centralb. f. Bakt., xxv Bd., 1899, PP- 379-38o. Author heats his filtered bouillon, to which agar has been added, for i hour on a sand bath, after which it filters readily if the sand temperature has been above 110 C. It will not filter satisfactorily if lower temperatures are used. ('99). BLIESENER. Ueber Gelatinekulturen im Briit- schrank. Zeitsohr. f. Hyg., Bd. xxxii, Heft i, 1899. Rev. in Centralb. f. Bakt., xxvn Bd., 1900, pp. 472-473- Author prepares a gelatin which remains solid at 27-30 C. ('99). CESARIS-DEMEL. Udber das verschiedene Verbal/ten einige Mikroorganismen in einem gefarbten Nahr-Mittel. Centralb. f. Bakt., xxvi Bd., 1899, pp. 529-540, with 2 plates. The medium recommended is liver broth with tincture of litmus. This serves, it is said, to differentiate certain species. (.00). GLAESSNER, PAUL. Ueber die Verwertbarkeit einiger neuer Eiweisspraparate zu Kultur- zwecken. Centralb. f. Bakt., XXVH Bd., 1900, pp. 724-732. Comparative tests of somatose, nutrose, etc. ('02). YENDO, K. Uses of marine Algae in Japan. Postelsia, Sit. Paul, Minn., 1902, pp. 3-18. 3 plates and 3 Japanese prints. Gelidium corneum (Japanese Ten-gusa) furnishes the agar-agar of commerce. ('02). WHIPPLE, GEORGE C. On the physical prop- erties of gelatin, with reference to its use in culture media. Technology Quarterly, Boston, Mass., vol. xv, pp. 127-160. Also a separate. ('04). GAGE, STEPHEN DE M., AND ADAMS, GEORGE O. Studies of media for the quantitative estima- tion of bacteria in water and sewage. Jour, of Infectious Diseases, vol. i, No. 2, 1904, PP- 358-377. Also a separate. 226 BACTERIA IN RELATION TO PLANT DISEASES. ('04). HESSE, GUSTAV. Beitrage zur Herstellung von Nahrboden und zur Bakterienziichitung. Ztschr. f. Hyg., 1904, Bd. XLVI, pp. 1-22. Discusses effect of adding various acids and alkalies, effect of heat on reaction, use of insoluble glass, etc. Most of the methods advised are already in use in many laboratories in the United States. XVII. Methods or Work, Apparatus, Etc. ('73). KLEBS, E. Beitraege zur Kenntniss der Micro- coccen. Arch. f. exp. Path. u. Plharmakol., 1873, Bd. i, pp. 31-64, with 4 plates. Klebs here outlines his fractional method of culture. See especially pp. 46-47. ('77). KOCH. See LV. ('78). LISTER, JOSEPH. On the lactic fermentation and its bearings on pathology. Trans. Path. Soc. of London, 1878, vol. xxix, pp. 425-467. Lister's dilution method for obtaining pure cultures is here described. See especially page 445 et seq. ('81). KOCH, ROBERT. Zur Untersuchung von path- ogenen Organismen. Mittheil. a. d. kaiser- lichen Gesundheitsamte, Bd. I, 1881. Ber- lin, pp. 1-48, 14 plates. In this paper Koch outlined his now universally used method of obtaining pure cultures (colonies) on solid was assumed that the quantity used to inoculate each flask contained only one organism (p. 19). ('83). KOCH, ROBERT. Ueber die Milzbrandimpfung, eine Entgegnung auf den von Pasteur in Genf gehaltenen Vortrag. Kassel und Ber- lin, Theodor Fischer, 1882. The same in French. Theo. Fischer, 1883. The celebrated Koch " rules of procedure " are given in this paper. ('84). HOFFMANN, F. W. Einfacher Einbettungs- apparat. Zoologisoher Anzeiger, vii Jahrg., 1884, No. 157-184, Leipzig, pp. 230-232, I fig. Makes use of a vacuum to hasten the infiltration of paraffin. ('84). ERRERA. Sur 1'emploi de 1'encre de phine en Microscopic. Bull. Soc. Beige d. Microscop., 1884. Not seen. The ink is used to form a contrasting back- ground. ('86). VON ESMARCH, ERWiN. Ueber eine Modifika- ition des Koch'sohen Plattenverfahrens zur Isolierung und zum quantitativen Nachweis von Mikroorganismen. Zeiitsoh. f. Hygiene, 1886, Bd. i, pp. 293-301, 3 figs. The method of roll cultures is here described. ('87). PETRI, R. J. Eine kleine Modification e differentiated in this way. ('88). BUJVVID, O. Neue Methode znm Diagnos- ticiren nnd Isoliren der Choierabakterien Centralb. f. Bakt., 1888, iv Bd., pp. 494-496. 11124 hours, in 2 per cent feebly alkaline peptone solu- tion at 37 C., the cholera organism gives a fine purple-red color on adding HC1. On longer cultivation other organ- isms give the same reaction. This depends on the forma- tion of indol and a trace of nitrite. ('90). PETRUSCHKY, JOHANNES. Die Farbenreaktion bakterieller Stoffwechselprodukte auf Lack- mus als Beitrag zur Charakteristik und als Mittel zur Unterscheidung von Bakterien- arten. Centralb. f. Bakt., vn Bd., 1890, pp. 1-8 and 49-53. ('92). Bujwip, ODO. Eine neue biologische Reaktion fur die Choierabakterien. CentraJb. f Bakt xn Bd, 1892, pp. 59S-S96. ('92). BEYERINCK, M. W. Notiz fiber die Cholera- rothreaktion. Centralb. f. Bakt, xn Bd 1892, pp. 715-718. ('93). SCHILD. Formalin zur Diagnose des Typhus- bacillus. Centralb. f. Bakt, Bd. xiv, 1893, pp. 717-718. The typhoid organism will not grow in bouillon con- taining as little formalin as 1:15,000. B. coli develops vigorously with as much formalin as 1:3000. ('93). MATHEWS, ALBERT P. On Wurtz's Method for the Differentiation of Bacillus typhi abdominalis from Bacillus coli communis, and its application to the examination of contaminated drinking water. Technology Quarterly, vol. vi, 1893, pp. 241-251. Litmus lactose gelatin or agar is reddened by B. coli and is unchanged or made deeper blue by B. typhosus. ('93). GORINI, KONSTANTIN. Anmerkung iiber die Cholerarotreaktion. Centralb. f. Bakt, Bd xni, 1893, pp. 790-792. A good peptone for this purpose must be white, without odor, entirely soluble in water, especially on warming The water solution must be clear, colorless, neutral or slightly alkaline, foamy on shaking. It must give a vio- let reaction with Fehling's solution, which does not change on boiling. It must give no nitrite reaction with Gnesz reagent, and finally must give, after about 5 min- utes with Diphenylamine, a faint but distinct narrow clear blue ring. ('94)- SCHNEIDER, PAUL. Die Bedeutung der Bak- terien farbstoffe fiir die Unterscheidung der Arten. (Inaug. Diss.) 8vo, 46 pp, 2 Taf, Basel, 1894. Rev. in Centralb. f. Bakt, Bd xvi, 1894, p. 633. See also Arbeiten a. d. bakt. Institut Karlsruhe I, 1894. ('94)- MARPMANN. Zur Unterscheidung des Bacillus typhi abdominalis vom Bacillus coli com- mune. Centralb. 1 Bakt, Bd. xvi, 1894, pp. 817-820. the first two. ('94). LUNKEWICZ, M. Eine Farbenreaktion auf die salpetrige Saure der Kulturen der Cholera- bacillen und einiger anderer Bakterien Centralb. f. Bakt, Bd. xvi, 1894, pp. 945-949. Describes the preparations of the Griess-Ilosvav re- agent. ('94). ABEL, RUDOLF. Ueber die Brauchbarkeit der von Schild angegebenen Formalinprobe zur Differential-Diagnose des Typhus bacillus Centralb. f. Bakt, Bd. xvi, 1894, PP. 1,041- 1,046. Finds some bacteria more tolerant of formalin than was stated by Schild. It is not a satisfactory method lor separating typhoid bacilli from B. coli 230 BACTERIA IN RELATION TO PLANT DISEASES. ('95). MACKENSIE, J. J. Opening discussion on "What new methods can be suggested for the separation of bacteria into groups, and for the identification of species." Jour. Am. Pub. Health Asso., Oct., 1895, Ann. vol. xx, pp. 419-431- Notes on synthetic media. ('97). STODDART. See xvi. ('97). Hiss. 'See xvi. ('98). HOUSTON, A. C. Note on four micro-organ- isms isolated from the mud of the river Thames, which resemble Bacillus typhosus. Centralb. f. Bakt., xxiv Bd., 1898, pp. 518- 525, colored diagram. ('98). FERMI. See xxxvi. ('98). ROTHBERGER, C. JULIUS. Differential diagnos- tische Untersuchungen mit gefarbten Nahr- boden. Centralb. f. Bakt., xxiv Bd., 1898, pp. 5I3-SI8. With neutral red (Toluidin red) in agar B. coli is said to cause a clearing of the color and a very decided fluor- escence. B. typhi leaves the medium unchanged. The best method is said to be to add 3 to 4 drops of a concen- trated water solution to lo cc. of fluid agar, and then % cc. of a 24-hour old bouillon culture. In agar stained with safranin, B. coli reduces the color ; B. typhi does not. Dead cultures of B. coli (cultures heated 2 hours at 78 C.) did not produce these changes. ('98). PACINOTTI, G., AND MUNIECKI, J. L'albume d'uovo colorito in verde-cupo dal caffe crudo, come mezzo diagnostico di svlluppi ibatterici. Gazz. degli ospedali e della dim'che, 1808, No. 31. Rev. in Centralb. f. Bakt., xxv Bd., 1899, p. 257. ('99.) ROTHBERGER, C. JULIUS. Differential diagnos- tische Untersuchungen mit gefarbten Nahr- boden. 11, Mittei-lung. Centralb. f. Bakt., xxv Bd., 1899, pp. 15-17 and 69-75. Author tested the effect of various bacteria on 35 anilin dves 13 of which proved useful in agar media. The loss of color in methylen blue, safranin, Toluidin blue, Or- seille extract and indigo carmin is due to reduction pro- cesses. In case of the indigo carmin, the blue was first changed to dark green. ('oo). SCHEFFLER, W. Das Neutrakot als Hilfsmittel zur Diagnose des Bacterium coli. Centralb. f. Bakt., xxvin Bd., 1900, pp. 199-205. Author says B. coli in neutral red, grape sugar agar gives regularly in 24 to 48 hours a beautiful green fluor- escence. He uses: fluid agar, loo cc., grape sugar, 0.3 gram ; concentrated watery solution of neutral red, ice. ('oi). HOELSCHER, WALTER. Ueber die Differenz der histologischen Wirkung von Tuberkel- bacillen und anderen diesen ahnlichen saurefesten Bacillen (Grasbacillus 1 1 Moel- ler, Butterbacillus Petri-Rabinowitsch, Thimotheebacillus Moller). Munchener med. Wochenschr., Bd. XLVIII, 1901, pp. 1,483-1,484- ('02). Hiss, PHILLIP HANSON, JR. New and simple media for the differentiation of the colonies of typhoid, colon, and allied bacilli. The Journal of Medical Research, June, 1902, vol. vin, pp. 148-167. Also a separate. 2 plates. Author describes a simple solid medium in which buried colonies of the typhoid bacillus send out thread- formed radiations, while those of B. coli, etc., do not bear any fringing threads. This medium, used in Petri- dish poured plates, consists of distilled water 1,000, agar 15, gelatin 15, dextrose 10, Liebig's extract meat 5, sodium chloride 5. ("02). FITZ GERALD, MABEL PUREFOY, AND DREYER, GEORGES. The unreliability of the neutral red method, as generally employed for the differentiation of B. typhosus and B coli. Festsksrift ved Indvielsen af Statens Serum- institut. Kjobenhavn, 1902, pp. 1-39. ('02). BUXTON, B. H. A comparative study of the bacilli intermediate between B. coli coni- inunis and B. typhosus. Jour. Med. Res. in (n. s.), pp. 201-230, 3 plates. Bibliography of 16 titles. ("03). GAGE, STEPHEN DEM., AND PHELPS, EARLE B. Notes on B. coli and allied forms, with special reference to the neutral-red 'reaction. Proceedings of the Thirtieth Annual Meet- ing, American Public Health Assn., New Orleans, La., Dec., 1002. Pub. Columbus, Ohio, 1903, vol xxvin, pp. 402-412. Also a separate, pp. 11. ('03). SCHUEDER. Zum Nachweis der Typhusbak- terien im Wasser. Zeitschr. f. Hyg., Bd. XLII, 1003, PP- 317-326. Describes differential methods. ('04). STOKES, WILLIAM ROYAL. A simple test for the routine detection of the colon bacillus in drinking water. Jour, of Infectious Dis- eases, vol. i, 1904, pp. 341-347. i plate. Neutral red reaction. XIX. Aerobism, Anaerobism. (See also various citations under XX.) ('61). PASTEUR, Louis. Animalcules infuspires vivant sans gaz oxygene libre et determinant des fermentation's. C. R. des se. de 1'Acad. des sci., Paris, 1861, T. LII, pp. 344-347- ('63). PASTEUR, Louis. Nouvel exemple de fermen- tation determinee par des animalcules in- fusoires pouvant vivre sans oxygene libre, et en dehors de tout contact avec 1'air de I'atmosphere. C. R., des se. -de 1'Acad. des sci., Paris, 1863, T. LVI, pp. 416-421. ('63). PASTEUR, Louis. Recherches sur la putrefac- tion. C. R. des se. de 1'Acad. des sci., Paris, 1863, T. LVI, pp. 1,189-1,194. " Je propose avec toute sorte de scrupules ces mots nouveaux aerobies et anaerobes pour indiquer 1'existence de deux classes d'6tres inferieures, les uns incapable de vivre en dehors de la presence du gaz oxygene libre, les autres pouvant se multiplier a 1'infini en dehors du con- tact de ce gaz.'' ('80). BUCHNER. Ueber die Lebensfahigkeit der Spailtpilze bei fehlenden Sauerstoff, 1880. Not seen. ('85). HESSE, W. UND R. Ueber Zuchtung der Bacillen des malignen Oedems. Deutsche med. Wochenschrift, 1885, 11 Jahrg., pp. 214-215. Describes a method of cultivating anaerobic organisms by sowing them in deep masses of solid media. ('86). LIBORIUS, PAUL. Beitrage zur Kentniss d. Sauerstoffbediirfnisses der Bakterien. Zeitschr. f. Hyg., Bd. i, 1886, pp. H5-I77- According to review by Zimmerman in Hot. Centralb. Bd. xxvin, 1886, p. 198, author found but little more oxy- gen in layers of agar buried 3 cm. and over under addi- tional agar than in vessels in which air was expelled by hydrogen, etc. He used " Indigotinloesung" as a test. AEROBISM, ANAEROBISM. 231 ('87). Roux, E. Sur la culture des microbes anaerobies. Ann. de I'lnst. Pasteur, Bd. I, 1887, pp. 49-62. ('88). BUCHNER, HANS. Eine neue Methode zur Kultur anaerober Mikroorganismen. Cen- tralb. f. Bakrt., 1888, iv Bd., pp. 149-151. ('88). FRAENKEL, CARL. Ucber die Kultur anaerober Mikroorganismen. Centralb. f. Bakt., 1888, in Bd., pp. 73S-740 and 763-768. I fig. ('89). FRAENKEL. See xxxvm. ('89). FRANKLAND, PERCY. On the influence of car- bonic anhydride or other gases on the de- velopment of micro-organisms. Proc. Roy. Soc., vol. XLV, 1889, pp. 292-301. See also Zeitsch. f. Hygiene, Bd. vi, 1889, pp. 13-22. The other gases tested were hydrogen, carbonic oxide, and nitrons oxide. Hydrogen had the least deleterious effect on the organisms tested, which were B. pyocy- aueus, Koch's comma bacillus, and Finkler's spirillum. Coo). SMITH. See xx. fyo). POPOFP. See XLvnr. ('92). OGATA, M. Einfache Bakterienkultur mit versohiedenen Gasen. Centralb. f. Bakt., xi Bd., 1892, pp. 621-623, mit i fig. Ogata's method, which is essentially that previously described by Heim , consists in softening the upper part of a test tube in the flame and drawing it out so that the tube consists of two normal portions connected by a nar- row isthmus. This part should be just below the colton plug, that is, in the upper one-third of the tube. A piece of glass tubing is now plugged at one end with sterile cotton, and is drawn out into a capillary tube, which must be long enough to reach down into the bottom of the culture medium. The broken end of this tube is now touched to the desired culture and passed into the test tube. The cotton-plugged end of the glass tube is now attached to a rubber tube connected with the gas appa- ratus. After the gas has bubbled through the medium for a sufficient time, the capillary tube is removed, ami the test tube is immediately sealed in the open flame by a further constriction of the Isthmus and a complete removal of the upper part of the test tube. Fluid cultures may also be made in such tubes by pre- paring the isthmus before the tubes are filled with the medium. ('92). HEIM, L. Zur Originalmittheilung von Ogata: "Einfache Bakterienkultur .mat verschiedenen Gasen." Bd. xi, p. 621. Cen- 'tralib. f. Bakt., xi, 1892, p. 800. ('92). VAN SENUS, A. H. C. Zur Kenntniss der Kultur anaerober Bakterien. Centralb. f. Bakt., xii Bd., 1892, pp. 144-145. CP3)- Now, F. G. Die Kultur anaerober Bakterien. Centralb. f. Bakt, Bd. xiv, 1893, pp. 581- 600, 2 figs. The "Novyjars" arc here figured and described, and 49 references to literature are given at the end of the article. ('93). BKYKRINCK, M. W. Ueber Atmungsfiguren beweglicher Bakterien. Centralb. f. Bakt., Bd. xiv, 1893, pp. 827-845, mit I Tafel. ('93)- SMITH. See xx. ('94). NICOLAIER. Bemerkung zu der Arbeit von Prof. F. G. Novy: "Die Kultur anaerober Bakterien" (Centralb. f. Bakt., Bd. xiv, 1893). Centralb. f. Bakt., Bd. xv, 1894, p. 227. (94). SMITH, THEOBALD. Further observations on the fermentation tube, with special reference to anaerobiosis, reduction, and gas produc- tion (abstract). Proc. Am. Ass. A. Sci., 42, Madison Meeting, 1893, Salem, 1894, p. 261. ('94). ENGELMANN, TH. W. Die Erscheinungsweise der Sauerstoffausscheidung chromophyllhal- tiger Zellen im Licht bei Anwendung der Baoterienmethode. Verhand. d. Kon. Akad. van Wetenschappen te Amsterdam (2 te sectie, deel in; No. 11, 1894), pp. 10, iv, with a folded plate, also a reprint, 17 pp. Gives a bibliography of 61 titles. See also Onderzoekingen Physiol. Laborat. Utrecht, iv Reeks, in deel., 1895. ('94). LUBINSKI, WSEWOLOD. Zur Methodik der Kultur anaerober Bakterien. Centralb. f. Bakt., Bd. xvi, 1894, pp. 20-25, mit 4 figs. ('94). NOVY, F. G. Die Plattenkultur anaerober Bakterien. Centralb. f. Bakt., Bd. xvi, 1894, pp. 566-571, with 3 figs. ('94). ARENS. Eine Methode zur Plattenkultur der Anaeroben. Centralb. f. Bakt., Bd. xv, 1894, pp. 15-17. The author makes his cultures in ordinary exsiccators having ground glass covers. ('95). SCHMIDT, AD. Eine einfache Methode zur Zitchtung anaerober Kulturen in fliissigen Nahrboden. Centralb. f. Bakt., xvii Bd., !895, pp. 460-461, i fig. ('95). BRAATZ, EGBERT. Einiges iiber die Anaero- biose. Centralb. f. Bakt., xvn Bd., 1895, pp 737-742, with i fig. ('95). KEDROWSKI, W. Ueber die Bedingungen, unter iwelchen anaerobe Bakterien auoh bei Gegcnwart von Sauerstoff existiren konnen Zeitsohr. f. Hyg., Bd. xx, 1895, pp. 358-375- They do this best when mixed with aerobes, but the absorption of the oxygen is not so important as Pas- teur supposed. The author believes that the aerobes ex- crete some special substance favorable to the growth of the anaerobes. This substance he did not determine. ('95). GERSTNER, R. Beitrage zur Kentniss obligat- anae'rober Bakterienarten. Arbeiten aus dem Bakteriologisohen Institut der Tech- nisohen Hochschule zu Carlsruhe, Bd i 1895. H. 2, pp. 148-183, with 2 Taf. ('96). KASPAREK, THEODOR. Bin einfacher Luftab- schluss flussiger Nahrboden beim Kulti- vieren anaerober Bakterien. Centralb. I. Bakt., xx Bd., 1896, pp. 536-537, 2 figs. ('96). DURHAM, HERBERT E. On a self-acting means of cultivating anaerobic microbes. Jour. Path, and Baot., vol. in, 1896, pp. 231-236. ('97). BECK, M. Zur Ziichtung anaerober Kulturen. Centralb. f. Bakt., xxn Bd., 1897, pp. 343- 345, with 2 figs. ('98). SCHOLTZ, W. Ueber das Wachstum anaerober Bakterien bei ungehindertem Luftzutritt. Zeitschr. f. Hyg., Bd. xxvii, 1898, pp. 132- 142. Review in Centralb. f. Bakt., xxrv Bd., 1898, p. 932. ('98). FERRAN, J. Ueber das aerobische Verhalten des Tetanusbacillus. Centralb. f. Bakt., xxrv Bd., 1898, pp. 28-29. Thinks his experiments show tetanus bacillus is not a strict anaerobe, but only a facultative anaerobe. 232 BACTERIA IN RELATION TO PLANT DISEASES. ie reviewer has not had that trouble with Novy's ap- itus complained of by Mr. Klein. The authors , r+. ... : ,. ; ,, , r thf> ru*irn*n water WltU ('98). KLEIN, ALEX. Ein Apparat zur bequemcn Herstellung von anaeroben Plattenkulturen. Centradb. f. Bakt., xxiv Bd., 1898, pp. 967- 971, 2 figs. The rev paratus compiameu ui uy xi. .^.... *- - siphon arrangement for mixing the potash water with the pyrogallol after exhaustion of the air appears to be very good . ('98) FESRAN, J. Ueber die Verwendung des Ace- tylens foei der Kultur anaerober Baktenen. Centralb .f. Bakt., xxiv Bd., 1898, p. 29. ('98) ZUPNIK, LEO. Ueber eine neue Methode anaerober Zudhtung. Centralb. f. Bakt., xxiv Bd., 1898, pp. 267-270, with i fig. fq8) OPRESCU Zur Technik der Anaerobenkultur. Hyg. Rundschau, 1898, No. 2. Rev. in Cen- tralb. f. Bakt., xxm Bd., 1898, p. 668. ('98) UCKE, ALEXANDER. Ein Beitrag zur Kenntnis der Anaeroben. Centralb. f. Bakt., xxm Bd., 1898, pp. 996-1,001. Co8) TRENKMANN. Das Wachstum der anaeroben Bakterien. Centralb. 'f. Bakt., xxm Bd., 1898, pp. 1,038-1,043 and 1,087-1,090. ('08). MARPMANN. Eine neue Methode zur Her- stellung von anaeroben Rollglasku.lturen out Gelatine oder Agar. Centralb. f. Bakt., xxm Bd., 1808, pp. 1,090-1,091. ('98). EPSTEIN, STANISLAUS. Apparat zur Ktiltur anaerober Bakterien. Centralb. f. Bakt., xxiv Bd., 1898, pp. 266-267, i fig. , ('99). KABRHEL, GUSTAV. Zur Frage der Zuchtung anaerober Bakterien. Centralb. f. Bakt., xxv Bd., 1899, pp. 55S-S6i, with I fig. As an oxygen indicator author uses methylene blue in sugar gelatin. ('99). SMITH, THEOBALD. Some devices for the cul- tivation of anaerobic bacteria in fluid media without the use of inert gases. Jour. Bost. Soc. Med. Sci., 1899, PP- 340-343- Also a separate, 4 pp. Coo) EPSTEIN, STANISLAUS. Em veremfachtes Ver- fahren zur Ziichtung anaerober Bakterien in Doppelschalen. Centralb. f. Bakt., xxvm Bd., 1900, p. 443, with i fig. Coo). PETRI. See xvii. Coo). KRAUSE. See xv. Coo). BULLOCK. See xvn. Coo). WRIGHT. See xvn. ('02) OMELIANSKI, W. Ein einfacher Apparat zur Kultur von Anaeroben im Reagenzglase. Centralb. f. Bakt., Abt. 2, Bd. vra, 1902, pp. XX. Fermentations, Gas-Formation, Enzymes, Etc. (See also XIX and XLVIII.) ('57). PASTEUR, L. Memoire sur le fermentation appelee lactique. C. R. des se. de 1'Acad. des sci., T. XLV, 1857, pp. 913-916. ('77). VINES, S. H. On the digestive ferment of Nepenthes. Journ. Linn. Soc. (Bot), vol. xv, 1877, pp. 427-43L ('79). v. NAEGELI, CARL WILHELM. Theone der Garung. Ein Beitrag zur Molecularphysiol- ogie. Miinchen, 1879, pp. iv, 156. ('79). PRAZMOWSKI, A. Zur Entwicklungsgeschichte und Fermentwirkung einiger. Bacterien- Arten. Vorlaufige Mittheilung. Bot. Zeit- ung, Bd. xxxvn, No. 26, col. 409-424, 1879 ('79). VAN TiEGHEM, P. E. L. Sur la fermentation de la cellulose. Bull, de la Societe Bot. de France, 1879, T. xxvi, pp. 25-30. ('82). MAYER, ADOLF. Die Lehre von den chetnis- chen Fermenten oder Enzymologie. pp. vi, 124. Heidelberg, 1882. ('82). BECHAMP, A. Sur les microzymas cornine cause de la decomposition de 1'eau oxygenee par les tissus des animaux et des vegetaux. C. R. des se. de 1'Acad. des sci., Paris, T. xciv, pp. 1,653-1,656. ('82). BERT, B., ET REGNARD, P. Action de 1'eau oxygenee sur les matieres organiques et les fermentations. C. R. des se. de 1'Acad. des sci., Paris, T. xciv, pp. 1,383-1,386. Jour, de Pharm. et de Chimie, T. vi, 5 serie, 1882, pp. 14-17. ('82). WORTMANN, J. Untersuchuii'gen uber das diastatische Ferment der Bacterien. Zeitschr. f. physiol. chem., Bd. vi, pp. 287-329, 1882. Also Jour. Chem. Soc., XLIV, pp. 930-938, 1883. ('82). MARCANO, V. Fermentation directe de la fecule. Mccanisme de cette metamorphose. C. R. des se. de 1'Acad. des Sci., Paris. T. xcv, 1882, pp. 856-859. ('83). BECHAMP, A. Les microzymas dans leurs rap- ports avec rheterogenie, I'histogenie, la pliysiologie et la pathologic, pp. xxxvni, 992, with 3 pi. Paris, 1883. Prior to 1883 Bechnmp isolated from yeast an enzyme capable of inverting cane sugar. See pp. 69 et seq. ('83). VIGNA, A. Ueber Bakteriengahrung des Glycerins. Ber. d. deutsch. chem. Gesells., xvi, pp. 1,438-1,439; Gazz. Ghim. Ital., vol. xm, pp. 293-296, 1883. ('83). TAPPEINER, H. Ueber Cellulosegahrungen. Ber. d. deutsch. chem. Gesells., Bd. xvi, 1883, pp. 1,734-1,740. ('83). TAPPEINER, H. Ueber die Sumpfgasgahrun im Schlamme der Teiche, Siimpfe und Kloaken. Ber. d. deutsoh. chem. Gesells., Bd. xvi, 1883, pp. 1,740-1,744. ('86). HOPPE-SEYLER, FELIX. Ueber Gahrung der Cellulose mit Bildung von Mebhan und Kohlensaure. Zeitschr. f. physiolog. Chemie., 1886, Bd. x, pp. 201-217 and 401-440. ('89). KRAMER, ERNST. Studien fiber schleimige Gahrung. Sitzungsb. d. k. Ak. d. Wissen- schaften. Math.-naturw. Classe, Bd. xcvni, 1889, iv Hft, Abt. ii b. Chemie, pp. 358-396. Wien. An interesting paper. ('89). FRANKLAND, PERCY P., AND Fox, J. J. On a pure fermentation of mannite and glycerine. Proc. Royal Soc., Lond., 1889, vol. XLVI, pp. 345-357- ('89). CURTMAN, CHAS. O. Naoh-weis der Glycose durch Safranin. Pharmaceutische Runds- chau, Bd. vn, 1889, p. 132. ('89). TISCHUTKIN, N. Die Rolle der Bakterien bei der Veranderung der Eivveissstoffe auf den Blattern von Pinguicula. Ber. d. deutsch. 'bot. Gessellsch., Bd. vn, 1889, pp. 346-355- ('90). SCLAVO, ACHILLE, UND Gosio, B. Ueber eine neue Garung der Starke. Staz. sperim. agrar. ital., 1890, vol. xix, p. 540. FERMENTATIONS, GAS-FORMATION, ENZYMES, ETC. 2 33 ('90). REINITZKR, FR. Uober die wahre Natur des Gummifermentes. Zeitschr. S. physiol. Ohcmie, Bd. xiv, 1890, pp. 453-470. This author states that Wiesner's ferment, which changes cellulose into gum and slime, does not exist. Nickel, of Berlin, has come to the same conclusion inde- pendently. (See Bot. Centralb., 1890, Bd. xi.in, p. 118). The "orciu salzsiiure " reaction of Reichl is a furfurol reaction. Coo). SMITH, THEOBALD. Das Gahrungskolbchen in der Bakteriologie. Centralb. f. Bakt., vn Bd., 1890, pp. 502-506, mit i Abbildung. ('90). SMITH, THEOBALD. Einige Bemerkungen fiber Satire- und Alkalibildung bei Bakterien. Centralb. f. Bakt., Bd. vm, 1890, pp. 389-391. ('90). FERMI, CLAUDIO. Die Leim und Fibrin losen- den und die diastatischen Fermente der Mikroorganismen. Centralb. f. Bakt., vii Bd., 1890, pp. 469-474. ('90). DUBOIS, R. Sur le pretendu pouvoir digestif du liquide de 1'urne des Nepenthes. C. R. des se. de I'Acad. des Sci. T. cxi, 1890, pp. 315-317. Maintains that the observed results are not due to any pepsin-like body secreted by the plant, but to the action of intruding bacteria, and concludes that the Nepenthes are not carnivorous. ('91). HERY. ,Sur une fermentation visqueuse de 1'encre. Annales de Micrographie, 1891, T. rv, pp. 13-21. (91). FERMI, CLAUDIO. Wekere Untersuchungen iiber die itryptischen Enzyme der Mikroor- ganismen. Centralb. f. Bakt., x Bd., 1891, pp. 401-408. ('91). RITSERT. Bakteriologische Untersuehungen iiber das Schleimigwerden der Infusa. Ber. d. pharm. Gesellsch., I Bd., 1891, pp. 389- 309. Rev. in Centralb. f. Bakt., 1892, xi, PP- 730-733. ('91). SUCHSLAND, E. Ueber Tabaksfermentationen. Ber. d. d. bot. Gesellsch., Bd. ix, 1891, pp. 79-8 1. ('91). BEYERINCK, M. W. Verfahren zum Nachwcise der Saureahsonderung bei Mikrobien. Cen- tralb. f. Bakt., ix Bd., 1891, pp. 781-786. This method consists in using with gelatin or agar, for plate-cultures, etc., a solid opaque substance which is converted into a soluble, transparent substance in the presence of acids. For this purpose Beyerinck adds to the culture- medium a small amount of water in which a very finely divided carbonate of lime has been shaken up. This makes a white opaqne plate. Colonies which secrete acids are soon surrounded by a transparent dif- fusion field. Other carbonates may be used, e. g , zinc carbonate. This method may also be used for the demon- stration of alkali production. ('91). TISCHUTKJN, N. P. Ueber die Rolle der Mikroorganismen bei der Ernahrttng der insektenfressenden Pflanzen. Arbeit, d. St. Petersburg naturf. Gesell., vol. for 1890, Sect. d. Bot. pp. 33-37, St. Petersburg, 1891. Abs. in Bot. Centralb., Bd. L, 1892, p. 304. C'jjt). CONN, H. W. Isolirung eines "Lab" fer- mentes aus Bakterienkulturen. Centralb. f. Bakt., Bd. xn, 1892, pp. 223-227. ('92?). FRANKLAND AND FREW. The fermentation of calcium glycerate by the Bacillus ethaceticus. Trans. Roy. Ohem. Soc., 1891. Rev. in Centralb. f. Bakt.. xn Bd., 1892, p. 724. ('92). FRANKLAND, P., AND MACGREGOR, J. Fermen- tation of arabinoM with the Bacillus etha- ceticus. Trans. Chem. Soc., 1892. Rev. in Centralb. f. Bakt., xn Bd., 1892, p. 725. ("92). FRANKLAND, P. P., AND FREW, W. A pure fermentation of mannitol and dulcitol. Trans. Chem. Soc., 1892, pp. 254-277. ('92). FRANKLAND, P. P., AND LUMSDEN, J. S. De- composition of mannitol and dextrose by the Bacillus ethaceticus. Trans. Chem. Soc., 1892, pp. 432-444. ('92). FERMI, CLAUDIO. Beitrag zum Studium der von den Mikroorganismen abgesonderten diastatischen und Inversionsfermente. Cen- tralb. i. Bakt., xn Bd., 1892, pp. 713-715. Of 62 bacteria investigated, 20 were acid producers, 2 in- verted cane sugar, 24 developed a proteolytic enzym, 20 a diastasic ferment. ('92). TISCHUTKIN, N. Ueber die Rolle der Micro- organismen bei der Erna/hrung insekten- fressender Pflanzen. Acta Horti Petro- politanie, Bd. xn, St. Petersburg, 1892, pp. 1-19. Abstract in Bot. Centralb., Bd. LIU, 1893, p. 322. ('92). FRANKLAND, P. P. Cantor lectures on recent contributions to the chemistry of bacteriol- ogy of the fermentation industries, gr. 8vo., 31 pp. London, W. S. Trounce, 1892. Not seen. ('93). HILDEBRANDT, H. Weiteres fiber hydrolytische Fermente, etc. Arch. f. path. Anat. u. Physiol, Bd. cxxxi, 1893, pp. 5-39. ('93)- GOEBEL. Pflanzenbiologische Sehilderungen ii, 1893, p. 186. Criticises views of Duhois and Tischutkin. ('93). HAPP. Bakteriologische und chemisohe Unter- suchungen fiber die sohleimige Garung, p. 31, these inaugurate. Berlin, 1893. Not seen. Describes Micrococcus gummosiis. ('93). CAVAZZANI, EMIL. Zur Kenntnis der diasta- tischen Wirkung der Bakterien. Centralb. f. Bakt., Bd. xm, 1893, pp. 587-589. ('93). PER, A. Sur la formation des acides lactiques isomeriques par 1'action des microbes sur les substances hydrocarbonees. Ann. de 1'Inst. Pasteur, T. vii, 1893, pp. 737-750. ('93). HESSE, W. Ueber die gasformigen Stoff- wechselproducte beim Waohsthum der Bak- terien. Zeitschr. f. Hyg., Bd. xv, 1893, pp. 17-37- ('93). SMITH, THEOBALD. The fermentation tube with special reference to anaerobiosis and gas production among bacteria. Wilder Quarter Century Book, Ithaca, N. Y., 1893, pp. 187-232. ('93). FRANKLAND, P., AND MACGREGOR, J. Sarco- lactic acid obtained by the fermentation of inactive Lactic acid. Trans. Chem. Soc., London, 1893, pp. 1,028-1,035. ('93). GRIMBERT. L. Fermentation anaerobie produite par le Bacillus prthobutylicus, ses variations sous certaines influences biologiques. Ann. de 1'Inst. Pasteur, T. vii, 1893, pp. 353-402. ('93). GREEN, J. R. On Vegetable Ferments. An- nals of Botany, 1893. Vol. vii, pp. 83-137. ('94). FERMI, CLAUDIO, AND PERNOSSI, LEONE. Ueber die Enzyme. Centralb. f. Bakt., Bd. XV, 1894, pp. 229-234. ('94). FERMI UND PERNOSSI. Ueber die Enzyme. Zeitschr. f. Hyg., Bd. xvin, 1894, pp. 83-127. Studies of the action of heat, light, and various chemi- cal substances on various enzymes. ('94). WOOD, JOSEPH T. Fermentation in the leather industry. Journal of the Society of Chemi- cal Industry, 1894, vol. xni, pp. 218-222. 234 BACTERIA IN RELATION TO PLANT DISEASES. ('94). GOTTSTEIN, ADOLF. Ueber die Zerlegung des Wasserstoffsuperoxyds durch die Zellen, imit Bemerkungen iiber eine makroskopische Reaktion fiir Bakterien. Virchow's Archiv., Bd. cxxxui, p. 296. Rev. in Centralb. f. Bakt., Bd. xvi, 1894, pp. 518-519- ('94). TIMPE, HERMANN. Ueber die Beziehungen der Phosphate und des Kaseins zur Milchsaure- garung. Die landwirtschaft. Versuchsstat., Bd. XLII, 1894, pp. 223-238. ('94). LASER, HUGO. Die makroskopische Wasser- untersuchung durch Anwendung von Was- serstoffsuperoxyd. Centralb. f. Bakt., Bd. xvi, 1894, pp. 180-182. This substance, which causes liberation of oxygen from H2O2, is said to be nuclein. Author failed to confirm Gottstein's results. (Virchow's Archiv., Bd. cxxxui, Heft 2.) ('94). GREEN, J. R. The influence of light on dias- tase. Abstr. of a paper read before the British Assoc. at Oxford, Aug., 1894. Ann. of Bot., vol. vin, 1894, pp. 370-373- ('94). Gosio, B. Ueber Links-Milchsaure bildende Vibrionen. Arch. f. Hyg., Bd. xxi, 1894, pp. 114-122. ('94). MACFAYDEN AND BLAXALL. See XXXIV. ('95). BAIER, EDUARD. Ueber Buttersauregalirung. Centralb. f. Bakt., 2 Abt., Bd. I, 1895, pp. 84-87 and 118-120. ('95). OMELIANSKI, V. Sur la fermentation de la cellulose. C. R. des se. de 1'Acad. des sci., T. cxxi, 1895, pp. 653-655. ('95)- JOERGENSEN, ALFRED. Les microorganismes de la fermentation. Traduit par Paul Freund. 8vo., 318 pp., 56 illst., Paris (Soc. d'editions scientifiques), 1895. Micro- organisms and Fermentation. English trans- lation bv Alex. K. Miller and A. E. Lenn- holm. Third edition completely revised, with 83 illustrations, pp. xm, 318. London, Macmillan & Co., Ltd. New York, The Macmillan Co., 1900. There is also a Ger- man -translation which has reached the 3d ed. Berlin, 1892. Orig., in Danish, not seen. ('95). Gosio, B. Zersetzungen zuckerhaltigen Nahrmateriales durch den Vibrio choleras asiaticas Koch. Arch. f. Hyg., Bd. xxn, 1895, pp. 1-27. ('95). FERMI UND MONTESANO. Die von den Mikro- ben bedingte Inversion des Rohrzuckers. Centralb. f. Bakt., 2 Abt., Bd. I, 1895, No. 13-14, pp. 482-487, No. 15-16, pp. 542-556. The author used Nylander's and Rubner-Penzoldt's reactions. Fehling's solution was not used, because it is not to be trusted in the presence of albumen. ('95). BEYERINCK, M. W. Ueber Nachweis und Ver- breitung der Glukase, das Enzym der Mal- tose. Centralb. f. Bakt., 2 Abt., Bd. I, 1895, pp. 221-229, pp. 265-271, and pp. 329-342. ('96). BOURQUELOT, EMIL. Les ferments solubles (diastases-enzymes), pp. yin, 220. 8vo. Paris, Societe d'editions scientifiques, 1896. ('96). PFEFFER, W. Ueber regulatorisehe Bildung von Diastase. Ber. u. d. Verhandl. d. K. saoh. Gesell'Sch. d. Wissenschaften zu Leip- zig. Mathematisch-Physik. Classe, 1896, pp. 513-518. Also a separate. ('97). BUCHNER, H. Die Bedeutung der activen loslichen Zellprodukte fur den Ohemismus der Zelle. Miinchen. med. Wochenschr., 1897, No. 12, pp. 209-302. ('97). BUCHNER, ED. Alkoholische Giiruiig ohnc Hefezellen. Ber. d. deutsch. chem. Ge- sellsoh., 1897, Jahrg. xxx, pp. 117-124 and 1,110-1,113. See also Ibid., Jahrg. xxxi, p. 568-574- " Eine Trenmmg der Gahrwirkuug von den lebendeu Hefezellen ist bisher nicht gelungen." (XXX, p. 117.) (*97)- VINES, S. H. The proteolytic enzyme of Nepenthes. Annals of Botany, vol. xi, 1897, PP. 563-584- ('98). PRESCOTT, S. C., AND UNDERWOOD, W. LYMAN. Contributions to our knowledge of micro- organisms and sterilizing processes in the canning industries. II The souring of canned sweet corn. Tech. Quarterly, vol. xi, No. i, 1898, 6 plates. Also a separate, 30 pp. ('99). OMELIANSKI, V. Sur la fermentation de la cellulose. Arch, des sci. biol. publiees par 1'inst. imp. de med. exper. a St. Petersbourg, T. vii, pp. 411-434. i heliotype plate. ('99). FERMI, CLAUDIO, AND BUSCAGLIONI. Die pno- teolytischen Enzyme im Pflanzenreiche. Centralb. f. Bakt., 2te Abt., Bd. v, 1899, No. i, pp. 24-27; No. 2, pp. 63-66; No. 3, pp. 91-95; No. 4, pp. 125-134; and No. 5, pp. 145-158. ('99). DUCLAUX, E. Traite de microbiologie. Tome ii. Diastases, toxines et venins. Paris, Masson et Cie, 1809, pp. ill, 768, large 8vo. The best treatise in French. ('99). SACHAROFF, N. Einige erganzende Angaben zur Mitteilung: "Ueber den Ohemismus der Wirkung der Enzyme und der bakterioiden Stoffe." Centralb. f. Bakt., xxv Bd., 1899, PP- 346-350. ('99). BULLEK, A. H. R. Die Wirkung von Bak- iterien auf tote Zellen. Inaugural. Disserta- tion. Univ. of Leipsic, pp. 47. Leipsic, 1899. ('99). BREDIG, G., UND MUELLER v. BERNECK, R. Ueber Platinkatalyse und die chemiische Dynamik des Wassers'toffsuperoxyds. Zeitschr. fiir Physikalische Chemie., Bd. xxxi, 1899, pp. 258-353, 3 'text figs. In many ways platinum black behaves like an enzyme. ('99). BUCHNER, E., UND RAPP, R. Alcoholisohe Gahrung ohne Hefezellen. Ber. d. deutsch. chem. Gesellsch., Jahrg. xxx (1897), 2,668; Jahrg. xxxi (1898), 209, 1,084, 1,090, i,S3i; Jahrg. xxxn (1899), 127-137. ('99). GREEN, J. REYNOLDS. The soluble ferments and fermentation, pp. xm, 480. Cambridge (England), at the University Press, 1809. 2d ed., 1901, pp. XV, 512, 'with a bibliography of 896 titles. An excellent book, and the only complete, authorita- tive one in English. Coo). DUCLAUX, E. Traite de microbiologie. Tome in. Fermentation alcoolique. Paris, 1900, pp. in, 760, 84 text figures, 8vo. Masson et Cie. Coo). TURRO, R. Zur Bakterienverdauung. Cen- tralb. f. Bakt., xxvin Bd., 1900, pp. 173-177- ('oo). MORGENROTH, J. Zur Kenntnis der Labenzyme und ihrer Antikorper. Centralb. f. Bakt., xxvn Bd., 1900, pp. 721-724. COT). SCHOENE, ALBERT. Die Mikroorganismen in den Saften der Zuckerfabriken. Berlin, Zs. Ver. D. Zuckerind., Bd. LI, 1901, techn. Tl, pp. 453-468. FERMENTATIONS, ENZYMES; PTOMAINES, TOXINS, ETC. 235 ('oi). BOUHFARD, A. Les maladies microbiennes des vins. Fermentation alcoolique; maladies microbiennes ; carre des vins ; hygiene des vins; traitement des vins malades. Nancy, 1901, I2mo., avec 6 planches et fig. Not seen. ('oi). DUCLAUX, E. Traite de microbiologie. Tome IV. Fermentations variees des diverses sub- stances ternaires. Paris, Masson et Cie., loor, pp. in, 768, 45 text figures. Valuable. ('oi). GKSSAKD, C. fitodes sur la tyrosinase. Ann. de I'lnst. Pasteur, T. xv, 1901, pp. 593-614. ('oi). BEIJERINCK, M. W. Anhaufungsversuche mil Ureumbakterien. Ureumspaltung duroh Urease und durch Katabolismus. Centralb. f. Bakt., Bd. vn, 1901, pp. 33-61, with I plate and 4 figures. ("02). RoLLY. Weiterer Beitrag zur Alkali- und Saureproduktion der Bakterien. Arch. f. Hyg., Bd. XLI, 1902, pp. 406-412. ('02). SCHREIBER, KARL. Fettzersetzung durch Mikroorgani&men. Arch. f. Hyg., Bd. XLI, 1902, pp. 328-347. ('02). SMITH, R. GREIG. The gum fermentation of sugar cane juice. Proc. Linnean Soc., N. So. Wales, for the year 1901, Sydney, 1902. Vol. xxvi, Pt. i, pp. 589-625, i plate. Also a separate. ('02). SMITH, R. GREIG. The deterioration of raw and refined sugar crystals in bulk. The acid fermentation of raw sugar crystals. Proc. Linn. Soc. of New South Wales, for 1901, Pt. 4, pp. 674-683, 684, Sydney, 1902. Also a separate (issued May 20, 1902). ('02). OMKUANSKI, W. Sur la fermentation fpr- menique de la cellulose. Arch, des sci. biol. publiees par .1'inst. imp. de med. exper. a St. Petersbourg, T. ix, 1902, pp. 251-278, I heliotype plate. ('02). EFFRONT, JEAN. Enzymes and their applica- tions. English translation by Samuel C. Pres- cobt, vol i. The enzymes of the carbohy- drates. The oxidases. New York, John Wiley i& Sons ; London, Chapman & Hall, Ltd., 1902, pp. xi, 322. ('02). SAWAMURA, S. On the liquefaction of mannan by microbes. Bulletin of the College of Agriculture, Tokyo Imperial University, vol. v, No. 2, 1902, pp. 259-262. Also a separate. ('02). WEISS, RICHARD. Uber die Baktcrienflora der Sauern Gahrung einiger Nahrungs-und Genussmittl. Arb. a. d. Bact. Institut der tech. Hochschule zu Karlsruhe, 11 Bd., 3 Heft, 1902, pp. 163-269. Forty-eight new species are described : Streptococcus citreus, S. maximus, Micrococcus pulcher, M. regularis, M. irregularis, M. umbilicatus, M. minimus, M. gummo- sus, M. mucitaginens, M. vulgaris, M. plliformis, M. II. eminans, B. ventricosus, B. citricus amygdaloides, B. robustus, B. tuberosus I!, fungosiis, B. flavescens, B. grnciles variosum, B. spinosum, B. crenatnm, B brevissimum, B. raiuificans, B. gibbosnm B. squanintinii, B. unifonne, B. insnlsum B. gramilosum, Pseudomonas lactica, Ps B. odoratus, B B. globulosus ens, Bacteriun plicativum, B B. gracillimum B. subcitrinuni Listed. ('02). GRAN, H. H. Studien iiber Meeresbakterien. 2. Ueber die Hydrolyse des Agar-Agars durch ein neues Enzym, die Gelase. Bergens Mus. Aarb., 1902, No. 2, p. 16. ('03). KELLERMAN, KARL. The effects of various chemical agents upon the starch-converting power of taka diastase. Bulletin Torrey Botanical Club, vol. xxx, 1903, pp. 56-70. Also a separate. ('03). VINES, S. H. Proteolytic enzymes in plants. Annals of Botany, vol. XVH, Jan., 1903, pp. 237-264. ('03). SMITH, R. GREIG. A gum (levan) bacterium from a saccharine exudate of Eucalyptus Stuartiana. Proc. Linnean Soc., N. So. Wales, vol. xxvn, for the year 1902, pp. 230- 236. i plate. Sydney, ioo2-'o3. ('03). LIPPMANN, E. O. VON. Zur Nomenklatur der Enzyme. Berichte d. deutsch. Chem. Gesell- schaft, 1903, Bd. xxxvi, p. 331. XXI. Ptomaines, Toxins, Antitoxins, Serums, Phagocytosis, etc. ('86). BKIECER. Untersuchungen iiber Ptomaine. Berlin, 1886. Traduotion par Roussy et Winter as Microbes, ptomaines et maladies. Paris, 1887, pp. xii, 235. ('87). METCHNIKOFF, ELIE. Sur la lutte des cellules de 1'organisme centre 1'invasion des microbes. Ann. de I'lnst. Pasteur, T. i, 1887, pp. 321-336. ('88). NUTTALL, GEO. Experiments fiber die bac- terienfeindlichen Einfluss des thierisohen Korpers. Zeitschr. f. Hyg., Bd. iv, 1888, pp. 353-394- ('88). GAERTNER. Ueber die Fleischvergiftung in Krankenhauser und den Erreger derselben Jena, 1888. Not seen. ('89). NISSEN, F. Zur Kenntniss der Bacterien- vernichtenden Eigensohaft des Blutes. Zeitsohr. f. Hyg., Bd. vi, 1889, pp. 487-520. ('89). BUCHNER, H. Ueber die baoterientodtende Wirkung des zellenfreien Blutserums. Cen- tralb. f. Bakt., Bd. v, 1889, pp. 817-823; and Bd. vi, 1889, pp. i-n. ('90). BRIEGER, L., u. FRAENKEL, CARL. Untersuchun- gen iiber Bacteriengifte. Berl. klin. Woch- enschr., Bd. xxvn, 1890, No. n, pp. 241-246; No. 12, pp. 268-271. ('90). LEHMANN. Ueber die pilztodtende Wirkung des frisohen Harns des gesunden Menschen. Centralb. f. Bakt., Bd. vn, 1890, pp. 457-460. ("90). JACQUEMART, F. Les ptomaines, histoire et caracteres chimiques, memoire oouronne par la Societe royale des sciences medicaks eit naturelles de Bruxelles. Jour, de med. de chir. et de pharm., Bruxells, 1890, No. 18. Rev. in Centralb. f. Bakt., ix Bd., 1891, pp. 107-110. ("90). CARBONE, TITO. Ueber die von Proteus vul- garis erzeugten Gifte. Centralb. f. Bakt., 1800, Bd. vm, pp. 768-773. ('90). LEWANDOWSKI, A. Ueber Indol- und Phenol- Wldung durch Bakterien. Deutsche mediz. Wochenschrft, Bd. xvi, 1890, p. 1,186. ('90). GABRITCHEVSKY, G. .Sur les proprietes chimio- tactiques des leucocytes. Ann. de I'lnst. Pasteur, T. iv, 1890, pp. 346-362. ('91). OGATA, M. Ueber die bacterienfeindliche Substanz des Blutes. Centralb. f. Bakt., Bd. ix, 1891, pp. 597-602. 236 BACTERIA IN RELATION TO PLANT DISEASES. ('gi ). TRAPEZNIKOFF. Du sort des spores de microbes dans 1'organisme animal. Ann. de 1'Inst. Pasteur, T. v, 1891, pp. 362-394, 2 plates. ('94). EHRLICH, P., UND WASSERMANN, A. Ueber die Gewinnung der Diphtherie-antitoxine aus Blutserum und Milch iinmunisirter Thiere. Zeitsch. f. Hyg., Bd. xvm, 1894, pp. 239-250. ('9=0 BRIEGER, L. Wetter* Erfahrungen uber Bak- teriengifte. Zeitsch. f. Hyg., Bd. xix, 1895, pp. IOI-II2. ('96) BRIEGER UND BOER. Ueber Antitoxine und Toxine. Zeitsch. f. Hyg., 1896, Bd. xxi, pp. 259-268. ('96). GAUTIER, ARM AND. Les toxines microbiennes et animates, 1896, pp. vn, 617. Soc. d'edi- tions scientifiques, Paris. ('96). EHRLICH, P. Die staatliche controle des Diph- theric serums. Berl. klin. Wochenschr., Bd. xxxm, 1806, pp. 441-443- ('96). VAUGHAN, VICTOR C., AND Now, FREDR. G. Ptomains, leucomains, toxins, and anti- toxins. 3d ed. Lea Bros. & Co., Philadel- phia and New York, 1896, pp. x, 604. Contains a bibliography of several hundred titles. The 4th ed. was issued in 1902, with the following title: Cellu- lar toxins, or the chemical factors in the causation of disease. Same publishers. Pp. vm. 495- ('96). METCHNIKOFF, EL., Roux, E., ET TAURELLI- SALIMBENI. Toxine et antitoxine cholerique. Ann. de 1'Inst. Pasteur, T. x, 1896, pp. 257- 282. ('97). SMITH, THEOBALD. A modification of the method for determining the production of indol by bacteria. Jour. Exper. Med., vol. n, 1897, pp. 543-547- Coo). BORDET, J. Les serums (hemolytiques, leurs antitoxines et les (theories des serums cytoly- tiques. Ann. de 1'Inst. Pasteur, 1900, T. xiv, pp. 257-296. Coo). METCHNIKOFF, E. Sur les cytotoxmes. Ann. de 1'Inst. Pasteur, T. xrv, pp. 36p-377, 1900. Coo). FISCHER, ALFRED. Die Empfindliohkeit der Bakterienzelle und das bakterieide Serum. Zeitschr. f. Hyg., Bd. xxxv, 1900, pp. 1-58, i plate. ('oi). ZABOLOTNY. See vn. ('oi). BORDET, J., ET GENGOU, O. Recherches sur la coagulation du sang et les serums anti- coagulants. Ann. de l"Inst. Pasteur, T. xv, 1901, pp. 129-144. ('oi). BORDET, J., ET GENGOU, O. Sur 1'existence de substances sensibilisatrices dans la plupart des serums antimicrobiens. Ann. de 1'Inst. Pasteur, 1901, T. xv, pp. 289-302. ('oi). BORDET, J. Sur le mode d'action des serums cytolytiques et sur 1'unite de 1'alexine dans un meme serum. Ann. de 1'Inst. Pasteur, T. xv, 1901, pp. 303-318. Coi). GRUBER, MAX. Zur Theorie der Antikorper. i. Ueber die Antitoxin-Immunitat. II. Ueber Bakteriolyse und Haemolyse. Miin- chener med. Wochenschr., Bd. XLVIII, 1901, pp. 1,827-1,830, pp. 1880-1884, pp. 1,924-1,986. Not seen. ('02). MARX, E. Die experimentelle Diagnostik, Serumtherapie und Prophylaxe der Infec- tionskrankheiten. [Bibliothek v. Coler, Bd. XL] Berlin (A. Hirschwald), 1902, pp. vn, 296, mit 2 Taf. ('04). WASSERMANN, A. Immune sera, haemolysins, cytotoxins, and precipitins. English trans- lation by Charles Bolduan. New York, John Wiley & Sons ; London, Chapman & Hall, 1904, pp. ix, 77. C8o). C8o). C8i). C8i). (81). C83). C87). ('89) ('93) ('94) ('95 ) ('03). XXII. Attenuation, Virulence. PASTEUR, Louis. De I'atJtenuation du virus du cholera des poules. C. R. des se. de 1'Acad. des sci., T. xci, 1880, pp. 673-680. CHAUVEAU, A. Des causes qui peuvent faire varier les resultats de 1'inoculation char- bonneuse sur les moutons algeriens. In- fluence des quantites des agents infectants. Applications a la theorie de I'inimnnite. C. R. des se de 1'Acad. des sci., T. xc, 1880, pp. 1,526-1,530. PASTEUR, Louis. Le vaccin des oharbon. C. R. des se. de 1'Acad. des sci., T. xcn, 1881, pp. 666-668. PASTEUR, CHAMBERLAND, ET Roux. De {'at- tenuation des virus et de leur retour a la virulence. C. R. des se. de 1'Acad. des sci., T. xcn, 1881, pp. 429-435. CHAUVEAU, A. De i'attenuation des effets des inoculations yirulentes par 1'emploi de^tres petites quantites de virus. C. R. des se. de 1'Acad. des sci., T. xcn, 1881, pp. 844-848. CHAMBERLAND ET Roux. Sur I'attenuation de la bacteridie charbonneuse et de ses germes sous 1'influence des substances antiseptiques. C. R. des se. de 1'Acad. des sci., T. xcvi, 1883, Paris, pp. 1,410-1.412. METCHNIKOFF, EuE. Sur I'attenuation des bacteridies charbonneuses dans le sang des moutons refractaires. Ann. de 1'Inst. Pas- teur, T. i, 1887, pp. 42-44. MACE. See xxm. D'ARSONVAL AND CIIARRIN. See XXXTI. ROGER. See xxxii. KLEPZOFF. See xxxni. FUHRMANN, FRANZ. Ueber Vinilenzsteigerung eines Stanwnes des Vibrio Cholerae asia- ticae. Sitzungsber. d. kaiserl. Akad. d. Wissensch. Mathematisdh-Naturwissensch. Klasse., Bd. cxn, Heft, vm, Abt. in, 1003, pp. 267-284. With bibliography of 15 titles. XXIII. Pigments. Green Bacteria. ('73). LANKESTER, E. RAY. On a peach-coloured bacterium, Bacterium rubescens, n. s. Quart. Jour. Micro. Sci., 1873, vol. xin, n. s., pp. 408-425. 2 plates (colored). ('80). VAN TIEGHEM, PH. Observations sur des bacteriacees vertes, sur des phycochro- macees blanches, et sur les affinites de ces deux families. Bull de la Soc. Bot. de France, Tome xxvn, 1880, pp. 174-179. ('82). ENCELMANN, TH. W. Zur Biologic der Schizomyceten. Bot. Zeitung, 40 Jahrg., 1882, col. 321-325 and 337-34L Describes a gi-een organism as Bacterium chlorinum. It is believed to contain chlorophyll. ('87). SCHOTTELIUS. See xv. ('87). PROVE, OSKAR. Micrococcus ocliroleucus eine neue chromogene Spaltpilzform. Beitr. zur Biol. der Pflanzen., Bd. iv, Hft. 3, 1887, pp. 409-439, i plate. PIGMENTS, GREEN BACTERIA. 2 37 ('89). MACK. Sur la recuperation de la vitalite cles cultures de bacteries par passages sur cer- tains milieux. Soc. des sc. dc Nancy pour 1888, Series n, T. ix, Fasc. xxll, pp. xxix- xxx and 79-83, Paris, 1889. The medium used was potato. Bacteria which have lost their power to form pigment on agar or to infect frogs will often regain these functions if cultivated for a time on potato. ('S'O. Zon>, W. Ueber Pilz farbstoffe. iv. Vor- kommen ei.ner Lipochroms bed Spaltpilzen. Dot. Zeitting, Bd. XLVII, 1889, col. 89-92. ('89). ZOPF, W. Ueber das niikrochemischen Ver- haken von Fettfarbstoffen und Fettfarbstoff- Jialtigen Organen. Zeitschr. f. wissensch. Mikroskopie, 1889, Bd. vi, pp. 172-177. ("89). SCHEIBENZUBER, D. Ein Bacillus mlt brauner Verfarbung der Gelatine. Allgemeinen Wiener medicinischen Zeitung, Jahrg. xxxiv, 1889, pp. 171-172, Wien, 1889. Also a separate, 7 pp. This organism liquefies gelatin. It was isolated from spoiled eggs. ('90). BEHR, P. Ueber eine nicht mehr farbstoff- bildende Rasse des Bacillus der 'blauen Milch. Centralb. f. Bakt., Bd. vni, 1890, pp. 485-487- ('90). CLAESSEN, HEINR. Ueber einen indigoblauen Farbstoff erzeugenden Bacillus aus Wasser. Centralb. f. Bakt., Bd. vn, 1890, pp. 13-17. ('91). DANGEARD, P. A. Contribution a 1'etude des Baoteriacees vertes (Eubacillus gen. nov.). Le Botaniste, Ser. n, 1891, fasc. 4, pp. 151- 160, avec. planche. Also C. R. des se. de 1'Acad. des soi., T. cxn, 1891, pp. 251-253. Review in Centralb. f. Bakt., x Bd., 1891, PP. 745-747- Daugeard found his green spore-bearing organism (Eubacillus miiltisporus) on the walls of a culture flask used for the growth of fresh-water algse, where it made a felt of very long slender flexible filaments. He states that it contains chlorophyll distributed through the pro- toplasm. The sporiferous filaments may be branched. The spores are a distinct green. Its habitat is among fresh-water algce in the vicinity of Caen, France. To the second section of his genus he would add the following forms ; described by Klein : Bacillus de Bary- anus, B. Solmsii, B. Peroniella, B. macrosporus, and B. limosus, all of which are sporiferous, the spores being blue-green. These spores are all said to be impregnated with chlorophyll. ("91). SI, KURT. Ueber die Einwirkung des elektrischen Stromes auf Bakterien. Centralb. f. Bakt., xxv Bd., 1899, pp. 650-655, with i fig. Results all negative. ('oo). KRAUSE. See xv. ELECTRICITY; ROENTGEN RAYS, ETC.; HIGH PRESSURE. 245 ('01). STREHEI., I IKUMANN. Untersuchungcn iiber die luktericide Wirkung des Hochspannungs- funkenliohtes nebst Angabe einer Methode zur besseren Ausntitzung der baktericiden Kraft des Voltabogenlichtes. D. med. Wochensclir., Berlin, Bd. xxvn, 1901, pp. 69-72, pp. 87-89. ('oi). UI.T.MANN, JOHANNES. Ueber die Einwirkung elektrischcn Bogenlichts auf Mikroorgan- iMiien in Gegenwart von fluoreszierenden Stoffen. Diss. Munch en (Druck v. M. Ernst), 1901, p. 17. XXXI. Action on Bacteria of Roentgen Rays, Ra- dium Rays, Etc. ('96). WITTLIN, J. Les rayons Rontgen exercent-ils une action quclconque sur les bacteries? Ann. de -micro., T. vm, 1896, pp. 514-515. Author fiuds that the Roentgen rays have no effect upon bacteria. ('96). MINCK, P. Zur Frage iiber die Einwirkung der Rontgen'sehen Strahle.n auf Baktericn und ihre eventuelle therapeutische Verwend- barkeit. Munchener mediz. Woohenschrift. 1806, Bd. XLIII, pp. 101-102 and p. 202. Author obtained only negative results. ('97). POTT, FRANCIS. Concerning .the action of X- rays on cultivation of tubercle Bacillus. The Lancet, London, vol. n, for 1897 (55th year), pp. 1,314-1,315. The tubercle bacillus was not affected by X-rays. ('97). BLAISE ET SAMBUC. De Faction des rayons X sur Je Pyocyaneus et la bacteridie char- bonneuse. C. R. des .se. et mem. de la soc. de biol., T. iv, ice serie, 1897, pp. 689-692. Little or no effect on these organisms. ('97). BEAUREGARD ET GUICHARD. Action des rayons X sur certains characteres biologique des microbes. C. R. des se. et mem. de la soc. de biol., T. iv, loe serie, 1897, pp. 803-804. The bacteria are much less sensitive than higher or- ganisms. ('98). RiEDER, HERMANN. Wirkung der Rontgen- strablen auf Bakterien. Munch, med. Wochensclir., 45 Jahrg., 1898, pp. 101-104, 2 text figures (exposed agar plates). Contrary to the statements of various other experi- menters, this writer says that he obtained positive germi- cidal results on seven pathogenic organisms by exposures lasting from 45 minutes to i hour. The earlier literature is cited. VolCohm's apparatus was used. The photo- graphs show the center of the agar Petri-dish cultures cleared of bacterial colonies. ('98). WOLFENDEN, MORRIS, AND FORBES-ROSS, F. W. A preliminary note on the action of the Roentgen rays upon the growth and activity of bacteria and micro-organisms. The Lancet, London, June 25, 1898, pp. 1,752- 1,753- Bacillus prodigiostis 011 potato was exposed to the rays for one hour on several occasions. Growth was much greater than in the control tubes, and more pigment was formed. ('98). RIEDER, H. Weitere Mittheilung iiber die Wirkung der Rontgenstrahlen auf Bacterien sowie auf die menschliche Haut. Munch, med. Wochenschr., 45 Jahrg., 1898, pp. 773- 774- foi). CASPARI, W. Ueber die bacterienschadigende Wirkung der Becquerelstrahlen. Nadi in Gemeinschaft mil Priv. Doc. Dr. Aschkinass ausgefuhrten Versuchen. Arch. ital. biol., Turin, T. xxxvi, 1901, p. 130. ('02). RIEDER, HERMANN. Nochmals die bakterien- todtende Wirkung der Rontgenstrahlen. Munchener med. Wochenschr., Bd. XLIX, 1902, pp. 402-406. ('04). PRESCOTT, S. C. The effect of radium rays on the colon bacillus, the diphtheria bacillus and yeast. Science, n. s., vol. xx, Aug. 19, 1904, pp. 246-248. " Radium rays have no effect upon fresh cultures of B. coli, B. diphtheria, or Saccharomyces cerevisiae at a dis- tance of one centimeter where the time of exposure is less than 90 minutes. XXXII. Effect of High Pressure on Bacteria. ('75)- BERT, P. Influence de 1'air comprime sur les fermentations. C. R. des se. de 1'Acad. des sci., Paris, 1875. T. LXXX, pp. 1,579-1,582. ('77). BERT, P. De 1'emploi de 1'oxygene a haute tension comme precede d'investigation physi- ologique ; des venins et des virus. C. R. des se. de 1'Acad. des sci., Paris, 1877, T. LXXXIV, pp. 1,130-1,133. ('91). SCHAFFER ET DE FREUDENREiCH. De la resis- tance des bacteries aux hautes pressions combinees avec une elevation de la tem- perature. Annales de Microg., T. iv, 1891, pp. 105-119. Milk subjected to a pressure of many atmospheres (78-90) for several hours at 45 to 63 C was not steril- ized. High pressure for a week also failed to sterilize it. ('93). D'ARSONVAL ET CHARRIN. Pression et microbes. La semaiine medicale, 1893, T. xin, p. 251. Rev. in Centralb. f. Bakt., Bd. xiv, 1893, p. 64. See also C. R. des se. et mem. de la soc. de Biol., Paris, 20 mai, 1893, pp. 532-533. B. pyocyaneus, in fresh bouillon cultures, was subjected to a pressure of 50 atmospheres under carbon dioxide. All were dead inside of 24 hours. Kven two hours' exposure interfered with the reproductive function, i. e., lessened the number of organisms capable of producing colonies, and in cultures made after four hours' pressure only traces of ability to form pigment remained. In cultures made after six hours exposure there was no formation of pigmeut, and generally no colonies when sown upon agar, but in one case there were a few. ('94). D'ARSONVAL AND CHARRIN. See xxxni. ('94). ROGER. Action des hautes pressions sur les microbes. C. R. des se. de '1'Acad. des sci., T. cxix, Paris, p. 963. Pressures ot 1,000 to 3,000 atmospheres were tried with, out destroying the bacteria. Certain functions, however- were destroyed, e. g., pathogenicity. ('97). MALFITANO, G. Sul comportamento dei micro- organism! a'll'azione dei gasi compressi. Boll, della Soc. medioo-chirurgica di Pavia, 1897. Rev. in Centralb. f. Bakt., xxm Bd., 1898, pp. 233-236. 246 BACTERIA IN RELATION TO PLANT DISEASES. XXXIII. Action of Heat and Cold on Bacteria. ("75). EIDAM, EDUARD. Die Einwirkung verschied- ener temperaturen und des Eintrocknens auf die Entwicklung von Bacterium termo Duj. Cohn's Beitrage z. Biol. d. Pflanzen, Bd. I, Heft 3, pp. 208-224, Breslau, 1875. ('77). FRISCH, A. Ueber den Einfluss niederer Tem- peraturen auf die Lebensfahigkeit der Bac- terien. Sitzungsber. der K. Acad. der Wissensch. Wien. Math.-natur.-wissenschaf- ten Classe, Mai, 1877, Bd. LXXV, in A'bt., pp. 257-269. ('77). TYNDALL, JOHN. On heat as a germicide when discontinuously applied. Proc. Roy. Soc., London, vol. xxv, 1877, No. 178, pp. 569-570. ('79)- CHAMBERLAND, CH. Resistance des germes des certains o.rganismes a la temperature de 100 degres; conditions de leur developpe- ment. C. R. des se. de 1'Acad. des sci., T. i,xxxviil, 1879, pp. 659-661. ('82). LEBEDEFF, A. Contribution a 1'etude de 1'action d la chaleur et de la dessication sur la virulence des liquides septique et sur les Torganismes inferieurs. Archives de Physiol. normale et Path., Ser. n, T. x, pp. 175-204, 1882. ('84). PICTET, R., ET YUNG, E. De 1'action du froid .sur les microbes. C. R. des se. de 1'Acad. des sci., T. xcvm, 1884, pp. 747-749. ('87). PRUDDEN. See XLVI. ('87) . ESM ARCH, E. Der Henneberg'sche Desinfector. Zeitschr. f. Hyg., Bd. n, 1887, pp. 342-368. ('88). GLOBIG. Ueber einen Kartoffel-Bacillus 9). MIQUEL, P. Title? Bull, de la statistique municipale de Paris, Decembre, 1879. Not seen. He discovered in the water of the Seine an immobile, rod-shaped Schizomycete capableof living and develop- ing at the temperature of 70 C. ('81). VAN TIEGHEM, PH. Stir des bacteriacees vivant a la temperature de 74 C. Bull Soc bot. de France, T. 28, 1881, pp. 35-36. This author cultivated several species of thennophilic bacteria at 70 C., and some at higher temperatures. C8r). MIQUEI.. Thermobacteria. Annuaire de 1'Ob- servatoire de Montsouris, pour 1881, p. 464. 248 BACTERIA IN RELATION TO PLANT DISEASES. ('86). CERTES, A., ET GARRIGOU. De la presence con- stante de micro-organismes dans ^les eaux de Luchon, recueillies au griffon a la tem- perature de 64, et de leur action sur la production de la baregine. C. R. des se. de 1'Acad. des sci., T. cm, I&S6, pp. 703-706. ('88) GLOBIG Ueber Bakterien-Wachsthum bei 50 bis 70. Zeitschr. f. Hyg., Bd. in, 1888, pp. 294-321. Rev. in Centralb. f. Bakt., Bd. in, 1888, pp. 366-368. Globig obtained 30 sorts of bacteria which grew on potato at 58 C. At 68 only a few of them continued to grow. At 70 C. there were only scattering colonies, and at higher temperatures there was no growth. These organisms were not pathogenic to mice. As a rule, growth began at about 50 C., i. e., about 13 degrees above blood-heat. One would not grow at 37 C. or 50 C., but grew at 60. One grew all the way from 15 or 20" C. to 68 C Spores were often formed in 24 hours. None of these were from feces or sewage. Most were bacilli (rods). ('88). MIQUEL, P. Monographic d'un baoille vivant a au-dela de 70 centigrades. Ann. de micro., 1888, T. I, pp. 3-10. This organism will not grow at temperatures under 40, nor above 72 C. Its optimum temperature is 65 to 70 C. ('90). COHN, FERDINAND. Ueber Warmeerzeugung durch Schimmelpilze und Bakterien. Vor- trag., Breslau, 1890. ('93). COHN, F. Ueber therniogene Bakterien. Ber. d. Deutsch. bot. Gesellsch., Bd. xi, 1893, Gen. Versarnlungs-Heft, pp. 66-69. Conn found that when cotton wool waste was moist- ened it reached a temperature of 67.2 C. in 24 to 36 hours, and then slowly cooled (6 days) to the air-temperature. When the same waste was sterilized there was no rise in temperature. ('94). MACFADYEN, ALLAN, AND BLAXALL, FRANK R. Thermophilic bacteria. Journal of Path- ology and Bacteriology, vol. in, 1894, pp. 87-99. See also Br. Med. Jour., No. 1,760, 1894, p. 644. These authors obtained from garden soil an abundant growth of thermophilic bacteria on agar at 60 to 65 C, They also isolated these organisms from feces, sewage, sea-water, dirt of London streets, Thames water, Thames mud, straw, surface soil, and soil 5 feet 4 inches down. These thermophilic bacteria are, therefore, most widely distributed. There were guile a variety of species at least twenty. All were bacilli ; all were spore-bearing. Some were actively motile. Active motility continued for three weeks in one hanging drop. The colonies developed very rapidly in agar-plates. Fifteen sorts were tested as fol- lows : None of them grew at 22^ or at 37 C. Horse-dung organisms grew at 40 to 42 C. Six sorts grew slowly at 50 to 52 C., and more abundantly at 60 to 6s C. Two grew first at 56, and four refused to grow at 56, but grew when the temperature was raised to 60 C. None would grow at 75. The lower limit of growth for nearly all was 50 C. and the upper near 75 C. Boiling for ten minutes did not destroy these organisms. The optimum temperature for growth is said to be 60 to 65 C. These organisms did not ferment sugars and did not thrive on substrata con- taining sugars, these substances seeming to retard growth. One changed starch to sugar. Query : How do these organisms exist in a climate as cold as that of Edinburg ? ('95). RABINOWITSCH, LYDIA. Ueber die thermo- philen Bakterien. Zeitschr. f. Hyg., Bd. xx, 1895, pp. 154-164. Leipsic. These thermophilic organisms were found in snow, in earth, in the dust of a street in Berlin. They were very abundant in the water of the Spree (7,000 to 8,000 per cubic centimeter). They are abundant in the dung of horses and cows, and also more or less so in the excrement of goats, rabbits, guinea pigs, dogs, mice, doves, hens, ducks, par- rots. They occur in the whole digestive tract ot man. and are found in certain fish, frogs, and some other cold- blooded animals. Miss R. also found them abundant in germinating barley in a brewery. They occur also in milk, even after it is boiled. She isolated and studied 8 species. All produced spores. None were pathogenic to mice or doves. The highest temperature at which any of them would grow was 75 C., and growth at this high tempera- ture was slight. There was an abundant growth at 58 to 68 C., and the optimum is said to be 60 to 70. They are very resistant to moist heat and also to dry heat. They were not killed by exposure to streaming sleam for 5 to 6 hours. While growing best at high temperatures, these organisms can grow slowly facultative-anaerobically at 33 to 40 C., and the author thinks that they multiply in- side warm-blooded animals. She found the temperature of dung-piles as high as 62 to 66 C. ('95). KARLINSKY, JUSTYN. Zur Kenntniss der Bac- terien der Thermalquellen. Hygienische Rundschau, 1895, Bd v, pp. 685-689. ('96). TEICH, M. Beitrag zur Kenntniss thermo- philer Bacterien. Hygienische Rundschau, 1896, Bd. vi, No. 22, pp. 1,094-1,095. ('98). LAXA, O. Ueber einen thermophilen Bacillus aus Zucker-Fabriksproduoten. Vorlaufige Mittheilung. Zeitschr. f. Zuckerindustrie in Bdhmen, Bd. xxn, 1898, p. 376. Not seen. ('98). OPRESCU. Studien iiber thermophile Bakterien. Arch. f. Hyg., Bd. xxxm, 1898, p. 164. Rev. in Centralb. f. Bakt., Bd. xxv, 1899, p. 360. ('99). TSIKLINSKY, MLI.E. Sur les microbes ther- mophiles des sources thermales. Ann. de 1'Ins.t. Pasteur, T. xni, 1899, pp. 788-795- Bibliog. of 13 titles. The author isolated five thermophilic organisms from hot springs. One form grew readily at 70 C. ('99). MICHAELIS, GEORG. Beitrage zur Kenntniss der therniophilen Bakterien. Arch. f. Hyg., Bd. xxxvi, Hft. 3, 1899, pp. 285-293. Rev. in Centralb. f. Bakt., xxvn Bd., 1900, p. 537. Describes and names four new thermophilic organisms. All have three specific names, and one has four, to-wit : Bacterium thermophilus aquatilis liquefaciens aerobius. ('99). CAMBIER. Thermophilic bacteria as ferments, action on glucose. Rev. de phys. et de chim., 1899, p. 223. Not seen. ('02). RUSSELL, H. L-, AND HASTINGS, E. G. A Micrococcus, the thermal death limit of which is 76 C. Centralb. f. Bakt., 2 Abt., Bd. vin, 1902, pp. 339-342, i plate. ('03). SETCHELL, WM. A. The upper temperature limits of life. Science, n. s., vol. xvn, 1903, PP- 934-937- Hot springs were studied in three localities in Cali- fornia and in Yellowstone National Park. Author found only CyanophyceaJ and Bacteria in strictly thermal waters. The Cyanophycese were found at 65 to 68 C., and spar- ingly up to 75" to 77 C. The bacteria were abundant at 70 to 71, and occurred in considerable quantity at 82 C. and 89 C. "The temperature of 89 C. is the highest at which I have been able to find any organisms Hying." Care was taken to determine the temperatures in the exact places frequented by the organisms, so as to remove the objection that lies against many of the earlier obser- vations. XXXV. Resistance to Dry Air. ('75). EIDAM. See xxxm. ('92). MOMONT, L. Action de la >dessication, de 1'air, et de la lumiere sur ila baeteridie char- ilxmneifse nlamenteuse. Ann. de 1'Inst. Pas- teur, 1892, T. vi, pp. 21-31. SWAN, ALLEN P. On the resisting vitality of the spores of Bacillus megaterium to the condition of dryness. Annals of Botany, vol. vn, p. 153-154, 1893- ('93). RESISTANCE TO DRY AIR; ACTION OF ACIDS AND ALKALIES, ETC. 249 ('94). WALLICZEK, HEINRICH. Die Resistenz des Bacterium coli commune gegen Eintrock- nung. Centralb. f. Bakt., Bd. XV, 1894, pp. 949-950. B. coli proved quite sensitive to dry air. The writer of this abstract has found great differences among bacteria e. g. B. tracheiphilus was killed by a few minutes' expos- ure ou cover-glasses, while Bact. hyacinthi lived under similar conditions for more than a month. Jones found his Bacillus carotovorus to be even more sensitive than B. tracheiphilus. See following citations. C'9S)- SMITH, ERWIN P. Bacillus tracheiphilus, etc. Centralb. f. Bakt., 2 Abt., Bd. i, p. ,370. ('97). MIQUEL, P. Sur la longevite des germes des bacteries dans les poussieres et dans le sol. Ann. de micr., 1897, T. ix, pp. 199-207 and 251-259. Coi). JONES, L. R. A soft rot of die carrot, etc. I3th Ann. Rep. Vt. Agric. Exp. Sta. for 1900. Burlington, Vt., 1901. Sec p. 328 for refer- ence to this subject. ('01). SMITH, ERWIN P. The cultural characters of Ps. hyacinthi. etc. Bull. 28, Div. Veg. Phys. and Path., U. S. Dep. Agr., Washington, D. C., 1901, p. 145. XXXVI. Action of Acids and Alkalies. ('86). ABBOTT, A. C. The germicidal value of some of the vegetable acids. The Medical News, Phil a., 1886, 9 Jan., pp. 33-34. ('92). DELBRUECK, M. Ueber das Verhalten der Cholerabacillen auf frischen Friichten, einigen Genuss- und Nahrungsmitteln. Son- derabdruck aus den Veroffentlichungen des Kaiserlichen Gesundheitsamte, 1892, No. 42, vom 19 October, Berlin. Verlag von Julius Springer, 1892, pp. 812-824. The per cent of malic acid in many fruits is given. This varies from 0.13 (certain pears) to 2.65 (red currantsV In the feebly acid fruits, the cholera bacilli were dead inside of 3 to 7 days : in the the tarter fruits thev retained their vitality only for a period measured by hours. Usually they were dead in from i to 6 hours. ('92). SCHLUETER, G. Das Wachstum der Bakterien auf saurem Nahrboden. Centralb. f. Bakt., Bd. xi, 1892, pp. 589-598. A dozen different bacteria were tested in " ordinary nu- trient gelatin " and in fish glue, with addition of lactic acid, aium, tartaric acid, citric acid, acetic acid, and hy- drochloric acid. Several organisms tolerated as much as i per cent of lactic acid, or i per cent tartaric acid, but their growth was slow and usually feeble. Several grew feebly in the presence of l /t per cent alum. Six grew abundantly in gelatin acidified with citric acid. so that H cc. of the gelatin required for its neutralization 4 cc. of sodium carbonate water of the strength 5.3:1000. In fish glue containing 0.15 per cent acetic acid, several grew, but only feebly. Six grew in fish glue containing 0.075 per cent hydrochloric acid. The anthrax organism grew better with 0.2 per cent alum than on a neutral substratum. (93). HESSE, W. Ueber den Einfluss der Alka- lescenz des Nahrbodens auf das Wachsthum der Bakterien. Zeitschr. f. Hyg., Bd. xv, 1893, PP. 183-191- ('9,3). VOCES, O. Ueber das Wachstum der Cholera- bacillen auf Kartoffeln. Centralb. f. Bakt., Bd. xm, 1893, pp. 543-550. Organism woulci uot grow on potato as ordinarily pre- pared, but grew well at 37 C. (and more slowly at 20) on the addition of a 2to 3 percent solution of sodium chlor- ide. Nearly as good results were obtained with % to % per cent sodium carbonate solution. Growth was also obtained on potato with J4 to % per cent sodium hydrate solution. ('97). DEELEMAN, M. Der Einfluss der Reaktion des Nahrbodens auf das Bakterienwachstum. Arbeit, aus dem Kaiserl. Gesundheitsamte, Bd. xin, 1897, Heft 3. Rev. in Centralb. f Bakt, xxn Bd., 1897, pp. 355-356. (98). FERMI, CLAUDIO. Die Mineral- und organ- ischen Sauren, die Alkali, die Alkaloide, das Jodkali und das arsensaure Kali zur Differ- enziierung der Mikroorganismen. Centralb. f. Bakt., Bd. xxin, 1898, pp. 208-217 and 266-273. Of the plant acids, oxalic was found to be the most deleterious to the Schizomycetes. The conclusions are given on p. 266 et seq. ('96) ('97) ('97) ('97) ('98) ('99) ('99) ('99) ('99) Coo) Coo) Coo) XXXVII. Agglutination and Precipitation. . WIDAI,, FERNAND. Sero^diagnostic de la fievre typhoide. Bull, et mem. de la soc. med. des hop. de Paris, 26 juin, 1896, pp. 561-566. . WIDAL, P., ET SICARD, A. Etudes sur le sero- diagnostic et sur la reaction agglutinante chez les typhiques. Ann. de 1'Inst. Pasteur, T. xi, 1897, pp. 353-432. . FLEXNER, S. A recently discovered property of the blood .serum in animals immune from certain diseases, and its application to the diagnosis of those diseases in human beings. Science (n. s.), vol. v, pp. 193-194, 1897. . MALVOZ, E. Reoherches sur '1'agglutination du Bacillus typhosus .par des substances chimique. Ann. de 1'Inst. Pasteur, T. xi, J897, pp. 582-590. . NICOLLE, CHARLES. Recherches sur la sub- stance agglutinee. Ann. de 1'Inst. Pasteur,, T. xii, 1898, pp. 161-191. . BORDET, JULES. Le mechanisme de 1'agglutina- tion. Ann. de 1'Inst. Pasteur, T. xm, 1899, pp. 225-250. . KRAUS, R. Ein Beitrag zur Kenntniss des Mechanismus der agglutination. Wiener Klin. Wochenschr. 1899, Jahrg. xn, pp. 1-4. . GRUBER. Zur Theorie der Agglutination. Munch, med. Wochenschr., 1899, No. 41. Rev. in Centralb. f. Bakt., xxvn Bd., igoo, pp. 285-286. . SABRAZES ET BRENGUES. Agglutinines chim- iques. C. R. de la Soc. de biol., 1899, No. 35. P- 930. Rev. in Centralb. f. Bakt., xxvn Bd., 1900, p. 756. . ZIKES. Ueber das Ausschleudern von Mikro- organismen unter Zuhilfenahme von Fal- lungsmitteln. Oesterr. Chemiker-Zeitung, 1900, No. 2. Rev. in Centralb. f. Bakt., xxvii Bd., 1900, p. 628. . SMITH, R. GREIG. The flocculation of bacteria. The mechanism of agglutination. Proceed- ings of Linn. Soc. of New South Wales, 1900, Part i, pp. 65-74, 75-83. Also a sepa- rate (issued Aug. 8, 1900). . DURHAM, HERBERT E. Some theoretical con- siderations upon the nature of agglutinins, together with further observations upon Bacillus typhi abdominalis, Bacillus enteri- tidis, Bacillus coli communis, Bacillus lactis aerogenes, and some other bacilli of allied character. Jour, of Exp. Med., vol. v, pp. 353-388. 250 BACTERIA IN RELATION TO PLANT DISEASES. ('01). WILSON, ROBERT J. Observations contributing to precision in the use of the Widal test for typhoid. N. Y. Univ. Bull, of the Med. Sci., vol. i, No. 2, 1901, pp. 87-92. ('or). DODGE, CHARLES WRIGHT. A short method for the Widal test. Jour, of Applied Micro., vol. iv, 1901, p. 1,565. Also a separate. ('02). NEUFELD, F. Ueber die Agglutination der Pncumokokken und iiber die Theorieen der Agglutination. Zeitschr. f. Hyg., 1902, Bd. XL, pp. 54-72. ('02). EISENBERG, PHILIPP, UND VOLK, RICHARD. Untersuchungen iiber Agglutination. Zeitsch. f. Hyg., 1902, Bd. XL, pp. 155-195- Bibliog. of 56 titles. ('02). Joos, A. Untersuchungen iiber den Mechan- ismus der Agglutination. Zeitsch. f. Hyg., 1902, Bd. XL, pp. 203-230. ('02). SMITH, R. GREW,. Further remarks upon the mechanism oi agglutination. Proc. Linnean Soc. of New South Wales, vol. xxvn, 1902, Part i, pp. 66-72. Also a separate (issued Aug. 22, 1902). ('03). FLEXNER, SIMON. An aspect of modern path- ology. Science, n. s., vol. xvin, No. 444, 1903, PP- 3-15- XXXVIII. Antiseptics and Germicides. (See also XXXVI.) ('70). LISTER, JOSEPH. On the effects of the anti- septic system of treatment upon the salu- brity of a surgical hospital. Edinburgh, Edmonston and Douglas, 1870, pp. 19. ('74). DAVAINE, C. Reoherches relatives a 1'action des substances antiseptiques sur le virus de la septicemie. Gaz. med. de Paris, 1874, p. 44. Reprinted in 1'Oeuvre de Davaine, Paris, 1889. ('75)- LEWIN, L. Das Thymol ein Antisepticum mid Antifermentativum. Virchow's Archiv., Bd. LXV, 1875, pp. 164-189. Polli's Annali di chimica applicata alia med. Milano, vol. LXII, 1876, pp. 321-324. ('77). POLLI, G. Sulle proprieta anti fermentative dell'acido boracico e sue applicazioni alia terapia. Mem. 1st. Lomb., vol. xm, pp. 453- 468. Journ. de Pharm., et de chimie, T. xxvi, 1877, 4 se., pp. 77-79. ('79). BOVET, V. Ueber die antiseptischen Eigen- sohaften der Pyrogallussaure. Journ. f. prakt. Chem. Neue Folge, Bd. xix, pp. 445-461, 1879. ('80). PAVESI, C. Del solfato di potassa, e special- mente della sua proprieta antisettica, anti- fermentativa. Polli. Annali, vol LXXI, serie 3a, 1880, pp. 110-115. C8o). ENDEMANN, H. Boracic acid as a preservative. Chem. News, vol. XLI, pp. 152-153, 1880. ('80). SCHWARTZ, NICOLAI. Ueber das Verbal-ten eini-ger Antisettica zu Tabacksinfusbacte- rien. Pharm. Zeitschr. f. Russhnd, Bd. xix, 1880, pp. 610-625, 641-658, 673-685. Tested chloroform and found it of little worth. Ex- periments were made with 40 substances. The tabular summary is on pp. 684-685. Picric acid heads the list for efficiency. ( ! 8o). REGNARD, PAUL. Influence de 1'eau oxygenee sur la fermentation. Gaz. med. de Paris T. n, 6 ser., 1880, p. 358. ('81). CHAPPUIS, E. Action de 1'ozone sur les germes conitenus dans 1'air. Bull, de la Soc. chim. de Paris, I sem., n. s., T. xxxv, Paris, 1881, p. 290. ('81). BARNES, J. B. The antiseptic properties of cinnamic acid. Pharmaceut. Jour, and Transactions, vol. xn, pp. 477- 478, 1881. ('81). JALAN DE LA CROIX, N. Das Verhalten der Bakterien des Fleischwassers gegen einige Antiseptica. Arch. f. exper. pathol. u. pharm., 1881, Bd. xm, pp. 175-255. Ber. d. deutsch chem. Gesellsch., Bd. xiv, pp. 2,835- 2,838. ('81). GOSSELIN, L., ET BERGERON, A. Recherches sur la valeur antiseptique de certaines substances et en particulier de la solution alcoolique de Gaultheria. Arch. gen. de med., Paris, 1881, vol. i, VHI, se., T. 7 (misprinted 6), 147 vol. de la collection, pp. 16-29. ('82). BERT, P., ET REGNARD, P. Action de 1'eau oxygenee sur les matieres organique et les fermentations. C. R. des se. de 1'Acad. des sci., T. xciv, 1882, pp. 1.383-1,386. ('82). BURCQ, V. Suir Faction desinfectante et anti- septique du cuivre. C. R. des se. de Facad. des sci., T. xcv, 1882, pp. 862-864. Workers in copper escape both cholera and typhoid fever. It is said that there has not been a single deatli from either disease in the Societe de Bonaccord (copper, bronze and brass workers) since its establishment in ;8ig. ('82). SCHIEFFERDECKER, P. Ueber eine neue Injec- tionsmasse zur Conservirung der Leichen fiir den Praparirsaal. Arch. f. Anat. u. Entwickeltingsgesch., 1882, pp. 197-198. ('82). VULPIAN. Etudes experimentales relatives a Faction que peut exercer le permanganate de potasse sur les venins, les virus et les maladies zymotiques. C. R. des se. de FAcad. des sci., T. xciv, 1882, pp. 613-617. Jour, de Pharm. et de Chimie, T. vi, 5 serie, 1882, pp. 100-104. ('86). UNNA, P. G. Ichthyol und Resorcin als Rep- rasentaten der Gruppe reduzicrender Heil- mittel. Hamburg, 1886. Unna's Derma- tologische Studien, 2 Heft, pp. 1-85. ('88). NUTTALL, GEO. Experimente iiber die bak- terienfeindlichen Einfliisse des thierischen Korpers. Zeitschr. f. Hyg., Bd. iv, 1888, pp. 353-394, i plate. ('88). SALKOWSKI, E. Ueber die antiseptische Wirk- ung des Chloroformvvassers. Deutsche med- ioin. Wochenschrift, 1888, Bd. xiv, pp. 309- 3". ('88). LOEW, O. Physiologische notizen iiber For- maldehyd. Miinchen. med. Wochenschr., 1888, Bd. xxxv, pp. 412-413. Physiol. Ges. Miinchen (1-6), 1888, pp. 39-41. Rev. in Oh. Centralb., 1889, LX Jahrg., Bd. i, p. 90. C88). BEHRING. Ueber QuecksilbersuWimat in ei-weisshakigen Fliissigkeiten. Central!), f. Bakt., 1888, Bd. in, pp. 27-30 and 64-66. ('89). FRAENKEL, CARL. Die Ei-nwirkung der Kohlen- saure auf die Lebensthiitigkcit der Mikro- organismen. Zeitsch. f. Hyg., Bd. v, 1889, PP- 332-362. ('90). ALTEHOEFER. Ueber die Desinfectionskraft von Wasserstoffsuperoxyd auf Wasser. Cen- tralb. f. Bakt., 1890, Bd. vm, pp. 129-137. ('90). KIRCHNER, M. Untersuchungen iiber der Ein- wirkung des Chloroforms auf die Bakterien. Zeitschr. f. Hyg., Bd. vm, pp. 465-488, 1890. ANTISEPTICS AND GERMICIDES. 251 Coo). SONNTAG, HERMANN. Ueber die Bedeutung des Ozons als Desinficiens. Zeitschr. fur Hyg., 1890, Bd. vm, pp. 95-136. ('91). TIZZONI, GUIDO, y. CATTANI, G. Ueber die Widerstandsfahigkeit der Tetanusbacillen gegen physikal-ische und chemische Einwirk- ungen. Archiv. f. exper. Path. u. Pharm., 1891, Bd. xxvin, pp. 41-60. ('91). FROELICH, O. Ueber das Ozon, dessen Her- stellung auf elektrischem Wege und dessen technische Anwendungen, inshesondere in der Gesundheitstechnik. Gesundsheits- Ingenieur, 1891, No. 16, pp. 543-551. ('91). FROELICH, O. Ueber das Ozon, dessen Her- stellung auf eloktrischem Wege und dessen technische Anwendungen. Electrotechnische Zeitschr., 1891, 12 Jahrg., pp. 340-344. Contains short paragraph on physiological action of ozone. Bacteria living in water are killed " siimmtlich." No experiments with pathogenic bacteria. It is still a question whether bacteria in the air are killed. CQI). f.nti.Acii. VAL. Ueber Lysol. Zeitschr. f. Hyg., Bd. x, 1891, pp. 167-196. Also a sepa- rate. Lysol is more active than carbolic acid or creolin. The hands maybe disinfected in a i per cent, solution without soap. Surgical instruments may be sterilized in \i per cent solution without the least injury. Walls may bedis- infected in a 3 per cent solution. It is to man tiie least poisonous of the antiseptics of its class. ('91). FISCHER. See XL. ('92). SCHLUETER. See xxxvi. ('92). RICHTET, CH. De Faction de quelques sels metaliques sur la fermentation lactique. C. R. des se. de 1'Acad. des sci., T. cxiv, 1892, pp. 1,494-1,496. f<;2). OHLMUELLER. Ueber die Einwirkung des Ozons auf Bakterien. Arbeiten aus dem Kaiserl. Gesundheitsamte, Bd. vm, 1892, I left i, pp. 229-251. Ozone in water is less effective as a germicide in pro- portion as the water contains more and more dead organic matter. It is not adapted to the disinfection of rooms. ('92). DELDRUECK. See xxxvi. ('92). HAMMER, HANS. Ueber die desinficirende Wirkung der Kresole und die Herstellung netrtraler wassriger Kresollosungen. II Mittheilung, Arch. f. Hyg., Bd. xiv, 1892, pp. 116-134. ('92). ARONSOHN, HANS. Ueber die antiseptischen Eigenschaften des Formaldehyde. Berl. klin. Wochenschr., 1892, Bd. xxix, No. 30, pp. 749-751- ('92). BERLIOZ, F., AND TRILLAT, F. Sur les pro- prieties des vapeurs du formal ou aldehyde formique. C. R. des se. de 1'Acad. des sci., 1892, T. cxv, pp. 290-292. ('92). HANKIN, E. L'action bactericide des eaux de la Jumna et du Gauge sur le microbe du cholera. Ann. de 1'Inst. Pasteur, T. x, pp. 5"-523. Cholera does not descend the rivers in India. Bacteria are much rarer in these rivers than in European rivers. The filtered, unboiled river water has a decided bacteri- cidal action on the cholera organism. When boiled tilt- water lost its germicidal property. ('93). UE CHRISTMAS. J. Sur la valeur antiseptique de 1'ozone. Ann. de 1'Inst. Pasteur, T. vn, 1893, PP- 776-78o. (*93)- SCHII.D. See xvni. (*93)- VOCES. See xxxvi. ('93). GREEN. Ueber den Werth der Kupfersalze als Desinfektionsmittel. Zeitschr. f. Hyg., Bd. xin, 1893, pp. 495-511. States that copper salts have considerable value as germicides, especially the soluble oiled. Cuprum bichlo- ratuni is considered most valuable. This is the only cop- per salt that is sufficiently active in solutions containing much albumen. For the treatment of wounds, copper bichlorate is much "belter than copper sulphate. ('93). LOEW, OSKAR. Ein natiirliches System der Giftwirkungen. Mtinchen, 1893, Wolff und Liineburg, pp. vm, 136. Rev. in Centralb. f. Bakt., 1893, Bd. xiv, p. 234. ('93). GRUBER, MAX. Ueber die Loslichkek der Kre- sole in Wasser und iiber die Verwendung ihrer wassrigen Losungen zur Desinfektion. Arch. f. Hyg., Bd. xvn, 1893, pp. 618-625. ('94). DIEUDONNE. See xxix. ('94). D'ARSONVAL ET CHARRIN. See xxxin. ('94). ABEL. See xvni. ('94). MIQUEL. De la desinfection des poussieres seches des appartements; and Contribution nouvelle a 1'etude de la desinfection par les vapeurs d'aldehyde formique. Ann. de micr., T. vi, 1894. See pages 257, 305, 396, 520, 588, and 621. ('94). POTTEVIN, HENRI. Recherches sur le pouvoir antiseptique de 1'aldehyde formique. Ann. de 1'Inst. Pasteur, T. vm, 1894. pp. 796-810. ('94). BOLTON, MEADE. The effect of various metals on the growth of certain bacteria. Internal. Med. Mag., December, 1894, PP- 812-822. Also a separate. Reviewed in Am. Nat., Oct., 1895, p. 933. ('94). SCHILOW, P. F. Ueber den Einfluss des Was- serstoffstiperoxydes auf einige pathogene Mikroorganismen. St. Petersb. med. Woch- enschr., 1894, No. 6. Rev. in Centralb. f. Bakt., Bd. xvi, 189.4, PP- 42-43. Cholera bacteria were destroyed in 3 minutes in 1:200; in 1:300 they were alive after I hour. Typhoid ba- cilli, in 1:100 to 1:200, were killed in 10 minutes ; in 1:1000, after i hour. Anthrax spores, in 14 per cent solution were killed in less than 3 minutes; a 2 per cent solution killed them in less than one hour; a l per cent solution did not kill in I hour. Staphylpcoccus pyogen. aureus, from cul- tures i day old, was killed in 1:100 in less than lo min- utes; in 1:200 it required more than 15 minutes. Diplo- coccus pneumoniae does not grow in bouillon to which hydrogen peroxide has been added in l:io,oooto 1:18,000. Solutions of 1:200 destroyed a one-day old culture in 15 minutes. ('94). WALLICZEK, HEINRICH. Die baktericiden Eigenschafiten der Gerbsaure (Tannin der Apotheken). Centralb. f. Bakt., Bd. xv, 1894, pp. 891-894. Tables showing effect of various per cents of tannin on B. coli, B. authracls, and Staphylococcus aureus. ('95). BURCKHARD, G. Zwei Beitrage zur Kenntnis der Formalinwirkung. Centralb. f. Bakt., xvni Bd., 1895, pp. 257-264. Twenty titles are cited at the end of this paper. ('95). VAN ERMENCEM, E. De la sterilization des eaux par 1'ozone. Ann. de 1'Inst. Pasteur, T. ix, 1895, pp. 673-709. Rev. in Centralb. f. Bakt., Bd. xix, 1896, pp. 836-838, 2 figs. Van Ermengem's report is favorable. ('95). SCHEPILEWSKY, EUGEN. Formaldeliyd als Desinfektionsmittel. (Dissert.) St. Peters- burg. 1895. (Russisch.) Rev. in Centralb. f. Bakt., Bd. xix, 1896, pp. 794-796. 252 BACTERIA IN RELATION TO PLANT DISEASES. ('95). GORIANSKY, G. J. Sur la disinfection des crachats phtisiques et des cultures tuber- culeuses par les solutions alcalines de good- ran et de vwiaigre de bois. Arch, des^ sci. biol., pub. par 1'Inst. imp. de med. exp. a St. Petersburg, Tome 3, 1895, pp. 148-166. Wood-vinegar is a very energetic disinfectant. lu quan- tity equal to the volume of sputum, and acting for 6 hours, it was found entirely efficient. Exposure of 4 hours is not sufficient in some cases to destroy B. tuber- culosis in sputum. In pure culture the organism is de- stroyed by exposure for l hour to this acid. ('95). D'ARSONVAL. Sur la production de 1'ozone concentre et sur ses effets bactericides. C. R. des se. et mem. de la soc. de foiol., Paris, 10 se., T. II, 1895, pp. 500-502. The writer's experiments were negative, and he is very skeptical as to germicidal power of ozone. ('95). FISCHER. See XL. ('96). WALTER, K. Zur Bedeutung des Formalins, bezw. Formaldehyde als Desinfekrions- mittel. Zeitschr. f. Hyg., Bd. xxi, 1896, pp. 421-451. Rev. in Cerrtralb. f. Bakt., xx Bd., 1896, p. 280. ('97). IWANOFP, W. A. Zur Frage iiber das Ein- dringen der Formalindampfe in die organ- ischen Gewebe. Centralb. f. Bakt., xxn Bd., 1897, pp. 50-58. Formalin vapor did not penetrate rapidly into the depths of the tissues tested (livers of rabbits and guinea pigs). ('97). WEYLAND, J. Desinfektionswirkung und Eiweissfallung chemischer Korper. Cen- tralb. f. Bakt., xxi Bd., 1897, pp. 798-802. ('97). FuERBRlNGER UND PREYHAN. Neue Unter- suchungen ii'ber die Desinfektion der Hande. Deutsche med. Wochenschr., 1897, No. 6. Rev. in 'Centralb. f. Bakt., xxi Bd., 1897, pp. 708-710. Authors recommend alcohol. A 2 per cent solution of mercuric chloride is still better. Both may be used, the latter preceded by the former. ('97). PODGORNY, K. M. Effect of iodine on patho- genic bacteria. Thesis of St. Petersburg, No. 36, 1897, pp. 74. ('97). SCHUMBURG. Ein neues Verfalhren zur Her- stellung keimfreien Trinkwassers. Deutsche med. Wochenschr., Bd. xxm, 1897, No. 10, pp. 145-146. Bromide treatment. ('98). MINERVINI, RAFAEL. Ueber die baktericide Wirkung des Alkohols. Zeitschr. f. Hyg., Bd. xxix, 1898, pp. 117-148. Bibliography of 18 titles. Ethyl alcohol has only a weak bactericidal action. It is most active in concentrations of 50 to 7o per cent. Al- coholic solutions of antiseptic substances are less active than water solutions. ('08). SCHULTZ, N. De I'actipn des antiseptiques sur le bac. pestis hommis et de la desinfection d'effeots et de locaux contamines par la peste bubonique. Arch, des >sci. biol. publiees par 1'inst. imper. de ined. exper. a St. Peters- bourg, T. vi, 1808, pp. 397-426, i plate. The appearance of the bacteria subjected to the anti- septics, as shown on the plate, strongly suggests the ap- pearance of organisms in old cultures, viz.: involution forms, and the two phenomena may be due to the same cause, the involution forms arising'from the harmful ac- tion of products excreted by the bacteria, or arising from the action of siibstances developed in the media as the result of bacterial occupation. ('98). Fi.uEGGE, C. Die Wohnungsdesinfektion durch Formaldehyd. Zeitschr. f. Hyg., Bd. xxix, 1898, pp. 276-308. ('98). FERMI. See xxxvi. ('98). POPOFF, S. P. Vergleichende Studien iiber die desinfizierende Wirkung reiner Sublimat- losungen und Kombinationen derselben mit anderen Desinficientien. (Diss.) St. Peters- burg, 1898. Rev. in Centralb. f. Bakt., xxv Bd., 1899, pp. 331-332. The power of i per cent sol. mercuric chloride is in- creased very decidedly by addition of i per cent hydro- chloric acid, or i per cent phenol, and by the addition of 2 per cent phenol a still more effective mixture is obtained. Addition of i to 2 per cent NaCl weakened the effect of the i per cent sublimate solution on some or- ganisms, but increased it on others. ('98). LUCAS-CHAMPIONNJERE. Sur la valeur anti- septique de 1'eau oxygenee. Bull, de I'acad. de med., 1898, T. xi,, serie 3, Paris, pp. 599- 6i 7 . ('99). MARMIER ET ABRAHAM. La sterilisation indus- trielle des eaux potables par 1'ozone. Rev. d'hyg. et de Police Sanitaire, Paris, 1899, T. xxi, pp. 540-554. Great things are claimed for this method. Only some specimens of Bacillus subtilis are said to have escaped destruction, and of these only one individual for each 15 cc. of water treated with a concentration of ozone equal to 6 milligrams per litre of air. ('99). STABLER, EDUARD. Ueber die Einwirkung von Kochsalz auf Bakterien, die bei den soge- nannten Fleischvergiftungen eine Rolle spielen. Arch. f. Hyg., Bd. xxxv, 1899, pp. 40-82. Rev. in Centralb. f. Bakt., xxvi Bd., 1899, p. 411. 2 to 3 clays tney were injured, nut alter tnai iney maae a luxuriant growth. The toleration limit for B. coli and B. enteritidis is between 7 and 8 per cent of NaCl, and that of B. morbificans bovis between 8 and 10 per cent. The writerof this abstract found certain plant bacteria much more sensitive to salt, e. g. Ps. hyacinthi was restrained by 1.5 per cent. ('99). BLISS, C. L., AND NOVY, PR. G. Action of formaldehyde on enzymes and on certain proteids. The Jour. Exp. Medicine, vol. iv, 1899, pp. 47-80. ('99). WEYL, TH. Keimfreies Trinkwasser mittels Ozon. Centralb. f. Bakt., xxvi Bd., 1899, o." Coi). GAZERT, HANS. Bakteriologische Aufgaben der deutschen Sudpolar-Expedition. Peter- martns geogr. Mitteil., Bd. XLVII, 1901, pp. I53-I55. Coi). BELLI, C. M. Chemische, mikroskopische und bakteriologische Untersuchungen iiber den Hagel. Hyg. Rdsch., Berlin, Bd. xi, 1901, pp. 1,181-1,187. ('02). BINOT, JEAN, fitude bacteriologique du massif du mont Blanc. Nature, Paris, (ler semest.), 1902, pp. 359-362, av. fig. C. R. des se. de 1'Acad. des sci., T. cxxxiv, 1902, pp. 673-676. 256 BACTERIA IN RELATION TO PLANT DISEASES. XLI1I. Soil-Organisms; Putrefactive Organisms. ('82). TYNDALL. See XLII. ('86). ADAMETZ. See XLII. ('86). BEUMER. See XLII. ('87). FRAENKEL. See XLII. ('89). REIMERS. See XLII. ('91). BERTHELOT, M., ET ANDRE, G. Sur 1'odeur prppre de la terre. C. R. de se. 1'Acad. des sci., Paris, 1891, T. cxn, 598. ('91). MANFREDI, LUICI. Sulla contaminazione della superficie stradale nelle grandi citta dal punto di vista dell'igiene e dell'ingegneria sanitaria. Recerche e studi fatti con speciale riguardo alia citta di Napoli. Atti della R. Accad. delle sci. fis. e mat. di Napoli. 2a serie, vol. IV, 1891, appendice, No. 4, pp. 1-79. ('93). DUCLAUX. See XLII. ('96). DUCLAUX. See xxvin. ('99). LEVIN. See XLII. ('01). SMITH, R. GREIG. Bacteria and the disintegra- tion of cement. Proc. Linn. Soc. of New South Wales, vol. xxvi, for the year 1901, Part i, Sydney, 1902, pp. 107-117. Also a separate (issued Aug. 13, 1901). Disintegration not due to the bacteria. ('02). KATAYAMA, T. On the general occurrence of Bacillus methylicus in the soil. Bull, of the College of Agr., Tokyo Imperial Univ., vol. v, No. 2, 1002, pp. 255-258. Also a separate. ('02). CHESTER, FREDERICK D. The bacteriological analysis of soils. Proc. 23d Ann. meeting of Soc. for Prom. Agric. Sci., 1902, pp. 173- 182. Also a separate. ('02). CHESTER, FREDERICK D. Bacteria of the soil in their relation to agriculture. Bulletin No. 98, Dept. of Agric. of Pennsylvania, 1902, pp. 88, with plates. A bibliography of 105 titles. ('02). REMY, TH. Bodenbakteriologische Studien. Centralb. Bakt., Abt. 2, Bd. vm, 1902, .pp. 657-662, pp. 699-705, pp. 728-735, pp. 761-769. ('04). CHESTER, FREDERICK D. Observations on an Important Group of Soil Bacteria. Organ- isms related to Bacillus .subtilis. Fifteenth Annual Report of the Delaware College Agrl. Exp. Sta., for 1903, Newark, Del., U. S. A. With 5 plates. Also a separate, pp. 1-54. Copy of separate received from author October 15, 1904. XLIV. Vinegar-Bacteria. ('61). PASTEUR. Acetic fermentation due to bac- teria. Ann. scient. de 1'Ecole normale supe- rieure, 1861. Not seen. ('68). PASTEUR, Louis. Etudes sur le vinaigre, sa fabrication, ses maladies, moyens de les prevenir; nouvelles observations sur la con- servation des vins par la chaleur. Paris, 1868. Gauthier-Villars, Imprimeur-Libraire. Victor Masson et Fils, Libraires, pp. vm, 1 19. ('86). BROWN. See XLI. ('86). ('87). (93). ('93). ('94). ('95). ('97). ('98). Coo). BROWN, A. J. The chemical actions of pure cultivations of Bacterium aceti. Jour. Chem. Soc. Trans., 1886, vol. XLIX, London, pp. 172-187. BROWN, A. J. Further notes on the chemical action of Bacterium aceti. Jour. Chem. Soc., London, 1887, vol. LI, Transactions, pp. 638- 642. HANSEN, EMIL CHR. Botanische Unter- suchungen iiber Essigsaurebakterien. Ber. d. deutsch. >bot. Gesellsch., Bd. xi, 1893, pp. (69) -(73). General Versammlungs-Heft. LAFAR, F. Physiologist-he studien iiber Essig- garung und Schnell-Essigfabrikation. Cen- tralb. f. Bakt., 1893, Bd. xni, pp. 684-697. Bibliography of 13 titles. HANSEN, E. C. Recherches sur les bacteries acetifiantes. Compt. rend. d. trav. du Lab. Carlsberg, T. in, Liv. 3, pp. 182-216. HANSEN, EMIL CH. Recherches sur les bac- teries acetifiantes. Ann. de micrographie, T. vi, 1894, No. 8, pp. 385-395; No. 9, pp. 441-470. Also a separate, pp. 41. 14 text figs. LAFAR. Physiologische studien iiber Essig- garung und Schnellessigfabrikation. Cen- tralb. f. Bakt., 2 Abt., Bd. I, 1895, pp. 129- ISO. LAFAR. See in. BEYERINCK, M. W. Ueber die Arten der Es- sigbakterien. Centralb. f. Bakt., 2 Abt., Bd. iv, 1898, pp. 209-216. HANSEN, EMIL CH. Recherches sur les bac- teries acetifiantes. (Troisieme memoire.) C. R. des travaux du laboratoire de Carls- berg, T. v, ire Livraison, 1900, pp. 39-46, I fig. Also a separate. Copenhagen, 1900. XLV. Silage-Bacteria, Fermentation of Tobacco, of Indigo, Retting of Flax, of Sisal Hemp, Etc., Softening of Pickles, Sauerkraut, Etc. (See also XX and XLIV.) ("87). ALVAREZ, E. Sur un nouyeau microbe, determ- inant la fermentation indigotique et la pro- duction de 1'indigo bleu. C. R. des se. de 1'Acad. des sci., Paris, 1887, T. cv, pp. 286- 289. ('89). BURRILL, T. J. The biology of ensilage. Bull. Ag. Exp. St. Univ. of 111., 1889, No. vn, pp. I77-I94. ('91). ALBERT, FRIEDRICH. Untersuchungen iiber Grunpressf utter. Jahrb. d. deutsch. Land- wirtsch. -Gesellsch., Bd. vi, Tl. i, pp. 149- 250, Berlin, 1891. This author says bacteria exert a preponderant influ- ence on the course of the fermentation. ('91). SUCHSLAND, EMIL. Ueber Tabaks fermenta- tion. Ber. d. deutsch. 'hot. Gesselsch., Bd. ix, Berlin, 1891, pp. 79-81. ('94). VAN LOOKEREN-CAMPAGNE, C. J. Bericht iiber Indigo-Untersuchungen, ausgefiihrt an der Versuchs-Station zti Klatten auf Java. D. landw. Vers.-Stat, 1894, Bd. XLIII, pp. 401- 426. SILAGE-BACTERIA, FERMENTATION OF TOBACCO, INDIGO, FLAX, ETC. 257 ('95)- WINOGRADSKY, SERGius. Sur le rouissage du lin et son agent tnicrobien. C. R. des se, de 1'Acad. des sci., Paris, 1895, T. cxxi, pp. 74^-745- A rsum of the principal results of work doue by Fribes iu Wiuogradsky's laboratory. 1. Stems sterilized under water'by a short heating at 100, repeated three days in succession, or by one heating at 115 for fifteen minutes, did not become retted. 2. Sterilized flax immersed in water and inoculated with any one of the maerobic and anaerobic organisms, first isolated from the macerations by means of gelatin plates, did not undergo, even after several mouths, a commencement of retting, nor was any liberation of gas noticed. On the contrary 3. If into tubes of water containing sterilized flax a small hit of straw of misterilized flax was thrown a very active fermentation commenced at the end of 12 to 15 hours, and at the end of two or three days the retting was completed. The specific organism was obtained for study from suc- cessive cultures upon steam -sterilized flax, protected from the air by immersion in deep tubes full of water, whose surface was covered by a layer of oil. After a long enough series of re-sowing under these same conditions, the microscopic study of these cultures has removed all doubt about the agent of this fermentation. It is found almost pure in the interior of the stem, and Fribes has succeeded iu isolating it in a completely pure state by cultivating it, in the absence of air, upon slices of cooked potato rubbed with chalk. It is a bacillus relatively large, forming spores in the terminal swellings (tadpole form). In the young state its rods are from 10 to 15 m long, with a th'ickuess of 0.8 M; often one finds articu- lated filaments much longer ; thev become later a little thicker { i ju), and form then ovoid swellings 3 ^ long by 2 n thick ; the ovoid spore which is formed there is 1.8 j* by 1.2 ft. Sterilized flax was retted in pure cultures of this bacillus, and, after undergoing the successive opera- tions of grinding (dressing) peeling and combing, yielded a fine silky flax of light color but a little too much retted and without consistence. In a general study of the bacteria of retting, Fribes dis- covered the following facts : 1. The bacillus ferments glucose, cane-sugar, milk- sugar, and starch, but only when the liquid contains pep- tone. With ammonia as the only source of nitrogen, the bacillus is absolutely void of action on these eminently fermentable substances. 2. Pectic matters, pectine, or pectic acid, extracted from flax, pears, carrots, white turnips, pure as they can be prepared, are decomposed, in presence of an am- monium salt as the sole nitrogenous food, with an extra- ordinary facility. 3. Cellulose, under the form of Swedish filter paper, or as an amorphous precipitate, can absolutely not he attacked by this bacillus. Gum arable is not fermented. 4. Vegetable substance, from flax, white turnips, ex- tracted cold by pure water and water slightly acid and alkaline, and submitted to fermentation by this bacillus, loses the greater part of the substances which are esti- mated as pectic matter ; also the loss of weight of the fermented substance corresponds sensibly to the content in pectic matter of the nufermented substance. As a result of these studies Winogradsky concludes that the retting of flax may be considered as a pectic fermen- tation in the micro-biological sense of the word, of which the bacillus described is the specific agent. 1/96). TOI,OMEI, Giuiyio. Ueber die Fermentation der Oliven und die Oxydation des Olivenoles. Atti R. Acad. dei Lincei Roma, se. v, Rendi- conti, Classe sci. fis., matem. e nat., vol. V, Feb. 16, 1896, pp. 122-129. Due to an enzyme. Not bacterial. ('96). VAN LOOKEREN-CAMPAGNE, C. J., y. VAN DER VEEN, P. J. Ueber Indigpbildung aus Pflanzen der Gattttng "Indigo fera." D. landw. Vers.-Stat., 1896, Bd. XLVI, pp. 249- 258- ('97). CONRAD, EUGEN. Bakteriologische und chem- ische studien iiber Sauerkrautgahrung. Arch. f. Hyg., Bd. xxix, 1897, pp. 56-9$- See also Zeitschr. f. Spiritusindustrie, xx Jahrg., 1897, No. 23, p. 188; No. 24, pp. 2OO-2OI. The fermentation of the " Weisskraut " is attributed to Bacterium brassica; acidse, Lehm. Conrad, nearly re- lated to Bacillus coli. This organism is motile, grows aerobically and anaerobically, produces acids, carbon dixoide, hydrogen and marsh gas. Gelatin is not liquefied. The surface colonies are gray white to gray yellow on gelatin and agar, and are bright yellow on potato. It ferments maltose, lactose and dextrose. Most of the acid is lactic acid. ('98). BREAUDAT, L. Sur le mode de formation de I'indigo dans les procedes d'extraction in- dustriels. Fonctions disastasiques des plantes indigpferes. C. R. des se. de 1'Acad. des sci., Paris, 1898, T. cxxvn, pp. 769-771. ('98). MOLISCH. Ueber die sogenannte Indigogahr- ung und neue Indigopflanzen. Sitzungsber. d. k. Akad. der Wiss. in Wien, July, 1898. Title only. ('98). PRESCOTT AND UNDERWOOD. See xx. ('99). BREAUDAT. Nouvelles recherches sur les fonc- tions diastasiques des plantes indigoferes. C. R. des se. de 1'Acad. des sci., Paris, T. cxxvni, 1899, pp. 1,478-1,480. ('99). VERNHOUT, J. H. Onderzoek over bacterien bij de fermentatie der tabak. Mededeelingen uit s'Lands Plantentuin, xxxiv, Batavia, G. Kolff & Co., 1809, p. 49, 2 plates. The fermentation of tobacco is ascribed to Bacillus tabaci-fermentationis Vernhout. ('99). LoEw, OSCAR. Curing and fermentation of cigar leaf tobacco. U. S. Dept. of Agric., Report No. 59, Div. Veg. Phys. & Path., 34 PP- ('oo). LOEW, OSCAR. Sind Bakterien die Ursache der Tabakfer.menta'tion ? Centralb. f. Bakt., 2 Abt., Bd. vi, 1900, pp. 108-112. The fermentation is strictly enzymic, and bacteria are not concerned in it. ('oo). LoEW, OSCAR. Physiological Studies of Con- necticut leaf tobacco. U. S. Dept. of Agric., Div. of Veg. Phys. & Path., 1900. Report 65. 57 PP- Coo). BEIJERINCK. Verdere onderzoekingen over de indigovorming uit weedl (Isatis tinctoria). Proc. K. Akad. Wetensch. Amsterdam, Deel ix, June 30, 1900, pp. 74-90. Abstr. in Bot. Zekung, 2 Abt., vol. 58, 1900, col. 188-189. The production of indigo blue is due to the action of the enzyme isatase upon isatan. Isatan occurs m the protoplasm ; isatase occurs in the chromatophores. In the living cell the author thinks reactions are prevented by the acidity of the cell-sap. He says : The action of isatase upon isatan is possible only in neutral or ampho- teric and very weakly acid solutions." A temperature of 48 to 50 C. is the optimum for this action. Coi) LOEW, OSCAR. Catalase, a new enzym of gen- eral occurrence, with special reference to the tobacco plant. U. S. Dept. of Agric. Rep. 69, Div. Veg. Phys. & Path., 1901, 47 PP- ('oi). RUSSELL, H. L., AND BABCOCK, S. M. Concern- ing the theories of silage formation. Science, n. s., vol. xni, p. 328, 1901. The conclusion reached is that bacteria do not play any very considerable role in the fermentation of silage. ('oi) PREYER, AXEL. Ueber Kakaofermemation. Tropenpflanzer, Berlin, Bd. v, 1901, pp. 157- 173. ('oi) SCHULTE IM HoFE, A. Zur Kakao-Fermenta- tion. Tropenpflanzer, Berlin, 1901, Bd. v, pp. 225-227. 258 BACTERIA IN RELATION TO PLANT DISEASES. ('02). BABCOCK, S. M., UND RUSSELL, H. L. Die bei der Herstellung von Garfutter (Silage) wirkenden Ursachen. Centralb. f. Bakt., Abt. 2, Bd. ix, 1902, pp. 81-88. ('02). HAUMAN, L. fitude microbiologique et chimique du rouissage aerobic du lin. Ann. de 1'Inst. Pasteur, T. xvi, 1902, pp. 379-385. ('02). BAIL, OSKAR. Untersuchung einiger bei der Verwesung pflanzlicher Stoffe thatiger Sprosspilze. Centralb. f. Bakt., Abt. 2, Bd. vin, 1902, pp. 567-584. ('03). WEHMER, C. Die Sauerkrautgiirung. Cen- tralb. f. Bakt., 2 Abt., 1903, x Bd., pp. 625-629. ('04). STOERMER, K. Ueber die Wasserroste des Flachses. Ceiitralblatt f. Bakt., 2 Abt., Bd. XHI, No. 1-3, Sept., 1904, pp. 35-45- Con- tinued. ('04). BEIJERINCK, M. W., AND VAN DELDEN, A. "On the bacteria which are active in flax-ret- ting." Koninklijke Akademie van Weten- schappen te Amsterdam. Proceedings of the Meeting of Jan. 30, 1904. Pub. Feb. 25, 1904, pp. 462-481, i .plate. Also a separate. ('04). OMLIANSKI, W. Die histologischen und chemischen Veranderungen der Leinstengel unter Einwirkurg der Mikroben der Pektin- und Cellulosegarung. Centralb. f. Bakt., 2 Abt., xii Bd., igo4, pp. 33-43, i plate. ('04). WEHMER, C. Die Sauerkrautgaerung. Be- richt des V. Internationale!! Kongresses f. angewandte Chemie zti Berlin, 1903. Sekt. VI, Bd. in, Berlin, 1904, p. 712. Also a separate, 5 pp. Obtains results decidedly different from Conrad. The ordinary sauerkraut fermentation is a mixed fermenta- tion. Gas-development is due to a bottom yeast; acid production to a non-liquefying, non-motile, non-gas- fonniug, lactic acid bacterium. XLVI. Bacteria in Water and Ice; Dung-Bacteria. (See also XXXIII and XXXIV.) ('76). WARMING. See xn. ('86). FRAENKEL, CARL. Ueber den Bakteriengehalt des Eises. Zeitschr. f. Hyg., Bd. i, 1886, pp. 302-314. (87). MACE. >Sur quelques bacteries des eaux de boisson. Ann. d'hyg. publ. et de med. leg., avril, 1887. 3d series, T. xvn, pp. 354-357- (87). PRUDDEN, T. MITCHELL. On Bacteria in ice and their relation to disease, with special reference to the ice supply of New York City. Med. Record, March 26 and April 8, 1887, Nos. 13 and 14; also a separate, 61 pp. Reviewed in Centralb. f. Bakt., Bd. I, 1887, pp. 650-652, and Ann. de J'Inst. Pasteur, T. I, 1887, pp. 409-410. Dr. Prudden tested tlie resistance of various bacteria to prolonged cold, in blocks of ice and to repeated freezings and thawiugs. Proteus vulgaris and Bacillus prodigiosus did not grow after 51 days freezing. A slender liquefying bacillus from Crotou water was killed in seven days The following withstood freezing : Staphylococcus pyo- genes (66 days) ; a fluorescent bacillus from ice (77 days) ; bacillus of typhoid fever (123 days) cultures made at intervals showed less and less living, but all were not destroyed. Repeated freezings and thawings were more fatal to the typhoid bacillus than a constant low tem- perature. Five freezings and thawings at intervals of three days destroyed tliis bacillus. ('87). BORDONI-UFFREDUZZI, GuiDO. Die biologiscbe Untersuchung des Eises in seiner Beziehung zur offentlichen Gesundheitspflege. Cen- tralb. f. Bakt., 1887, Bd. H, pp. 489-497. ('88). SCHMELCK, L. Eine Gletscherbakterie. Cen- tralb. f. Bakt., 1888, Bd. iv. pp. 545-547. A green fluorescent organism was the commonest form This was a short rod which liquefied gelatin. ('91). VIRON, L. Du role des Schizophytes dans les reactions qui se passent dans les eaux dis- tillees. Jour, de Pharm. et de Chini., 1891, T. xxm, series 5, pp. 586-593. ('91). NORDT MEYER. See xvn. ('94). LASER. See xx. ('94). HOUSTON. See xvn. ('94). FRANKLAND, P., AND MRS. P. Micro-organ- isms in water. London, 1884, pp. xr, 532. ('95). SEDGWICK UND PRESCOTT. On the influence of variations in the composition of nutrient gelatin upon the development of water bac- teria. Am. Pub. Health Asso., vol. xx, 1895, pp. 450-458. Rev. in Centralb. f. Bakt., xix Bd., 1896, p. 222. ('95). RABINOWITSCH. See xxxiv. ('95)- SMITH, THEOBALD. Notes on bacillus coli communis and related forms; together with some suggestions concerning the bacterio- logical examination of drinking water. The Amer. Journal of the Med. Sci., Sept., 1895. Also a separate, pp. 20. ('95). SMITH, THEOBALD. Ueber den Nachweis des Bacillus coli communis im Wasser. Cen- tralb. f. Bakt., xvin Bd., 1895, pp. 494-495. ('95). SEVERIN, S. A. Die im Miste vorkommenden Bakterien und deren physiologische Rolle bei der Zersetzung derselben. Centralb. f. Bakt., 2 Abt., Bd. I, 1895, pp. 97-114 and 709-817. ('97). HESSE, FRIEDR. Ueber die Verwendung von Nahragar-Agar zu Wasseruntersuchungen. Centralb. f. Bakt., xxi Bd., 1897, pp. 932-937. Finds agar-media better than gelatin on account of number of liquefying organisms constantly present in water. ('97). KERN, HEINRICH. Beitrag zur Kenntniss der im Darme und Magen der Vogel vorkom- menden Bacterien. Arb. a. d. bact. I list. d. tech. Hochschule zu Karlsruhe, Bd. i, Heft iv, 1897, pp. 379-532. Many bacteria are described at length. CgS). WARD, H. MARSHALL. Some Thames Bac- teria. Annals of Botany, vol. xn, 1898, pp. 287-322. Two double plates in color. This paper treats of (i) A short colorless bacterium forming stearine-like colonies : type of Bacterium ureae (Jaksch); (2) A colorless capsuled coccus or bacterium ; (3) rose-pink Micrococcus : type of M. carneus (Zimni.) ; (4) A pseudo-bacillus. ('99). KASANSKY. See xxxm. Cc9). FULLER, GEORGE W., AND JOHNSON, GEORGE A. On the differentiation and classification of water bacteria. Jour. Exp. Med., vol. iv, 1899, PP- 609-626; also a separate. Coo). KOHLBRUGGE, J. H. F. Vibrion-Studieii. i. Die Ubiquitat choleraahnlicher Wasser- vibrionen. Centralb. f. Bakt., xxvm Bd., 1900, pp. 721-726. it. Panmorphismus und erbliche Variationen. Ibid, pp. 833-842. Coo). SEUGWICK AND WINSLOW. See xxxm. Coi). PARK .See xxxm. Coi). HORHOCKS, W. H. An introduction to the bac- teriological examination of water. London, J. & A. Churchill, 1901, pp. x, 300, 5 plates. MILK-HACTERIA, BUTTER-BACTERIA, CHEESE-BACTERIA, MEAT-BACTERIA. 259 ('01). GAGE, STEPHEN DE M. Bacteriological Studies at the Lawrence Experiment Station, with special reference to the determination of B. coli. 33d An. Rep. St. Bd. of Health of Mass., for 1901, pp. 397-420. Also a sepa- rate, pp. 26. Coi). Micur.A, W. Compendium der bakteriologis- chen Wasseruntersuchung nebst vollstaen- diger Ucbersicht dcr Trinkwasserbakterien. O. Nemnich, Wiesbaden, 1901, pp. vn, 440, with 2 plates. ('02). ('03). Coj). (03). (03). ('04). (04). MACFADYEN. See xxxm. JORDAN, EDWIN OAKES. The kinds of Bacteria found in river water. Journal of Hygiene, vol. in, No. i, 1903. Also a separate, pp. 1-27. IMMENDORF, H. Ueber Stallmist-Bewahrung (Konservierung) mil Chemischen Mitteln. Berlin, Mitt. d. Landw. Ges., Bd. xvm, 1903. pp. 99-101. SCHUEDER. See xvm. WlNSLOW AND NlBECKER. See XVII. GAGE AND ADAMS. See xvi. STOKES. See xvm. XLVII. Milk-Bacteria; Butter-Bacteria; Cheese- Bacteria; Meat-Bacteria. C8i). JALAN DE LA CROIX. See xxxvm. I'Sj). ScHMiDT-MuEHLHEiM. Untersuchungeti iiber fadenziehende Milch. Pfliiger's Archiv., 1882, Bd. xxvii, pp. 490-510, i fig. ('84). HuEi'PE, FERDINAND. Untersuchungen iiber die Zersetzungen der Milch durch Micro- organismen. Mitth. a. d. K. Gesundheits- amte, Bd. II, Berlin, 1884, pp. 309-371. ('89). MENGE, KARL. Ueber rothe Milch. Centralb. f. Bakt., vi Bd., 1889. pp. 596-602. ('89). BAGINSKY, ADOLF. Rote Milch. Deutsche Medizinal-Zeitung ,1889, No. 9, pp. 106-107. ('89). BAGINSKY, ADOLF. Zum Grotenfelt'schen Bacillus der roten Milch. Deutsche mediz. Wochenschrift, 1889, Bd. xv, p. 212. Tliis organism was isolated from feces. It liquefied gelatin slowly and colored milk a dirty red or red-brown. ('91). CONN, H. W. Ueber einen bittere Milch erzeugenden Micrococcus. Centralb. f. Bakt., ix Bd., 1891, pp. 653-655. ('91). ADAMETZ, L. Untersuchungen iiber Bacillus lactis viscosus, einen weitverbreketen milch- wirthschaftliohen Schadling. Berliner land- wirthschaftlichc Jahrbiicher, 1891, Bd. XX, pp. 185-207, i plate. Rev. in Centralb. f. Bakt., ix Bd., 1891, pp. 698-700. ('9-0. NENCKI, L., ET ZAWADZKI, J. Sur la sterilisa- tion du lait. Arch, des Sci. Biol. publiees par L'institut Imperial de Med. Exp. a St. Petersbourg, T. I, 1892, pp. 371-397. Contains a bibliography of 50 numbers. ('93). DUCLAUX, E. Sur le role protecteur des microbes dans la creme et les fromages. Ann. de I'lnst. Pasteur, T. vn, 1893, pp. 305-324- ('93). BLEISCH, MAX. Ueber bittere Milch mid die Sterilisierung der Milch durch Erhitzen unter Luftabschluss. Zeitschr. f. Hyg., 1893, Bd. xin, pp. 81-99. ('94). BORDONI-UFFREDUZZI, GUIDO. Ein Fall von fuchsinahnlicher Bakterienfarbung des Fleisches. Hygien. Rundschau., 1894, Bd. iv, pp. 12-14. ('94). v. KLECKI, VALERIAN. Ueber einige aus ran- ziger Butter kultivierte Mikroorganismen. Centralb. f. Bakt., Bd. xv, 1894, pp. 354-362. ('94). LEICHMANN, G. Ueber eine schleimige Garung der Milch. Landw. Ver.-Stat., Bd. XLIII, 1894, pp. 375-398. Rev. in Centralb. f. Bakt., Bd. xvi, 1894, pp. 122-123. ('94). RussELL, H. L. Outlines of dairy Bacteriology, pp. vi, 186, 1894. Pub. by author. Madison, Wisconsin. ('94). PAMMEL, L. H. An aromatic bacillus of cheese (Bacillus aromaticus n. sp.). Extracts from the Iowa Agricultural Exp. Station, Bull. No. 21, 1894, pp. 1-5. Rev. in Centralb. f. Bakt., Bd. xvi, 1894, p. 128. ('94). WEIGMANN, H., UND ZIRN, GG. Ueber "seifige" Milch. Centralb. f. Bakt., Bd. xv, 1894, pp. 4 3-47 O > -mit 2 Abbildungen. ('94). DUCLAUX, EMILE. Le lait; etudes chimiques et microbiologit|iies. 2 tirage, augmente de notes sur le role des microbes et sur les phosphates du lait. 376 pp., I2mo. Paris, J. B. Bailliere & fils., 1894. ('94). HENRICI, H. Beitrag zur Bakterienflora des Kiises. Arb. a. d. bact. Inst. d. tech. Hoch- schule zu Karlsruhe, Bd. i, Heft i, 1804, pp. i-iio. ('95). RABINOWITSCH. See xxxiv. ('95). JOLLES, MAX, u. WINKI.ER. FERDINAND. Bak- teriologische Studien iiber Margarin und Margarinproductes. Zeitschr. f. Hyg., Bd. xx, 1895, pp. 60-108. The bacterial content of margariu products is slight in comparison with that of natural butter. ('95). CONN, H. W. Bacteria in the dairy, vi. Ex- periments in ripening cream with Bacillus - No. 41. 7th Ann. Rep. of the Storrs Ag. Ex. Station for 1894, pp. 57-68. Middletown, Conn., 1895. ('96). CONN, H. W. The relation of pure cultures to the acid, flavor, and arorna of butter. Cen- tralb. f. Bakt., 2 Abt., Bd. 11, 1896, pp. 409- 415. ('97). CONN, H. W. Butter arorna. Centralb. f. Bakt., 2 Abt., Bd. ill, 1897, pp. 177-179. ('59). MAASZEN, ALBERT. Fruohtatherbildende Bak- terien. Arb. a. d. k. Gesundheitsamte, Bd. xv, Berlin, 1899, pp. 500-513, 3 pi. from photomicrographs. The plates and text deal largely with Bacillus esterifi- caus, B. ester fluoresceus aim B. prjepolleus. There are numerous references to literature. ('59). MOORE, V. A., AND WARD, A. R. An inquiry concerning the source of gas and taint-pro- ducing bacteria in cheese curd. Bull. No. 158, Cornell Univ. Agr. Exp. Station, 1899, pp. 221-237, I plate. ('99). WARD, ARCHIBALD R. Ropiness in milk and cream. Cornell Univ. Agr. Exp. Sta. Dairy Div. Bui. 165, 1899, pp. 395-412, 4 figs. Ropiness attributed to Bacillus lactisviscosus. ('99). STADLER. See xxxvui. Coo). WEBER, A. Die Bakterien der sogcnannten sterilisirten Milch des Handles ihre biolo- gischen Eigenschaften, etc. Arb. a. d. k. Gesundheitsamte, Bd. xvn, 1900, pp. 108-155. With bibliography of 225 titles. 260 BACTERIA IN RELATION TO PLANT DISEASES. ('oi). CHODAT, R., ET HOFMAN-BANG, N. O. Les bacteries lactiques et !eur importances dans la maturation du fromage. Ann. de 1'Inst. Pasteur, T. xv, 1901, pp. 36-48. Discusses relation of tyrothrix to ripening of cheese. ('oi). WARD, ARCHIBALD R. Further observations upon ropiness in milk and cream. Cornell Univ. Agr. Exp. Sta. Dairy Div. Bull. 195, 1901, pp. 29-39, 2 figs. ('oi). PARK, WM. HALI.OCK. The great bacterial contamination of the milk of cities. Can it be lessened by the action of Health authori- ties? 'The Journ. of Hygiene, vol. I, 1901, pp. 391-406. See also N. Y. Univ. Bull, of the Med. Sci., vol. i, 1901, pp. 7i-6. ('02). FISCHER, BERNHARD. Zur Aetiologie der sogenannten Fleisohvergiftungen. Zeitschr. f. Hyg., Bd. xxxix, 1902, pp. 447-510, 2 plates. With a bibliography of 31 titles. ('02). EPSTEIN, ST. Untersuchungen iiber die Reif- ung von Weichkasen. Arch. f. Hyg., Bd. XLIII, 1902, pp. 1-20. ('02). CONN, H. W., AND ESTEN, W. M. The com- parative growth of different species of bac- teria in normal milk. Fourteenth Ann. Rep. Storrs Agr. Exp. Station, Storrs, Conn., 1001, pp. 13-80. Middletown, Conn., 1902. Relates to those bacteria occurring naturally in milk and not to those introduced by the bacteriologist. ('02). GRUBER, TH. Ueber einen die Milch rosafar- benden Bacillus. Bacillus lactorubefaciens. Centralb. f. Bakt, Abt. 2, Bd. vin, 1902, pp. 457-462. ('02). CRUDER, TH. Ueber eine in der Milch Riiben- geruch tind Rubengeschmack erzeugende Bakterie. Molk. Zeitung, Hildesheim, Bd. xvi, 1002, pp. 351-353- ('02). HARDING, H. A., UNO ROGERS, L. A. Rost- flecken in Cheddarkase. Centralb. f. Bakt., Abt. 2, Bd. vin, 1902, pp. 442-443. Ascribed to Bacillus rudensis. ('02). RICHTER, ALBRECHT P. F. Bakterielles Ver- halten der Milch bei Boraxzusajtz. Arch. f. Hyg., Bd. XLIII, 1902, pp. 151-156. ('02). GRIMM, MAX. Ueber einen neuen aroma- bildenden Bacillus nebst einigen Bemer- kungen i\l>er Reinkulturen fur Exportbutter. Centralb. f. Bakt., Abt. 2, Bd. VIH, 1902, pp. 584-590. ('02). ROSAM, A. Ueber Konservierung der Milch mittels WassersiofTsuperoxyd. Centralb. f. Bakt., Abt. 2, Bd. vin, 1902, pp. 739-744, pp. 769-774. ('03). HARRISON, F. C., AND GUMMING, M. The bac- terial flora of freshly-drawn milk, Part rv. Journal of Applied Microscopy, vol. VI, 1903, No. 2, p. 3,181. Bibliography of 25 titles. ('03). SWITHINBANK, HAROLD, AND NEWMAN, GEORGE. Bacteriology of Milk. With special chapters by Dr. Newman on the spread of disease by milk and the control of the milk supply. With chromo-lithographs, Woodfoury type reproductions of photographs of cultures, and other illustrations of bacteria and of apparatus, and also charts illustrating epi- demics. London, 1903, John Murray, pp. xx, 605. ('03). CONN, H. W., AND STOCKING, W. A., JR. Com- parison of bacteria in strained and un- strained samples of milk. Rep't of Storrs Agr. Exp. Station, Conn., 1902-3, pp. 33-37. ('03). CONN, H. W., AND STOCKING, W. A., JR. Series n. Strained and unstrained milk preserved at 70 and 50. Rep't of Storrs Agr. Exp. Sta., Conn., 1902-3, pp. 38-51. ('03). CONN, H. W., AND STOCKING, W. A., JR. Series HI. Aseptic milk. Rep't of Storrs Agr. Exp. Station, Conn., 1902-3, pp. 52-62. ('03). CONN, H. W., AND ESTEN, W. M. Qualitative analysis of bacteria in market milk. Rep't of Storrs Agr. Exp. Station, Conn., 1902-3, pp. 63-91. ('03). CONN, H. W. Bacteria in freshly drawn milk. Rep't of Storrs Agr. Exp. Station, Conn., 1902-3, pp. 92-98. ('03). CONN, H. W. "The relation of temperature to the keeping property of milk." Storrs Agr. Exp. Station, Storrs, Conn. Bull. 26, Oct., 1903, PP. 3-I5- The author's summary is as follows : 1. Variations in temperature have a surprising influ- ence upon the rate of multiplication of bacteria. At 50 these organisms may multiply only 5-fold in 24 hours, while at 70 they may multiply 7,so-fold. 2. Temperature has a great influence upon the keeping property of milk. Milk kept at 95 (heat of the cow's body) will curdle in 18 honrs, while the same milk kept at 70 will not curdle for 48 hours, and if kept at 50 F. the temperature of an ice-chest, may sometimes keep without curdling for two weeks or more. 3. So far as the keeping property of milk is concerned, the matter of temperature is of more significance than the original contamination of the milk with bacteria. 4. Milk preserved at 50 or lower will keep sweet for a long time, but it becomes filled with bacteria of a more unwholesome type than those that grow at higher tem- peratures. Old milk is not fit for market, even though it be perfectly sweet. ('03). WILHELMY. Die Bakterienflora der Fleisch- extracte und einiger verwandter Praparate. Arb. a. d. Bact. Inst. der techn. Hochschule zu Karlsruhe, in Bd., i Heft, 1903, pp. 1-42, with 3 plates (18 photomicrographs). Most of the bacteria exist in form of spores. Twelve new species are described: Micrococcus carniphilus, M. margiuatus, Streptococcus debilis. Bacterium naveum, B. insulosum, Bacillus carniphilus, B. canaliculatus, B. car- nis, B. iutertnittens, B. anthraciformis, B. glaciformis, B. micans, B. kaleidoscopicus. B. carniphilus occurs in most meat extracts. Other species found were : Bacte- rium rusticum Kern, Bacillus meseutericus Fliigge, B. vulgatus Fliigge, B. cereus Frankland, B. laevis Frank- land, and B. vegetus Keni. XLVIII. Bacteria in Bread. ('85). LAURENT, EMILE. La bacterie de la fermenta- tion panaire. Bull, de 1'Acad. roy. de Bel- gique, 3 ser., T. x, 1885, pp. 765-775. (,'&&). ARCANGEH, G. Sulla fermentazione panaria. Atti della Societa toscana di scienze natural! residente in Pisa. Memorie. Pisa, 1888, vol. ix, pp. 190-211. Bibliog. 29 titles. ('89). KRATSCHMER. UND NIEMILOWICZ. Ueber eine eigentiirhliiche Brotkrankheit. Wiener klin- ische Wochenschrift, 1889, Bd. H, pp. 593- 594- Authors ascribe the stringy bread to Bacillus mesen- tericus vulgatus Fliigge. They did not determine from what source the bread was infected, but state that the or- ganism will not grow in acid dough or acid bread, but that it grows luxuriantly in feebly alkaline dough or bread. BACTERIA IN BREAD; IRON BACTERIA; SULPHUR BACTERIA. 26l ('89). PETERS, W. L. Die Organismen des Sauer- teiges und ihre Bedeutung fur die Brot- gahrung. Bot. Zeitung, 1889, Bd. XLVII, col. 405-419, 421-431, 437-449- ('90). UFFELMANN, J. Verdorbenes Brot. Centralb. f. Bakt.. vin Bd., 1890, pp. 481-485. (.'90). POPOFF, M. Sur un bacille anaerobie de la fermentation panaire. Ann. de 1'Inst. Pas- teur, 1890, T. iv, pp. 674-676. ("94). LEHMANN, K. B. Ueber die Sauerteiggarung und die Beziehungen des Bacillus levans zum Bacillus coli communis. Centralb. f. Bakt., Bd. xv, 1894, pp. 35O-354- ('94). WALDO, F. J., AND WALSH, DAVID. Does bak- ing sterilize bread? Tihe Lancet, London, 1894 (n),pp. 906-908. The general conclusion is that baking does not fully sterilize. The authors cultivated numerous kinds of bacteria from the interior of baked loaves. ('97)- VOCEL, J. Beitrag zur Kenntniss des "faden- ziehenden Brotes." Zeitschr. f. Hyg., Bd. xxvi, 1897, pp. 398-416. (*99)- JUCKENACK, ADOLF. Beitrag zur Kenntniss des "fadenziehenden Brotes." Zeitschr. f. Untersuoh. d. Nahrungs- und Genussmit- teln, ii Jahrg., 1899, pp. 786-788. ('oo). THOMANN, J. Beitrag zur Kenntniss des "fadenziehenden Brotes." Centralb. f. Bakt., 2 Abt., Bd. vi, 1900, pp. 740-743. Stringy bread was found due to bacteria introduced with the flour. The bacillus isolated by the author out of bread and two kinds of flour is called Bacillus panis viscosi (Vogel). Its cultural characters are given as follows : It is sporiferous, actively motile, stains by Gram's method, liquefies gelatin rapidly, forms a dry. gray-white growth on agar, spreads widely and is wrinkled and gray- white on potato, grows in grape-sugar bouillon without gas-formation, produces a thick pellicle on peptone bouillon with a clear fluid under it, and grows best at 40 to 42 C. ('01). DUCLAUX. Pain filant. See Traite, T. iv, pp. 513-SiS. ('01). BEULSHAUSEN, FRIEDRICH. Zur Kenntnis der Ursaohe des Klebrigwerdens von Brot. Diss. Rostock (Druck v. C. Hinstorff), 1901, p. 24. ('02). MICHELS, WOLFGANG. Zur Entsitehung des fadenziehenden Brotes. Diss. Kiel. Konigs- berg i. Pr. (Durck v. Hantung), 1902, p. 15. ('02). LEHMANN, K. B. Hygienische Untersuchun- gen iiber Mehl und Brot. x. Neue Studien iiber die Aciditat des Brotes, ihre Ursachen und i'hre beste Bestimmungs-methode. Arch. Hyg., Mtinchen, Bd. XLIV, 1902, pp. 214-237. XLIX. Iron-Bacteria. ('88). WINOGRADSKY, S. Ueber Eisenbacterien. Bot. Zeitung, 46 Jahrg., 1888, col. 261-270. ('92). MOLISCH, HANS. Die Pflanze in ihrer Bezie- hung zum Eisen. Eine physiologische Studie. Jena, 1892, Gustav Fischer, 119 pp., i table. ('95). MACALLUM, A. B. On the distribution of as- similated iron compounds, other than -haemo- globin and haematins, in animal and vege- table cells. Quarterly journal of microsc. science, 1895-06, vol. xxxvm, new series, No. 150, pp. 175-274, with 3 plates. The part relating to the bacteria begins on page 254. ('97)- MARPMANN, G. Bakteriolqgische Mitteilungen. i. Ueber einen neuen Nahrboden fur Bak- terien. n. Ueber ferrophile Bakterien. in. Ueber den Zusanimenhang von pathogenen Bakterien mit Fliegen. Centralb. f. Bakt., xxn Bd., 1897, pp. 122-132. The new substratum is raw silk. Author has found a bacterium which stores iron in its cell-contents. It is non-motile, 2-3 x 0.8-2.0 >, ends rounded, form plump, with black polar chromatophores and intermediate gray gran- ules. Many cells are entirely black and opaque. The pigment is insoluble in alcohol, ether, carbon bisulphid and benzine. It becomes bluish opalescent with am- monia, and bleaches with HC1, giving off hydrogen sul- phid. On adding ferricyauide of potash after HC1, or with it, the bacteria become an intense blue. On pep- tone-gelatin the organism was white, but when a trace of iron sulphate was added it became black. ('97). MIYOSHI, MANABU. Ueber das massenhafte Vorkommen von Eisenbacterien in den Thermen von Ikao. Journ. of the Coll. of Science, Imperial Univ., Tokyo, Japan, vol. x, Pt. 11, 1897, pp. 139-142. ('97). MIGULA. See in. ('97). LAFAR. See in. ('04). SCHORLER, B. Beitrage zur kenntnis der Eisen- bakterien. Centralb. f. Bakt., 1904, Bd. xn, pp. 681-695. L. Sulphur-Bacteria. C86). CERTES AND GARRIGOU. See xxxiv. ('87). WINOGRADSKY, SERGIUS. Ueber Schwefelbak- terien. Bot. Zeitung. 1887, Bd. XLV, col. 489, 513, 529, 545, 569, 585, 606. 3 figs. ('88). WINOGRADSKY, S. Sur la morphologic et la physiologic des sulfobacteries. Beitr. z. Morphol. und Physiol. d. Bakterien., fasc. I. Leipzig, 1888. Not seen. ('89). WINOGRADSKY, S. Recherches sur les sulfo- bacteries. Ann. de 1'Inst. Pasteur, T. in, 1889, pp. 49-60. ('89). DE TONI AND TREVISAN. Sulphur bacteria. See Saccardo's Sylloge Fungorum, vol. 8, p. 1,027. Species granula sulphuris secernentes. ('93). ZELINSKY, N. D. Ueber Schwefelwasserstoff- garung im Schwarzen Meere und den Limans von Odessa. Fortschr. d. russ. chem. u. phys. Gesellsch., Bd. xxv, Part V, 1893, PP- 298-303. (In Russian.) ('95)- YEGOUNOW, M. Sur les sulfo-bacteries des limans d'Odessa. Archiv. des sci. bio. de 1'Inst. imperial de med. exper. de St. Peters- bourg, vol. in, 1895, pp. 381-397. Rev. in Ann. de Micr., T. vn, 1895, pp. 281-282. ('95). BEYERINCK, M. W. Ueber Spirillum desul- furicans als Ursaohe von Sulfatreduotion. Centralb. f. Bakt., 2 Abt., Bd. i, 1895, pp. 49-59 and 104-114. ('97). MIGULA. See in. ('97). LAFAR. See in. ('97). MIYOSHI, MANABU. Studien iiber Schwefel- rasenbildung und die Schwefelbacterien der Thermen yon Yirmoto bei Nikko. Jour. College Sci., Imp. Univ. Tokyo, vol. x, Pt. 11, pp. 143-173, 1897, i plate. Rev. in The American Naturalist, vol. xxxn, 1898, pp. 456-457- (*0i). CONN. See in. 262 BACTERIA IN RELATION TO PLANT DISEASES. ('02). NATHANSOHN, ALEXANDER. Ueber eine neue Gruppe von Schwefelbacterien und ihre Stoffwechsel. Mitt. a. d. zool. Stat. Neapel., Bd. xv, 1902, pp. 655-680. ('03). HINZE, G. Thiophysa volutans, ein neues Schwefel-Bacterium. Ber. der d. hot. Gesellsch., Bd. xxi, Hft. 6, July, 28, 1903, PP. 309-316. ('03). VAN DELDEN. See xxiv. ('79). ('95). ('95). ('95). (95). (96). (96). ('96). ('96). ('96). ('96). ('96). ('97). (97). (97). ('97). ('98). LI. Bacteria in Prehistoric Times. VAN TIECHEM, PH. Sur la fermentation buty- rique (Bacillus amylobacter) a 1'epoque de la houille. C. R. des se. de 1'Acad. des sci., Paris, 1879, T. LXXXIX, pp. 1,102-1,104. RENAULT, B. Sur quelques bacteries des temps primaires. Bull, du Museum d'histoire naturelle, Paris, annee 1895, T i, No. 4, pp. 168-172, 4 figs. RENAULT, B. Sur quelques Bacteries due Dinantien (Culm). C. R. des se. de 1'Acad. des Sci., Paris, T. cxx, 1895, pp. 162-164. RENAULT, B. Sur quelques inicrococcus du Stephanien, terrain houiller superieur. C. R. des se. de 1'Acad. des sci., Paris, T. cxx, 1895, pp. 217-220. RENAULT, B. Sur quelques bacteries anciennes. Bull, du Mus. d'Hist. nat, Paris 1895, T. i, pp. 247-252, 6 figs. RENAULT, B. Sur quelques bacteries de- voniennes. C. R. des se. de 1'Acad. des sci., Paris, T. cxxn, 1896, pp. 1,226-1,227. RENAULT. Houille et Bacteriacees. Soc. d'nat. d'Autun, Bull, ix, Autun, 1896, pp. 475-500, i pi. RENAULT, B. Les bacteriacees de la houille. C. R. des se. de 1'Acad. des sci., Paris, T. cxxin, 1896, pp. 953-955. RENAULT, B. Les Bacteries devoniennes et le genre Aporoxylpn d'Unger. Bull, du Mus. d'Hist. nat., Paris, 1896, T. n, pp. 201-203. RENAULT, B. Notes sur quelques nouvelles bacteries fossiles. Bull, du Mus. d'Hist. nat., Paris, 1896, T. 11, pp. 285-288, 4 figs. RENAULT, B. Recherclies sur les bacteriacees fossiles. Ann. des sci. nat. hot., vin serie, T. ii, 1896, pp. 275-349, with 46 figures. RENAULT, BERNARD. Les bacteries devonienne et le genre Aporoxylon d'Unger. Bull. d. 1. Soc. d'Hist. nat. d'Autun, T. ix, 1896, pp. 139-142, of the Proces-verbaux des se. RENAULT, B. Les bacteriacees des bogheads. C. R. des se. de 1'Acad. des sci., Paris, T. cxxiv, 1897, PP- 1,315-1,318. RENAULT, B. Les bacteriacees et les bogheads a Pilas. Bull. d. Mus. d'Hist. nat., Paris, T. in, 1897, pp. 33-39, 4 figs. RENAULT, B. Les bacteriacees des bogheads. Bull. d. Mus. d'Hist. nat., Paris, T. in, 1897, pp. 251-258, 6 figs. RENAULT, BERNARD. Bogheads et bacteriacees. Soc. d'histoire naturelle d'Autun. x Bulle- tin, 1897, PP- 433-469, 18 text figures. RENAULT, B. Les microorganismes des lig- nites. C. R. des se. de 1'Acad. des sci., Paris, T. cxxvi, 1898, pp. 1,828-1,831. ('98). RENAULT, B., ET ROCHE, A. Du mode de propagation des bacteriacees dans les com- bustibles fossiles et du role qu'elles ont joue dans leur formation. Soc. d'histoire nat. d'Autun, ix Bull., 1898, pp. 133-147, in the Proces-Verbaux d. se. ('oo). LEMIERE, L. Transformation des vegetaux en combustibles fossiles. Essai sur le role des ferments. Congres geologique international de 1900, Paris, T. I, 1901, pp. 502-520. LII. Preparation of Slides, Cultures, Etc., for Museums, &c. ('80). KAISER, EDUARD. Verfahren zur Herstellung einer tadellosen Glycerin-Gelatin. Bot. Central!)., Bd. I, 1880, pp. 25-26. ('83). GROVE, W. B. New methods of mounting for the microscope. (Hillhouse's method for glycerine mounting.) Midland Naturalist, vol. vi, 1883, p. 166. Journal of the R. Microscop. Soc., London, August, 1883, p. 599- Accordiugto Hillhouse, as reviewed by Dippel in Botan. Centralb., p. 159, Vol. xvi, 1883, glycerin mounts are readily made tight by substituting Canada balsam dis- solved in turpentine for ordinary cements. Ring in ordi- nary way. Hillhouse says that a drop of glycerin on glass can" be covered by a drop of balsam, and the latter will spread over it and adhere firmly to glass around it on all sides, inclosing it completely. ('87). SOYKA, J. Ueber ein Verfahren, Dauerpr;ip:i- rate von Reinkulturen auf festem Nahrboden herzustellen. Centralb. f. Bakt., 1887, Bd. i, PP. 542-544- ('88). JACOBI, ED. Hartung und Farbung von Plat- tenkulturen. Centralb. f. Bakt., 1888, in Bd., pp. 536-538. ('88). SOYKA, J., UND KRAL, F. Vorschlage und Anleitungen zur Anlegung von bacteriolo- gischen Museen. Zeitschr. f. Hyg., 1888, Bd. IV, pp. 143-150. ('89). KRAL, FRANZ. Weitere Vorschlage und An- leitungen zur Anlegung von bakteriologis- chen Museen. Zeitschr. f. Hyg., Bd. v, 1889, PP. 497-505. ('89). SCHILL. Kleine Beitrage zur bakteriologischen Technik. i. Konservirung von Flatten- und Reagensglaskulturen. 6. Schimmelpilze hindert man i.m Waohsthum. Centralb. f. Bakt., 1889, v Bd., Marz i, No. 10, pp. 337- 340. Cultures are covered for 24 hours with a fluid consisting of equal parts of alcohol and glycerin, to which has been added i part per :oo of a I per cent solution of mer- curic chloride. Preparations treated in this way are said to remain unchanged for years. Camphor is said to hinder thegrowth of molds without interfering seriously with bacteria. ('92). DAWSON, CHARLES F. Eine Methode, Dauer- kulturen von Bakterien hermetisch zu ver- schliessen. Centralb. f. Bakt., xn Bd., 1892, pp. 720-721. STOCK-CULTURES, ETC.; COLOR-CHARTS; PHOTOGRAPHY, ETC. 263 ('93)- HAUSER, G. Ueber Verwendung des For- malins zur conservirung von Bacteriencul- turen. Miinchen. nied. Wochenschr., 1893. Bd. xr., pp. 567-568. Rev. in Centralb. f. Bakt, Bd. xiv, 1893, p. 290. Ten or fifteen drops of fresh formalin are put on filter paper and placed tinder the cover of the Petri-dish cul- ture. This is then exposed to the vapor of formalin in a close room lined with wet filter paper, 15 drops of forma- lin being 1 placed on cotton and introduced for each 1000 cc. of air space. As the formalin penetrates the deeper layers of gelatin only slowly, a thin layer should be used for liquefying: organisms. The formalin should be allowed to act for several days, and be renewed once or twice. The gelatin appears to be permanently disin- fected, will not melt at any temperature, and is un- changed in appearance. To have permanent preparations it is only necessary to keep them from drying out. fy.3). HAUSER, G. Weitere Mitteilungen iiber Ver- wendung des Formalins zur conservirung von Bacterienculturen. Miinchen. med. Wochenschr., 1893, Bd. XL, pp. 655-656, No. 35- Rev. in Centralb. f. Bakt., Bd. xiv, 1893, pp. 468-469. Describes a method of fixing and mounting colonies taken from gelatin plate cultures. ('94). KRUECKMANN. EMU.. Eine Methode zur Her- stellmig bakteriologischer Mttseen ttnd Kon- servierung von Bakterien. Centralb. f. Bakt, Bd. xv, 1894, pp. 851-857. Fixes cultures with mercuric chloride, etc., and pre- serves them in formalin, air-tight, in the dark. ('97). FAKES, W. C. C., AND EYRE, J. W. Formalin as a preservative for cultivations of bac- teria. Jour, of Path, and Bact., vol iv, 1897, pp. 418-420. Also a separate, 3 pp. ('oi). CONN, H. W. How can bacteria be satisfac- torily preserved for museum specimens? Science, n. s., vol. xin, 1901, p. 326. LIII. Stock-Cultures, How Best Kept; Vitality on Media. ('89). CZAPI.EWSKI, E. Zur Anlage bakteriologischer Museen. Centralb. f. Bakt., vi Bd.', 1889, pp. 409-411. This method consists in limiting the amount of air which can reach the culture by saturating the upper part of the cotton plug with melted paraffin. The chief objec- tion to it is the increased difficulty of cleaning the dis- carded tubes. ('98). LUNT. On a convenient method of preserving living pure cultures of water bacteria. Rev. in Centralb. f. Bakt., xxm Bd., 1898, pp. 795-796. Certain water-bacteria may be kept alive for two years or more in sterile water, i. e. much longer than in ordi- nary culture-media. ('oo). BOLLKY, HENRY L. The duration of bacterial existence and [in ?] trial environments. Centralb. f. Bakt., 2 Abt, vi Bd., 1900, pp. 33-38. Reports getting a good growth of Bacillus amylovorns and Bact. dianthi in agar and bouillon by transfers from cultures which had been hermetically sealed for 9 years. Tests of the pathogenic power of these cultures appear not to have been made. ('oi). SCHUI.TZ. See vi. LIV. Color-Charts ; Nomenclature of Colors. ('86). RIDGWAY, ROBERT. Nomenclature of colors for naturalists. 195 water colors on ten plates, with rules for making the same and a general discussion of colors. Boston, Lit- tle, Brown & Co., 1886. Valuable, but out of print. Especially useful on account of the number of colors. Another edition in preparation. ('94). SACCARDO, P. A. Chromotaxia sen nomen- clator colorum polyglottus additis speci- minibus coloratis ad usum botanicorum et zoolpgortim. 2d ed. Padua. Typis Semi- narii, 1894, 8vo., 22 pp., with two tables con- taining 50 colors. A cheap and useful color scheme for botanists. ('95). Color chart under Spectrum, in the Standard Dictionary, Funk and Wagnalls, New York, 1895- This may be had separately. ('95). SHUTTLEWORTH, E. B. Nomenclature of col- ors for bacteriologists. Jour. Am. Pub. Health Asso., Oct., 1895, Annual vol. xx, pp. 403-407. ('98). PRANG, Louis. The Prang standard of color. Popular ed., Boston, 1898. Folio. C ) Chart of Spectrum Scales made from the Bradley colored papers. Milton Bradley Co., Springfield, Mass. A small sheet (about 3/4x754 inches) with 90 colors. Also a large folded chart (11x28 indies). Colors are bright, but must be carefully protected from the light. LV. Photography and Photomicrography. C/7). KOCH, ROBERT. Verfahren zur Untersuchun- gen, zum Conserviren und Photographiren der Bacterien. Cohn's Beitrage, II Bd., 3 Heft, Breslau, 1877, pp. 399-434, with 24 photomicrographs on 3 plates. ('81). KOCH, R. Zur Untersuchung von pathogenen Organismen. Mitth. aus dem Kais. Gesund- heitsamte, Bd. I, 1881, pp. 1-48. The paper is illustrated by 84 heliotypes from photomi- crographs. ('83). STERNBERG, GEO. M. Photomicrographs and how to make them ; pp. xv, 204, with twenty plates of photomicrographs. James R. Os- good & Co., Boston, 1883. ('87). CROOKSHANK, EDGAR M. Photography of bac- teria. Illustrated with 86 photographs re- produced in autotype, pp. xix, 64, London, H. K. Lewis, 1887. ('87). Roux, E. La photographic appliance a 1'etude des microbes. Ann. de 1'Inst. Pasteur, T. I, 1887, pp. 209-225. ("88). ZETTNOW, E. Das Kupfer-Chrom-Filter. Centralb. f. Bakt.. 1888. Bd. iv, pp. 51-52. This light filter is well adapted to photographing bac- teria, whether they are stained red, blue or violet. This filter is made as follows: For use with sunlight, 160 grams copper nitrate and 14 grams pure chromic acid diluted with water to 250 cc. More convenient to prepare and suitable for most purposes in a layer l to 1 cm. thick is 175 grams sulphate of copper and 17 grams bichromate of potash dissolved in I litre of water. Thecopper-chrom- filter transmits only a small portion of the spectrum , viz., those yellow-green rays which act most strongly upon erythrosin plates. For the concentrated solution these rays are from wave length 580 to 560 : more diluted from 590 to 545. 264 BACTERIA IN RELATION TO PLANT DISEASES. ('88). NEUHAUSS, R. Verschiedenes uber Micro- photographie. Zeitschr. f. Mikr., Bd. v, pp. 484-486, 1888. ('90). NEUHAUSS, RICHARD. Lehrbucb der Mikro- photographie. pp. xi, 273, with 61 wood cuts, 4 autotypes, 2 collotype plates, and I photogravure. Braunschweig, Harald Bruhn, 1890. A very useful book. ("90). PRINGLE, ANDREW. Practical photo-micro- graphy; by the latest methods. The Scovill & Adams Co., New York, 1890, pp. 183, ix, 42 figs. Frontispiece and 6 plates. ('90 to '99). VOGEL, H. W. Handbuch der Photo- graphic. Vier Theile enthaltend die photo- graphische Chemie, Optik, Praxis und Kunstlehre. Berlin. I Thiel., 1890, pp. XVI, 351, 12 plates; n Theil., 1894, pp. xi, 367; in Theil., Abt. I, 1897, pp. x, 311, Abt. II, 1899, PP- x, 159. ('99). The photo-miniature. Tennant and Ward, New York. Begun in 1899. A series of small, inexpensive volumes, by various autliors, on various phases of photography. Some of them excellent. About 70 numbers up to beginning of 1905- ('99). HUBBARD, J. G. Color screens as applied to photomicrography. Jour. Bost. Soc. Med. Sci., vol. in, 1899, pp. 297-301. ("99). WRIGHT, JAMES H. Examples of the applica- tion of color screens to photomicrography. Jour. Bost. Soc. Med. Sci., vol. in, 1899, pp. 302-307. ('01). ABNEY, SIR WILLIAM DE WIVELESLIE. A treatise on photography. Tenth ed. thor- oughly revised, with 134 illustrations. Long- mans, Green & Co., 39 Paternoster Row, London, New York, and Bombay, 1901, pp. xvii, 425. An excellent book, butsubjects treated very briefly. ('02). WALMSLEY, W. H. The A B C of photo- micrography, pp. viii, 155. Tennant and Ward, New York, 1902. LVI. Methods and Systems of Classification. (See also III and X.) ('38). EHRENBERG. See v. ('41). DUJARDIN, FLIX. Histoire naturelle des zo- ophytes, infusoires comprenant la physiolo- gie et la classification. Paris, 1841. ('65). DAVAINE. See v. C5-'67). TRECUL, A. Urocephalum. C. R. des se. de 1'Acad. des sci., 1865, T. LXI, p. 156 and 432. Ibid. 1867, T. LXV, p. 513. The form of bacterium with a spore at one end, which is swollen, was called Urocephalum by Trecul. ('72). COHN. See v. ('79). TREVISAN. Introduzzione allo studio die bac- teri. Atti d. Inst. Lombardo, 1879. ('80). WINTER. See in. ('81). ZOPF, W. Ueber den genetischen Zusammen- hang von Spaltpilzformen. Monatsbericht d. Konigl. preuss. Akad. d. Wissenschaften, Berlin, 1881, pp. 277-284, i plate. See also various editions of Zopf s "Spaltpilze." ('85) DE BARY. See in. ('85). KUENTSLER, J. De la position systematique des bacteriacees. Jour, de micr., T. ix, 1885, pp. 295-307. According to this author the bacteria are of animal origin, the nearest relatives being the Flagellata, espe- cially the astomous forms. There are transition forms. ('86?). SCHROETER. See in. ('86). HUEPPE, FERDINAND. Die Formen der Bak- terien und ihre Beziehungen zu den Gattun- gen und Arten. With 24 wood-cuts. Wies- baden, C. W. Kreidel's Verlag, 1886, pp. vin, 152. ('89). DE TONI AND TREVISAN. Sylloge Schizomy- cetarum. Forms a portion of vol. vin of Saccardo's Sylloge Fungorum. Padua, 1889, pp. 923-1,087. Those volumes of Saccardo's Sylloge Fuugorum which are out of print have been reproduced in fac-simile (zincography) by R. Friedlander and Sohn, Berlin. ('90). MESSEA, AL. Contribuzione allo studio delle ciglia dei batterii e proposta di una classifica- zione. Revista d'igiene e Sanita Pubblica, Anno I, 1890, pp. 513-528, I plate. Bibliog- raphy of 19 titles. Rev. in Centralb. f. Bakt., etc., Bd. ix, 1891, pp. 106-107, and in Baum- garten's Jahresbericht, Bd. vn, p. 344. The bacteria are classified as Gymnobacteria and Trich- obacteria. The latter are subdivided into four groups : Monotricha (one polar flagellum), Lophotricha (a tuft at one pole), Amphitricha (one flagellum at each end), and Peritricha (flagella from various parts of the body). These names are not used in a generic sense. ('92). WARD, H. MARSHALL. On the characters or marks employed for classifying the Schizo- mycetes. Annals of Botany, vol. 6, 1892, p. 103- ('94). MIGULA, W. Ueber ein neues System der Bak- terien. Arbeiten aus dem Bakt. Institut der Technische Hochschule zu Karlsruhe, Bd. I, Hft. 2, 1894, pp. 235-238. ('95). MIGULA, W. Schizomycetes. Engler and Prantl's Die Naturlichen Pflanzenfamilien, Leipzig, Wilhelm Engelmann, 1895. ('95). FISCHER, ALFRED. Untersuchungen iiber Bak- terien. Jahrb. f. wissensoh. Botanik, Bd. xxvn, 1895, Hft. i, pp. 1-163. 5 plates. ('96). MEz, CARL. Der heutige Stand der bak- teriologischen Systematik. Botanisches Centralb., Bd. LXVIII, 1806, pp. 203-211. ('97). CHESTER, FREDERICK D. A preliminary arrange- ment of the species of the genus Bacterium. Oth Ann. Rept. of the Delaware Coll. Agr. Exp. Sta., 1897. Also a separate, pp. 93. ('97). MIGULA. See in. ('97). FISCHER. See in. Coo). MIGULA. See 111. ('02). THAXTER. See x. ("03). KENDALL, ARTHUR I. A proposed classifica- tion and method of graphical tabulation of the characters of bacteria. Proc. Amer. Pub. Health Asso., Thirtieth annual meeting, held at New Orleans, La., Dec., 1902, vol. xxvin, pp. 481-493. Also a separate, pp. 3-15. Pub. 1003. ('03). FORD, WILLIAM W. The classification and dis- tribution of the intestinal bacteria in man. Studies from the Royal Victoria Hospital, Montreal, vol. i, No. 5 (Pathology n), 1003, PP- 3-95. 3 tables. Also a separate. CATALOGUES. 265 ('03). GAGE, STEPHEN DE M, AND PHELPS, EARLE B On the classification and identification o< bacteria, with a description of the card sys- tem in use at the Lawrence Exp. Station for Records of Species (Lawrence, Mass.). Proceedings of thirtieth annual meeting- of American Public Health Assn., New Or- leans, La., Dec., 1902, vol. xxvm, pp. 494- 505. Pub. Columbus, O., 1903. Also a separate, pp. 12-23. ('04). PERKINS, ROGER G. Bacillus Mucosus Capsu- latus. A study of the group and an attempt at classification of the varieties described. Jour, of Infectious Diseases, vol. I, No. i, 1904, pp. 241-267. Also a separate. ('05). WINSLOW, C. E. A., AND ROGERS, ANNE P. A revision of the Coccaceae. Preliminary communication. From the Biological labo- ratories of the Massachusetts Institute of Technology. Science, n. s., Vol. xxi, 1905, pp. 669-672. The 445 described forms are reduced to 31 types. Five genera are recognized, viz, Diplococcus and Streptococ- cus, belonging to the sub-family Paracoccaceae, and Mi- crococcus, Sarcina and Ascococcus, belonging to the sub- family Metacoccaceae. LVII. Useful Catalogues. Catalogues and addresses of instrument makers, manufacturers of chemicals, etc. : CARI, ZEISS, Jena. (1) Microscopes and microscopical accessories, 32 ed., 1902. (2) Photographic objectives and photo-optical ap- pliances, 1901. (3) Catalogue of instruments and appliances for projection and photomicrography, fourth ed., 1899. ERNST LEITZ, Wetzlar. Microscopes and accessory apparatus. Cat. 39. U. S. Branch: Wm. Krafft, 80 East i8th st, near Broadway, New York. BAUSCH AND LOME, Rochester, N. Y. (1) Optical apparatus, microscopes, photographic lenses. (2) Chemical apparatus, bacteriological apparatus. EIMER AND AMEND, New York. Chemical and physical apparatus, 1903. Am. agents for Zeiss. WHITALL AND TATUM, Philadelphia, New York, and Boston. Glassware. F. AND M. LAUTENSCHLAEGER, Berlin. Cat. No. 60. Bacteriologie, Chemie, Asepsis. MAISON WIESNEG (P. Lequeux), 64 Rue Gay-Lussac, Paris. Catalogue des appareils de bacteriologie et d'hygiene. M. SCHANZE, Leipzig. Preisverzeichnis von Mikrotome. DR. HERMANN ROHRBECK, Berlin. Brood-ovens, thermo-regulators, etc. DR. G. GRUEBLER AND Co., Leipzig. Preislisten von Farbstoffe und Reagentien. Griibler's stains may be obtained in the U. S. from Wm. Krafft, 80 East i8th st., near Broadway, New York. Koenigliohe Pprzellan-Manufactur, Berlin. Preis-Verzeichniss, No. v. Gerathschaften zu chemischen und pharmaceutischen Zwecken. i Jan., 1899. Price list of chemical apparatus manufactured and sold by C. Gerhardt, Bonn am Rhein, Ger- many. P. J. KIPP u. ZOHNEN, Delft, Netherlands. Makers of the Reinhold-Giltay microtome. DR. ROB. MUENCKE. Catalog fiber chemische Apparate und Gerathschaf- ten, 1900, Berlin, N. W., Luisen-Strasse 58. Preis 5 mark. pp. 600. DR. PETERS & ROST. (1) Preis-Liste fiber Apparate und Utensilien fur elektrochemische und elektrolytische Ar- beiten. Liste No. 29. Berlin, 1900, pp. 48. (2) Preis-Liste fiber Apparate und Utensilien fur Bakterioloeie, Hygiene, Mikroscopie. Rea- gentien, Farbstoffe, Nahrboden, Reincul- turen, mikroscopische Praparate. Liste No. 30. Berlin, 1900, pp. 123. (3) Preis-Liste fiber chemische Apparate und Uten- silien fur wissenschaftliche und Fahriks- Laboratorien, Chemicalien, Reagentien, Normal-Losungen. Liste No. 28. Berlin, 1002, pp. 534. (4) Preis-Liste fiber physikalische Apparate. Liste No. 37. Berlin, 1902, pp. iv, 383. MAX KAEHLER AND MARTINI, Berlin. Chemical and Bacteriological Apparatus. The above five catalogues or their equivalent may now be obtained from the following: Vereinigte Fabriken ffir Laboratoriumsbedarf, ges. M. B. H., ChauseestT. 3. Berlin. N., formerly Max Kaehler & Martini. Dr. Peters & Rost. New York Office : Laboratory and School Supply Co., 20-24 East 20th street, New York. EMU, GRF.INER, New York. Bacteriological apparatus, glassware, high-grade glass blowing. Very reliable. GREINER & FRIEDRICHS, Stutzerbach, Germany. Glassware. C. P. GOERZ. Price list of double-anastigmat lenses and of the Goerz-Anschutz camera. Berlin-Friedenau, and 52 East Union Square, New York. FOI.MER AND SCHWING, 407 Broome street, New York. High-grade cameras. HENRY HEIL CHEMICAL Co., 298-312 S. Fourth street, St. Louis, Mo. Illustrated catalogue and price list of chemical and physical apparatus and instruments for labora- tories, chemists, iron and steel works, smelters, assayers, mines, sugar refineries, schools, col- leges, universities, etc. SPENCER LENS Co., Buffalo, N. Y. Microscopes. Excellent lenses, especially low- power apochromatics and a new 2 mm. oil im- mersion achromatic. 2 66 BACTERIA IN RELATION TO PLANT DISEASES. BECKER, CHRISTIAN. Price list of balances and weights of precision. Factory : New Rochelle, N. Y. ; New York office : 7 Maiden Lane. THE KNY-SCHEERER Co., 225-233 Fourth avenue, New York. (1) Illustrated Catalogue of Surgical Instruments, 1902, pp. LVI, and pp. 1,001-1,061, 2,001-2,140, 3,001-3,192, 4,001-4,102, and 5,001-5,180. (2) Aseptic Surgical Furniture and General Hos- pital Supplies, 6 ed., 1902, pp. 232. Fully illustrated. E. H. SARGENT, & Co., 143 and 145 Lake street, Chi- cago. Importers and makers of laboratory supplies, price list of apparatus, chemicals, reagents, tissue-stains, microscopical and bacteriological supplies, etc. JAMES T. DOUGHERTY, 409 and 411 W. Fifty-ninth street, New York. Scientific apparatus. Sole United States agent for Carl Reichert, Vienna, Austria, microscopes, microtomes, and polarizing apparatus. THE VblGTLAENDER & S*ON OPTICAL Co., 137 W. Twenty-third street, New York. Collinear and telephoto lenses, porro prism binoc- ulars, etc. WARNER & SWASEY, Cleveland, Ohio. Excellent binocular field glasses. CHARLES J. Ross Co., 1525 Fairmount avenue, Phila- delphia, Pa. Excellent quality of drawing-board, heavily coated, so as to permit of any number of erasures. Rec- ommended by Dr. Roland Tfaaxter and by the writer. THE CENTURY CAMERA Co., Rochester, N. Y. Excellent cameras embodying many convenient, time-saving devices. STANLEY PHOTOGRAPHIC DRY PLATE Co. Cheap dry-plates of good quality. M. A. SEED DRY PLATE Co., St. Louis, Mo. Standard dry-plates. Non-curling films. Isochro- matic, ordinary, and non-halation plates of good quality. HAMMER DRY PLATE Co., St. Louis, Mo. Both Seed and Hammer publish interesting little free handbooks on negative making, etc. G. CRAMER DRY PLATE Co., St. Louis, Mo. New York Depot : 32 East loth street. CADETT SPECTRUM DRY PLATE Co., London, England. THE ILFORD CHROMATIC PLATE Co., London, England. The 3^4 by 4^4 plates are highly recommended by Dr. Jeffrey for photomicrographic work. They cost 20 cents per dozen, exclusive of duty. THE EASTMAN KODAK Co., Rochester, N. Y. Roll and cut films, Kodoid films, non-curling films, solio paper, velox paper, etc. WILLIS AND CLEMENTS, Philadelphia, Pa. Platinotype paper. DR. J. C. MILLER, Denver, Colo. Blue-print papers of high grade, known as "French Satin, Jr." G. GENNERT, 24 and 26 East ijth street, New York. Hauff's Ortol developer, white glass for lantern- slide covers, etc. MALLINCKRODT CHEMICAL WORKS, St. Louis, Mo., and New York, N. Y. Photographic chemicals of a high grade. KEUFFEL & ESSER Co., New York. 127 Fulton street and 42 Ann street. Catalogue and price list of drawing materials and surveying instruments. Drawing paper of any quality de- sired converted into blue-print paper. Branches in Chicago, St. Louis, and San Francisco. E. SCHERING, manufacturing chemist, Berlin, Ger- many. KRAL'S LABORATORY, Prag, Austro-Hungary. Der gegenwartige Bestand der Kral'schen Samm- lung von Mikroorganismen, Oct., 1902. Krai's Bac- teriologisches Laboratorium, Prag, I. Kleiner Ring 11. Telegramm-Addresse : Krai's Laboratorium. Cultures of several hundred sorts of bacteria and of some fungi may be had from this laboratory. Authors are urged to send their new species to Krai. W. P. Stender, Leipzig, Gerichtsweg 10. Fabrik ttnd Lager von Glasgegenstaenden zur Anfertigung mikroskopischer Praeparate, u. s. w. EAGLE OXYGEN Co. (See p. 81.) DEFENDER PHOTO SUPPLY Co., Rochester, N. Y. Argo and other photographic papers. ADDEN DA. Page 46, paragraph b, for " fat " read casein. The fluid has a soapy feeling, but " saponification " also is probably not the proper term. What actually occurs is a matter for the chemist to determine. The inoculated milk shows no change at first, but gradually becomes intensely alkaline and clears synchronously, without coagula- tion or precipitation. By addition of acids, or concentrated solutions of sodium chloride, copper sulphate, etc., the transparent fluid may now be filled with a white flocculence, which slowly settles to a bulky caseous precipitate, leaving a superna- tant clear, pale whey. It usually requires a month or six weeks for the inoculated milk to become entirely transparent, but a similar transparency may be produced at once in check tubes of milk by adding a few drops of ammonia-water. On testing cultures grown for a few days in " nitrate bouillon," as described on p. 63, it happens frequently that there is no nitrite reaction. It is then neces- sary to know whether nitrate is actually present in the bouillon. Usually cultures of Bacillus coli, or some other known nitrate-reducing organism serve this purpose. If such cultures have not been provided, the test for nitrates may be made with diphenylamin dissolved in strong sulphuric acid. On addition of a few drops of this reagent there is an immediate, evanescent, deep-blue reaction if nitrates are present. The reagent is prepared by slowly dropping 90 cc. of c. p sulphuric acid into 10 cc. of pure water, after which i gram of diphenylamin is added, and the solution preserved in a glass-stoppered bottle. 267 I NDEX. Page. Abbe camera 130 Abstracts, making of 114 Acclimatization to high temperatures 222 Acetic alcohol. as a fixing fluid 202 with mercuric chloride 202 Acetone, for use in fermentation-tubes 52 Acid-fast bacteria 188 Acid-forming bacteria, demonstration by plate cultures 51,233 Acid gelatin, effect on growth 30 Acid, hydrochloric, in culture-media 98 oxalic, actively germicidal 249 Acids, action on enzymes 68 effect on bacteria 249 formation of, in milk-cultures 46 method of estimating in plants, when not free 207 spilled, neutralization of 107 toleration of, by bacteria 70, 249 vegetable, sensitiveness of bacteria to 69 Achromatium 162 Acrolein, germicidal action of 252 Achromatic objectives, disadvantages of 140 with excellent definition 265 Ae'robe, Pasteur's definition of 230 Agar-agar, combined with bouillon and sugars 33,5 1 commercial, source of 31, 225 composition of 32, 223 cultures on, for flagella staining 20 filtration of 33, 224, 225 glycerinated 50, 196 litmus lactose 196 melting point of 32 methods of making 223, 224, 225 nutrient, centrifuging of 225 nutrient, temperature at which poured 105 per cent used for media 35 rate of diffusion in 224 roll-cultures of 36 softening of, by bacteria 32 solidification of 32 source of 31 standard nutrient, preparation of 33,195 sterilization of 98, 99 variation on, of same organism 180 with neutral red 230 without nutrients 229 - Page. Agar-block method for study of antagonism. . 73 Agar-plates, for detection of acid-forming colonies 51 Agfa-intensifier 142 Air, bacteria infrequent in Arctic 255 bacteria rare in upper strata of 255 of mountains, bacteria rare in 255 over the sea, bacteria rare in 255 Air-currents, danger of 103, 106 Air-shaft, for ventilation of dark-room 149 Albumen, egg, Mayer's 119 preparation of, for culture-medium 48 Alcohol, as a fixing fluid 8, 202 ethyl, for use in fermentation-tubes 52 methyl, for use in fermentation-tubes 52 weak germicidal action of 252 Alkalies action on enzymes 68 formation of, in cultures 61 sensitiveness of bacteria to 69 toleration of, by bacteria 70 Alkaline methylene blue 188 Amaryllis, inoculated with B. hyacinth! 66 Amiclo-bacteria 175 Amins, test for 64 Ammonia-bacteria 175 Ammonia, oxidation of, by bacteria 64 test for 64 Ammonium, citrate, relation to pigment-production 65 lactate, relation to pigment-production 65 oxalate, solvent of middle lamella 67 salts, oxidation of, by bacteria 64 salts, sterilization without loss of ammonia. 52 succinate, relation to pigment-production. . . 65 Amoebobacter 163 Anaerobe, Pasteur's definition of 230 Anaerobes 51 Arens' method of cultivating 231 Buchner's method for growing 231 Bulloch's method of obtaining 228 Heim's method 231 Hesse's method 230 Klein's apparatus for 232 - on culture-media in hydrogen, etc 57 present in milk 46 Wright's methods of cultivating 228 269 2-JO BACTERIA IN RELATION TO PLANT DISEASES. Page. Anilin-dyes, differential diagnosis with 230 watery solutions of '87 Analin-dyes (basic), affinity of bacteria for 2 7 use of, in vegetable tissues 29 Anilin fuchsin '^7 Anilin gentian violet : 7 Anilin methyl violet ^7 Anilin stains, alcoholic solutions of 187 Anilin-water T 7 Animal-fluids for culture-media, beef-broth 45 blood-serum 48 egg-albumen 4 egg-yolk 49 litmus-milk 4, 196 milk 46 rice cooked in milk 48 Anschiitz, normal thermometers 78 Antagonistic action of bacteria 73 Anthrax organism, asporogenous 222 behavior when injected into plants 89 branching in 216 generic name for !7' Anthrax spores, substratum influences forma- tion of 219 Antiseptics, effect on form of bacteria 252 kinds of 74 literature on 250 Aplanobacter, species included under genus. . 171 Apochromatic objectives, in photomicrography 139 recommended for bacteriological work 130 Spencer 14 Zeiss 130 Apparatus necessary in laboratory 95 Apple blight, due to B. amylovorus 202 Arnold steam-sterilizer 47 Arthrospores, disputed existence of. .. 21, 158,218 Asparagin, in fermentation-tubes 52 relation to pigment-production 65 Atkinson, method for photographing poured plates 134 Aujeszky, spore-stain 219 Autoclave, for sterilization of culture-media. 84, 98 Bacillus, Cohn's genus 158 Fischer's genus 157 in Migula's classification 160 Bacillus amylovorus, absence of pigment in 65 distributed by insects 215 effect of direct sunlight on 72 enters plant through nectaries 92 flagella hard to stain ; 190 hosts of 87 nitrates not reduced by 113 non-sporiferous 158 useful for comparison 29 Page. Bacillus aroideae 115 hosts of 86 soft rot due to 115 stain produced by 65 thermal relations of 86 variation in shape of colonies at different temperatures 180 Bacillus Biitchlii, inner structure of 279 Bacillus carotovorus, soft rot due to 103 stain produced by 65 thermal relations of 86 tissues occupied by 5, 6 Bacillus coli, effect of formalin on 229 how differentiated from B. amylovorus. . 51, 113 method of differentiating from B. typhosus. 229 useful for comparison 29 vitality of 72 Bacillus denitrificans, size of 18 Bacillus hortulanus, isolation of, by heat. 106,211 Bacillus oleraceae, maximum temperature for. 86 Bacillus oligocarbophilus 241 Bacillus prodigiosus, culture-media for 238, 239 for study of production of pigment 64 magnesium sulphate necessary for pigment. 238 pigmented and non-pigmented races of. . 64,222, 238 reaction of color to acids and alkalies 65 Bacillus rosaceus metalloides, rosette-like groupings 218 Bacillus tracheiphilus, absence of pigment in 65 action of block-tin on 97 behavior in fermentation-tubes 53 behavior toward acids and alkalies 70 distributed by insects 178, 215 effect of f rezing on 82 effect of sodium chloride on 70 susceptibility to dry air 71 viscidity of 20 Bacillus typhosus, action of copper on 74, 97 behavior when injected into living plants. . . 89 effect of formalin on 229 Hiss' special medium for differentiating. .. . 230 non-sporiferous 158 Wurtz's method of differentiating 229 Bacteria, action on photographic plate 242 amount of acid tolerated 70 animal origin of 264 blue pigment forming, in cheese, etc 237 branched forms 177, 217, 218, 237 effect of chemicals on 74, 250 effect of freezing 79, 246, 247, 258 effect of gases on 58, 231 effect of heat on 75, 246 effect of ions on 244 effect of light on 71, 243 effect of pressure on 245 hanging-block cultures 22 INDEX. 271 Page. Bacteria continued. hanging-drop cultures 22 in bread, baking does not destroy all 261 in milk 196, 260 isolation from diseased tissues, methods of. n limits of size 18 long vitality of 72, 214, 263 monotonous morphology of 25 move against a stream 253 non-nitrifying associated with nitrifying. . . 241 oxidation of ammonia and ammonium salts by 64 photographing in stained tissues 136, 140 prehistoric 362 presence in diseased tissues, how deter- mined 10 rapidity of movement 26 rare in Arctic 255 rare in mountain air 255 sensitiveness to acids 69 sensitiveness to alkalies 69 species found in meat-extracts 260 thermal range of 75, 246 toleration limit for sodium chloride. .. . 70,252 Bacterial ash, alkaline 42 Bacterial disease communicated by beetles.... 92, 178,215 Bacterial soft rots, similarity of 8 Bacteriology, milestones in progress of 151 prominent workers in 152 Bacteriosis of walnut 174, 1/6 prevention of 93 Bacterium, Cohn's genus 166 Ehrenberg's genus 165 iron stored by 261 plant parasites belonging to genus 171 Migula's use of the word 160, 165 substituted for Pseudomonas 166, 171 triloculare 165, 169 Bacterium campestre, communicated by slugs, larvae, etc 215 effect of desiccation on 71 enters plant through water-pores. 92,94, 102, 124 enzymes produced by 68 relation to soft white rots 73 tissues occupied by 7, 10, n, 12 useful for comparison 29 Bacterium hyacinth!, effect of desiccation on 71 effect on Amaryllis atamasco 66 Bacterium malvacearum, leaf spots due to 95 stomatal infection 126 Bacterium pediculatum, branching of 221 Bacterium phaseoli, beans infected with, by way of stomata 92 effect of direct sunlight on 72 pigment of 179 Bacterium pericarditidis, fiagellum of 237 green fluorescence of 65 Page. Bacterium pericarditidis continued. viscidity in fluid culture-media 42 vitality of 73 Bacterium solanacearum, distributed by insects 215 optimum temperature for 86 plants inoculated with 17, 202 relation to soft white rots 73 stain produced by 65 Bacterium syncyaneum, blue color of, in acid milk 65 Bacterium syringae 64, 66, 135 Bacterium Stewart!, stock-cultures of, how best kept 72 tissues affected by 4, 90 Bacterium termo, Cohn's and de Bary's account of 166 Dallinger & Drysdale's conception of 170 flagella of 20, 170, 219 Bacterium triloculare, flagella of 20,169 Bacterium vascularum, relation to red stain of sugar-cane 66 stock cultures of, how best kept 72 Bactridium, as a genus name 158 Balsam, for mounting sections 119 Banti, isolations on slant media 227 Barfoed's reagent 208 Bechamp, views concerning morphology 176 Beef-bouillon, proper sterilization of 98 standard peptonized 45, 195 Beetles, disease spread by 92, 178, 215 Beggiatoa 162 Bell-jars, for use in inoculations 108 Benda's iron-haematoxylin 188 Benzoic acid 74 Beyerinck, agar for detection of acid-forming colo- nies 51, 233 agar for cultivation of nitrite bacteria 199 Bacillus oligocarbophilus 241 blue pigment bacteria in cheese 237 fermentation of indigo 257 Kapillarhebermikroscopirtropfenflasche 226 root-tubercle bacteria, study of 153 thermotaxis 254 Bilirubin as a test for free oxygen 57 Billroth, views concerning morphology 176 Birds, intestinal tract of, bacteria in 258 Blood-serum 48 Blue prints, making of, for drawing purposes. 150 Bokorny, amount of nutrients needed by bac- teria 225 Bolley, on longevity of bacteria 263 Boni's method of staining capsules 194 Bouillon, cultures in, for flagella staining 20, 190 lead acetate in 51 neutral red in 51 of varying degrees of alkalinity and acidity. 51 peptonized standard 195 salted 51 sterilization of 98, 99 272 BACTERIA IN RELATION TO PLANT DISEASES. Page. Bougie, Berkefeld's, for filtration of culture-fluids. . 43 Chamberland's, for filtration of culture- fluids 43 Chamberland's, sterilization of, necessity for frequent 44 Bowhill's flagella stain I9 2 Boxes for sterilization of pipettes 41 Branched forms 23, 177, 215, 216, 217, 218 Bread, Bacteria cause stringiness in 260, 261 baking does not fully sterilize 261 Bredig und Miiller, enzymic action of plati- num black 234 Brenner, black rot of cabbage, communicated by aphides 215 Bromide-prints IS 1 Broomcorn, natural infection in 92, 150 red blotches and stripes, relation of bacteria to 66 Brownian movement, deceptive nature of. . 10, 26 Brown pigment, bacteria producing. 65, 21 1, 214,237 Brunner and Zawadzki, counting plate 227 Bubonic plague, nature of organism causing.. 214 Buchner, effect of light on bacteria 244 method of growing anaerobes 231 Bujwid, cholera red 229 comparison of Chamberland and Berkefeld filters 227 Bunge's flagella stain 191 Burcq, effect of metallic copper on bacteria. . . 250 Biitschli, nature of central body in bacteria. 216, 217 Butter, bacteria in 259 Cabbage, natural infection in. . . 92, 93, 94, 102, 124 Cages for inoculation 108 Calcium carbonate, effect of, on longevity 61 Calcium chloride, effect on luminous bacteria. 60 Camera, Abbe, for drawing 130 centering of horizontal 137 enlarging, reducing, and copying 146 photographing with horizontal 137 Cankers produced by bacteria 8 Capillary drop flask 21, 226 Capsule, branching caused by one-sided development Of 221 examination of unstained 19 Friedlaender's stain 220 methods of staining for demonstration of . 19, 194 Moore's contrast stain 221 Carbol-fuchsin 187 Carbol-methylene blue 188 Carbolic acid 74 for sterilizing surface of diseased material . . 14 Carbon dioxide, assimilation of, by bacteria 64 behavior in 59, 223 cultures in, factors to be considered 59 generation of 54, 55 Page. Carbon dioxide continued. removal of all oxygen from 57 test for, in fermentation-tubes 61 test for purity of 55 washing of 55 Carbon monoxide, effect on bacteria 58 Carney's fixing fluid 202 Casein, non-precipitated, clearing of 267 precipitated, re-solution of 46 separation of, in milk-cultures 46, 67 Cavities produced by bacteria 8 Cell-nucleus 216, 217, 219 Cellulose, in bacteria 219, 254 pure, fermented by bacteria 209 Chains, occurrence of bacteria In 22 Chamberland autoclave 85 Chamberland filter, cleaning of 45, 227 enzymes entangled in walls of 68 penetration of, by bacteria 45, 226 used in study of antagonism 73 Characters, cultural, value of 178 decimal system of recording 175 morphological, value of 176 variation in, due to environment 182, 183 Check-plants, importance of 14, 186 proper behavior of 15, 16 Checking work, necessity for, and methods of. 184 Chemicals, for use in culture-media 97 Chemotropism 27 Chester, decimal system 175 terminology for description of colonies 30 Chlamydothrix 162 Chloroform 74, 250, 254 Chlorozinc-iodide, use in staining anthrax spores 218 Chodat, lactic acid bacteria in cheese 260 Cholera vibrio, cultivation on potato 249 destroyed by river waters in India 251 effect of copper on 250 method of diagnosing and isolating 229 names for 173 Chondromyces 165 Chromatium 164 Chromatium Okenii, cells connected sidewise. 216 Chromic acid, cleaning mixture 200 use in staining spores 218 Chromo-aceto-osmic acid for fixing fluid 202 " Chromo-agars " for differentiation 229 Chromogens, medium for study of 48, 224 Chromoparous bacteria 237 Cladothrix 162 Classifications 154 Classification, iMigula's system 159 Fischer's system 157 INDEX. 2 73 Page. Classification continued. water-bacteria 203 Winslow & Rogers 265 with reference to nitrogen-nutrition 175 Cleaning, glassware 100, 226, 227 Cleaning mixture, chromic acid 200 Clostridiutn pasteurianum, separation by heat. 106 Cloth, impervious to light 143 Clouding, nature of, in culture-fluids 42 rapidity of, in culture-fluids 42 Coccaceae, revision of 265 Coccobacteria septica 176 Coconut-water, as a culture-medium 41 Cocothrix 172 Cohn, Bacillus of 158 Bacterium of 166 nutrient solution of 197 rise of temperature in cotton-wool waste. . . 248 Collections, the making of 117 Collodion, sac-method for study of antagonism 73 sacks, preparation of, for dialyzing 37, 229 Colonies, acid-forming, special agar for detection of. 51 characters to be noted on solid media 29 counting of 36 effect of physical conditions on appearance of 182, 183 measurement of 115 photographing of 134 Color-scales, importance of 109 kinds recommended 263 Comma bacillus, classification of 173 Koch's paper on 212 Conditions which are unfavorable, influence on cell-unions 22 Conrad, fermentation of sauerkraut 257 Constancy of characters 183 Contact-irritation 27 Contamination, how to avoid 103 Control-plants, proper behavior of 15, 16 Coplin's staining jar 119 Copper-chrom-filter 201 Copper salts, germicidal value of 251 Copper sulphate 74 Corallin, test for slime derived from starch.. 221 Cotton, dry heating in bulk 99, 101 natural infection in 95 surgeons' absorbent, sterilization of 101 waste, for laboratory use 107 Counting-plates, ruled 36, 227 Cover-glasses, cleaning of 227 Cover-glass preparations 20, 28 Covers, discarded, care of 107 Cramer, resistance of spores to dry heat, cause of 218 Crenothrix 162 Cristiani, bacteria in upper air 255 Page. Cross-inoculations 186 Cross-level 149 Crystals, formation of, in milk-cultures 46 in old cultures 66 prevent making thin sections 122 Cucumber, natural infection in 92,178,215 Cultural characters, value of 178 variations in, due to environment 182, 183 Culture-fluids, cold sterilization of 43, 52 examination of, before inoculation 42 growth in, characters of, to be noted 42 non-synthetic 195 synthetic 197 time required for clouding 42 Culture-media 223 agar-nutrient 31, 195 beef-broth 45, 195 blood-serum 48 containing sugars, sterilization of 98 cooked vegetables for 40 Dunham's solution 195 egg-albumen 48 egg-yolk 49 for luminous bacteria 60, 198 gelatin 29, 196 hydrochloric acid in 98 litmus milk 48, 196 milk 46 non-synthetic 195 plant juices used as 41 preparation and care of 97 protection from light 53 raw, preparation of 41 record-book for 109 silicate-jelly 36, 198 special, for differential purposes 229 starch-jelly 50, 196 sterilization of, in autoclave 98 storage of 99 synthetic 197 titration of 69 Culture-rooms 104 Cultures, elective 106 involution forms in 23 method of keeping notes on 113 methods of testing purity 184, 185 plate, stab and streak 29, 30 preservation of, in museums 262 storage of stock 123, 263 treatment of spilled 107 Cyanophyceae, cilia on side walls of 211 Cystobacter 165, 218 Cytase 67 Dallinger, measurement of unstained flagella, 170, 219 Dallinger & Drysdale, Bacterium termo, fla- gella of 20 Dangeard, green bacteria 237 Dannappel, spores sensitive to heat 22, 246 274 BACTERIA IN RELATION TO PLANT DISEASES. Page. Dark-room, arrangement of 15 ventilation of 149 d'Arsonval, negative results with ozone 252 osmotic pressure of bacteria 247 Darwin's wax-mixture 200 Dawson, root-tubercles of legumes 64 Davenport & Castle, acclimatization to high temperatures 222 Decimal system of recording characters 175 de Freudenreich, bacteria in mountain air 255 penetration of Chamberland filter by bac- teria 226 Degeneration-forms 23, 177, 252 de Koninck, gas-generator 54 de Lagerheim, macaroni as a culture-medium. 224 Delbrueck, acid fruits, effect on cholera or- ganism 249 Delphinium, bacterial disease of 92, 139 Denitrifying organisms, Giltay & Aberson's culture-medium for 198 Desiccation, effect of 70, 248 Developer, hydrochinon 146, 201 lantern-slide 201 ortol 140 pyrogallol 200, 201 Development of negatives 140 Dewar glasses, for liquid air 80 Dextrin for use in fermentation-tubes 52 Dialyzing, collodion sacks for 37 Diastase 67 Diastasic action, medium for study of 50, 196 Dieudonne, part of spectrum harmful to bac- teria 244 Differentiation of species 25,225,229,230 Dilution method for obtaining pure cultures. . 226 Diphenylamine-test for nitrates 267 Direct-infection experiments, value of 9 Disease, conditions favoring the spread of 93 geographical distribution of 7 meaning of term 4 signs of 7 varietal resistance to 93, 186 Disinfectants 74 for sterilizing surface of diseased material. . 14 literature of 250 Disinfection of hands, wounds, floors, tables, etc 107 Distilled water, of a high degree of purity 129 made in large quantities 124 made in small quantities 128 storage of I2 6, 129 Distribution, geographical 7 Divided-plate method for study of antago- nism 73 Division of cells, arrangement after 22, 218 Downes & Blunt, discover germicidal action of light 243 Page. Drawings, on photographic prints 151 Dreyer, staining of bacteria in tissues 222 Dreyfuss, cellulose in bacteria 254 Dry air, sensitiveness of bacteria to 71,249 Dubois, fluid medium for luminous bacteria. . 242 Duckwall, method of staining flagella 193 Dunham's solution 19=; Eau de Javelle 207 Economic aspects, conditions favoring spread of disease 91 methods of prevention 93 natural methods of infection 91 need of statistics relating to plant diseases . . 90 Egg-albumen, for culture-media 48 Mayer's i ig Eggs as a culture-medium 224, 225 Egg-yolk, for culture-medium 49 Ehrenberg, Bacterium of 165-169 Spirillum of 173 Ehrlich's anilin-water gentian violet 187 Ehrlich-Weigert anilin methyl violet 187, 188 Elective culture 106 Embedding, method of, for microtome-sec- tions 1 18 Engelmann, green bacteria 236 photometric bacteria 243 Enlargements, photographic 147 Environment, influence on cultural characters . 178, 182, 183, 222 response morphologically to change in . 176, 222 Enzymes, filtering of 68 kinds of 66 nature of 66 not in j ured by dry heat 68 number produced by one organism 68 substances inhibiting action of 68 thermal relations of 67, 233 Errera, India ink in microscopy 226 spirillum of large size 19 Error, methods of guarding against 184 Ether, sulphuric 74, 254 Euspirosoma 161 Ewart, green bacteria 238 Ewell, anaerobic apparatus 56 Exposure scales 143 Exposure, time of, for isochromatic plates 143 for lantern slide 146 for Petri-dish poured plates 134 for photomicrographs 143 for velox paper 151 Faber's pencils for glass no Farmer's reducing solution 142 Favorable influence of one organism on an- other 72,74 Fehling's solution, reaction of peptone with 229 when not usable 208, 234 INDEX. 275 Pane. Feinberg, on existence of nucleus 217 Fermentation, causes of 60 commercial importance of 60 determination of gases produced by 61 effect of calcium carbonate on 6r isolation of products of 61 literature of 232 meaning of term 60 measurement of volume of gas produced. . . 61 observations to be made on 61 of cellulose 209 pectic 257 products of 60, or Fermentation-tubes, absorption of air into closed end of 52 fluids to be used in 52 observations to be made on cultures in 52 style preferred 53 substances to be tested in 52 test for presence of air in closed end 54 wooden rack for 52 Fermented foods, bacteria of 235 Fermi, concentrated solution for silicate-jelly... 39, 197 effect of acids, etc., on bacteria 249 studies of ferment-bacteria 233 Fermi und Montesano, reagent for sugar 234 Fermi und Pernossi, action of heat, light, etc., on enzymes 233 Ferran, aerobic behavior of tetanus 231 Picker, glass, influence of substances dis- solved out of 223 Filaments, bacteria in form of 22 Filters, Berkefeld and Chamberland, for cold steril- ization of fluids 43 Zettnow's copper-chrom 201 Filter-papers, enzymes held in meshes of 68 most convenient form of 34 reaction of 34 Filtration, by means of compressed air, etc 44 inside-out method 43, 226 of milk for culture-media 46 of nutrient-agar 33-35 of thick fluids 69 Fiocca's spore-stain 194 Fischer, A., classification of 157 experiments on plasmolysis 22, 254 flagella-stain 191 subfamilies distinguished by flagella 20 Fischer, B., bacteria in sea-air 255 germicidal action of light through water 244 Fish-parasites 213,214 Fixation of plant material containing bacteria. 8 Fixing fluids 202 Flagella 219 classifications based on 20, 157 earliest demonstrations of 20 stains 20, 190 Page. Flagel la-staining, cultures best suited for 190 literature on 219 technique 189 Flasks, for storing distilled water 120 Flax, retting of 257, 258 Flemming, fixing fluid 202 triple stain 188 Flexner's anilin gentian violet 187 Flocculence in fluid-cultures 22 Florideae, source of agar 32, 224 Fluorescine 65 Focus-difference, with achromatic objectives. 140 Foerster, Chromatium Okenii, cells connected sidewise 216 Folmer & Schwing, enlarging, reducing, and copying camera 146 Formaldehyd 74 Formalin 74 differential effect on bacteria 229 germicidal action 252, 253 preservation of cultures by use of 263 Formulae 187 Forster, gelatin with high melting point 225 Foth's spore-stain 218 Fractional method of culture. 226 Fraenkel & Voges' solution 197 Frankland, effect of gases on bacteria 231 photographic action of bacteria 242 Frankland & Ward, method for study of an- tagonism 73 Freer & Novy, germicidal action of organic peroxides 253 Freezing 246 effect on anthrax, etc 247 effect on B. typhosus 247, 258 effect on bacteria of pest and diphtheria 247 osmotic pressure a factor in 247 with liquid air 79-83 with salt and ice 83 Frost, antagonism 73 gasometer 61 Friedlaender, acid-fast stain 187 capsule-stain 220 Fribes, retting of flax 257 Fruit-ether-forming bacteria 259 Fuchsin, anilin 187 Fuller's scale 69 Funck, cleaning of cover-glasses 227 Fiirbringer und Freyhan, disinfection of hands 252 Gabbett's stain 188 Gas-analysis 55 Gases, compressed 81 Gas-generator, de Koninck 54 hydrogen, with wash-bottles 55 Kipp 54 Gasometer, Frost s 61 Gas-pressure regulator, Murrill's 78 276 BACTERIA IN RELATION TO PLANT DISEASES. Page. Gauze, surgeon's, uses in laboratory 102 Gelatin, antiseptic salts in 3 brands recommended for bacteriological work 3 r composition of 30 impurities in 30 melting point of 30 nitrates in 224 per cent recommended for media 30 plate-cultures, stabs, streaks 29 standard nutrient, preparation of 196 sterilization of 98 variation of same organism on 180 with cane-sugar 51 with high melting point 225 with malic acid 51 with soluble starch, etc 51 Gelatinization, of old milk-cultures 46 Gelidium, species, source of agar-agar 31 Generic characters based on morphology 156 Generic names rejected 174 Gentian violet, Ehrlich's anilin-water 187 Flexner's anilin 187 Gerlach, germicidal action of lysol 251 Germicides 74 literature on 250 Germination, spores should be subjected to. . . 21 Gessard, green-fluorescent bacteria 237 Giltay & Aberson, culture-medium for denitri- fying organisms 198 Glassware, cleaning and sterilization of 100 pencils for writing on in solubility of 129, 223 Globig, thermophilic bacteria 248 Glue, blue-pigment bacteria injurious to 237 Glycerin, agar 33, 196 gelatin 262 mounts, sealing of 262 Goriansky, wood-vinegar as a disinfectant. . . . 252 Gram's stain 188 Nicolle's modification 222 Grape-sugar harmful effect of 223 test for, in plant tissues 208 Green, germicidal value of copper salts 251 Green bacteria 237, 238 Green-fluorescent bacteria 65 Gessard's studies 237 Jordan's studies 238 Thumm's studies 238 Griess-Ilosvay's reagent 229 Grimbert, on value of synthetic media 51 Growth of bacteria, manner and rapidity of 27 on media exhausted for other organisms. . . 211 rates of, in fluid cultures 42 restrained by acid gelatin 30 Grove, glycerin mounts rendered air-tight 262 Gruber, resistance of spores 246 Page. Guillebeau & de Freudenreich, agar made without filtering 223 Haegler, oentrifuging agar 225 Halation, means of avoiding 136 Hallier, views concerning morphology 176 Hands, disinfection of 252 Hanging-drop cultures 22 containing single organism 27 manner and rapidity of growth at given temperatures 27 Hankin, germicidal action of river water 251 Harris, collodion sacs, method of making 229 Haswell, method of substituting alcohol for water 226 Hauser, spore-stain 194 preservation of cultures for museums 263 Heat, as a means of separating organisms 106 as a sterilizer for surface of tissues 13 dry, cause of resistance of spores to 218 effect on pigmentation, pathogenicity, spor- ulation 87 enzymes injured by moist 67 influence of substratum on resistance to. 219, 247 Heidenhain's iron-haematoxylin 189 Heim, anaerobic cultures of 231 Hellstroem, grape-sugar, harmful effect of . . . 223 Hempel, burette for gas-analysis 55 pipette for liquid reagents 55 Henle, on proofs of pathogenesis 9 Herter & Foster's test for indol 201 Hesse, media for water-bacteria 196, 258 method of cultivating anaerobes 230 Hill's hanging-block method 22, 228 Hinterberger, method of staining flagella 220 Hiss, media of, for differentiation of motile bacteria 26 for differentiation of typhoid, colon, and allied bacilli 230 Kitchens, on autoclaving bouillon containing sugar 99 Hoffmann, infiltration in vacuo 226 Hoods, improvised 104 in laboratories 104 Host, color changes in, due to injury other than that caused by parasite 66 due to oxidation phenomena 65 due to parasite 65 Host, reaction to parasitic attack 8 Host-plants, kinds attacked by a single para- site 87 Hydrochloric acid, in culture-media 98 Hydrogen, compressed, where obtained 81 cultures in 56, 57 test for, in fermentation-tubes 61 Hydrogen-cellulose ferment 106, 209 INDEX. Page. Hydrogen generated with zinc 54, SS hydrogen sulphide in, means of avoiding evolution of 56 impurities in 55 removal of last traces of oxygen from 57 washing of 56, 57 Hydrogen peroxide, use in staining spores 218 substance causing liberation of oxygen from 67, 234 Hydrogen sulphide 62, 242, 243 Hypo for fixing 142 Tee, bacteria in 247, 258 India oil-stone 121 Indigo-carmine, reduction of, by bacteria 62 Indigo-blue, production of 257 Indol, production of, media suitable for 62 tests for 62, 201 Infection, carriers of 91, 178, 215 conditions favoring 16 due to ultramicroscopical organisms.... 18,211 natural methods of 8, 92 Infection, stomatal 84, 86, 90, 92, 108, 126 Infection-experiments, value of direct 9 Infectious material, final disposal of 106 Infiltration, in vacuo 8, 226 with paraffin 118 Injection-needle 101, 227 Inoculated organism, method of proving viru- lence 16, 185 Inoculated plants, labeling of 112 Inoculation, cages 108 checks on 16, 186 cross 186 methods of 108 with more or less exact numbers of bac- teria 226 Inoculation-experiments, conditions favoring 16 how carried on 15 importance of 14 where best made 16 with mixed organisms 9, 72 Insects, inoculation by means of. . 91, 108, 178,215 Instruments, sterilization of 100, 107 Intensifying negatives 142 Invertase 66 Investigation, training necessary for 181 Involution-forms 23, 177 Iodine-starch test for nitrites 63 Iron-haematoxylin 188, 189 Irritation, contact 27 Isochromatic plates, for photographing stained sections 140 time of exposure with 143 uses of 136 Isolation of bacteria, from diseased tissues, methods of n on slant agar, etc 227 Page. Twanoff, penetrating power of formalin 252 Jeffer, ruled counting plate 36 Jennings, contact-irritation 27, 254 Johan-Olsen, branching forms 217 Jolles und Winkler, bacteria in oleomarga- rine and butter 259 Jones, enzymes from soft-rot organisms 68 Jordan, pigments produced by green-fluores- cent bacteria 65 Kabrhel, methylen blue as oxygen indicator. . 232 Kaiser, glycerin-gelatin 262 Karten, chemical analysis of agar-agar 32 Kasansky, effect of winter cold on bacteria. . . 247 Kaufmann, method of staining capsules 194 Kedrowski, anaerobes grown with aerobes. . . . 231 Kendall, decimal system 176 Kephir 214 Kern, two endospores in a cell 22 Kipp gas-generator 54 Kitasato flask 68 Klebs, fractional method of culture 226 Klein, A., anaerobic apparatus of 232 spore-stain 219 Klein, L., bacilli bearing green spores 218 Klepzoff, freezing experiments of 246 Knauer, cleaning slides and cover-glasses.... 226 Knives, microtome 122 Koch, A., branching of B. pediculatum 221 Koch, R., comma bacillus of 173 demonstration of flagella by stains 20 importance of photography in microscopic works 23 magnification recommended for photomicro- graphs 24 methods of isolating parasite from tissue, n, 226 paper on tuberculosis 153 rules of proof 18, 226 streak-method of isolating bacteria u, 226 views concerning morphology 176 Konradi, germicidal action of soaps 253 Kraemer, germicidal action of copper 253 Kratschmer und Niemelowicz, stringy bread . . 260 Krause, Bacillus pyocyaneus 223 Krueckmann, preservation of cultures for museums 263 Kuehne, carbol methylene blue 188, 222 malachite green 222 Ktintsler, animal origin of bacteria 264 Kuntze, medium for Bacillus prodigiosus 238 Kuprianow, device for filling test tubes 227 Labels, for tubes, flasks, etc no, in Lab ferment 67 Laboratory, care of 96 equipment of 94 things to be considered in location of 94 Lamella, middle, dissolution of 67 Lamprocystis 163 Lantern-slides, apparatus for making 143, 146 2 7 8 BACTERIA IN RELATION TO PLANT DISEASES. Page. Lantern-slides continued. development of 146, 201 exposure of 146 Larkspur, natural infection in 92, 139 Laser, on action of hydrogen peroxide 234 Lautenschlager oven 100 Lead acetate paper 62 Leguminosae, root-tubercle bacilli of, fixation of nitrogen by 64 Moore's culture-medium for 197 Lenses, achromatic, defects of 14 apochromatic, in photomicrography 139 for bacteriological work 23 Lenses, photographic, Goerz 130 Voigtlaender 130 Zeiss Double- Protar 130, 132 Zeiss Planar 132 Zeiss Protar 132 Zeiss Unar 132 Lenticels, natural infection through 92 Lepeschkin, branched form described 218 Leucin 63 Leuconostoc mesenterioides, isolation of 106 Levin, bacteria rare in Arctic 255; Levy, physical properties of enzymes 68 Lewandowski, test for phenol 63 Liborius, oxygen in depths of agar 230 Light, action on bacteria 71, 243, 244 bactericidal action ascribed to hydrogen peroxide 244 germicidal action on dust greatest when moistened 244 germicidal in hydrogen 244 germicidal action in water 244 Light filter for photomicrography, dry 24 Zettnow's 24, 201, 263 Lilac blight 64 Liquefaction in gelatin stab-cultures, characters to be noted 29 interferences of various substances with. ... 29 Liquid air, effect of, on bacteria 79, 247 temperature of 80 Lister, dilution method for obtaining pure cultures 27, 226 Litmus-lactose-agar 33, 196 Litmus milk, cultures in, observations on 48 preparation of 48, 196 Litmus paper, for testing acidity 61 Litmus, reduction of, by bacteria 62 Loeb, typhoid bacillus, branching forms in... 218 Loeffler, alkaline methylene blue 188 extraction of enzymes 68 flagella-stain icjo Loew, culture-medium for Bacillus prodigiosus. . . 239 enzymic fermentation of tobacco 257 Page. London, resistance to starvation 223 Loops, platinum-indium, for making transfers . 43 Loquat, bacterial disease of 88 L6 wit's flagella-stain 192 Luminous bacteria 60, 241 cause of luminosity of 60 culture-medium for 198 Dubois' fluid medium for 242 effect of chlorides on 60 effect of magnesium sulfate on 60 effect of manganese sulfate on 60 effect of potassium iodide on 60 effect of potassium nitrate on 60 effect of potassium sulfate on 60 in or near salt water 60 in sand-fleas 60 Molisch's work on 60 on meat exposed in markets 60 on synthetic media 60 spectrum of 242 thermal relations of 60 Lunt, stock-cultures of water-bacteria, how best kept 263 Lysol, for sterilizing surface of diseased material. 14 germicidal action of 251 Maassen, branching forms produced at will by 23 culture-fluid of 198 fruit-ether-forming bacteria 259 reducing powers of bacteria 62, 243 Mace, recuperation of pigmentation and pathogen- icity 237 ruled counting-plate 36 Macfadyen, effect of liquid air on bacteria 247 thermophilic bacteria 248 Mackensie's culture-fluid 198 Magnesium chloride, effect on luminous bac- teria 60 Magnesium sulfate, effect on luminous bac- teria 60 Magnification, determination of 115 Maize, artificial infection of 90 cross-section of stem 4 natural infection in 92 Malachite green, use of 189, 222 Maltose-agar 33 Manganese sulfate, effect on luminous bac- teria 60 Marmier et Abraham, germicidal action of ozone 252 Marpmann, " chromo-agars " for differentiating 229 germicidal action of sodium fluoride 253 iron bacteria 261 Marsh gas 61,209 Maximum temperature, how determined 75 range of 87 Mayer's culture-fluid 197 Maze, root-tubercle bacteria 240 INDEX. 279 Page. Meat-extracts, bacteria in 260 Media for cultures, agar 31, 195 animal fluids 45 gelatin 29, 196 milk 46, 196 non-synthetic 195 plant juices 41 silicate-jelly 41 special 49 starch-jelly 5. T 96 sterilization of 85 synthetic 49, 197 vegetable media, solid 39 Media free from spores, easy sterilization of. 40,85 Mercaptan 62, 243 Mercuric chloride 74 for sterilizing plants before inoculation. 108, 109 for sterilizing surface of diseased material. 14 germicidal action of 232 in hot alcohol, as a fixing fluid 8 preservation of cultures by use of 263 Messea, classification of 20, 264 Mctcalf, bacillus softening agar 32 Meters, for photographic exposures 143 Methane-cellulose ferment 106 Methods of work, checking 184 literature on 226 Methylene blue, as a test for free oxygen 57, 232 Loeffler's alkaline 188 reduction of, by bacteria 62, 239 Methyl violet, as a germicide 74 Meyer, effect of liquid air on bacteria 247 Michaelis, thermophilic bacteria 248 Micrococcus 160 Micrococcus progrediens, small size of 18 Micrometers, stage 115 Micro-organisms, cell-life without 211 Microscope, achromatic objectives for 140, 265 apochromatic objectives for 130 for recording exact location of desirable fields 129 Leitz 129 oculars 130 Spencer 129 Zeiss, for bacteriological investigations 129 Zeiss, for photomicrographic work 129 Microspira 161, 172 Microtome, kind preferred 122 Microtome-knives 122 Microtome-sections, cutting and care of 119 mounting of 119 staining of 119, 120 Microzymas of Bechamp 176 Migula, classification of 159 Milk, a good culture-medium 47 anaerobes in 46 Page. Milk continued. clearing of 46, 267 coagulation of, by bacteria 67 enormous numbers of bacteria in 196 litmus 196 observations to be made on cultures in 46 preparation of, for culture-medium 46 reddened by bacteria 259 resistant spores in 46 ropiness in 259, 260 sterilization of 46, 98 temperature governs bacterial multiplica- tion in 260 Minervini, bactericidal action of alcohol 252 Minimum temperature, for growth, how determined 76 range of 87 Miquel, bacteria in sea-air 255 thermophilic bacteria 247, 248 Mixed cultures, behavior of 72 Moller's spore-stain 218 Molisch, on luminous bacteria 60 Monas 173 Moore, A., capsule-stain 221 Moore, G. T., copper sulphate as a germicide 74 culture-medium for root-tubercle bacilli. ... 197 root-tubercle bacteria 241 soil-inoculation for legumes 64 Moore, V. A., flagella-stain 190 Moore & Kellerman, action of copper on B. typhosus in water 97 Morphological characters, change in, due to changed environment 222 value of 176 Morphology 19 extreme views concerning 176 general account of 18 generic characters should be based on 156 insufficient for differentiation of many spe- cies 25 literature on 215 modern views concerning 177 Morton, flagella-stain 220 Motility of bacteria 26 Motor-reflex in bacteria 254 Mueller, bacterial reduction processes 239 Miiller, genus Vibrio 172 Muir, capsule-stain 194 Murrill, gas-pressure regulator 78 Museums, preservation of cultures for. . . 262, 263 Mustard, natural infection in 92 Mycobacterium 172 Myconostoc 161 Myxobacter 165 Myxobacteriaceae, characters of 164 Myxococcus 165 Naegeli's nutrient solution 197 Nakanishi, nucleus, existence of, in bacteria. . 217 Nectaries, natural infection through 92 Needle-punctures, inoculation by 108 280 BACTERIA IN RELATION TO PLANT DISEASES. Page. Needles, platinum-iridium, for making trans- fers 43 Negatives, development of 14 records on 142 Nessler*s test, how used 61 Neisser's spore-stain 104 Neumann, variability of pigment- formation . . . 238 Neutral red 230 Nicolle, modification of Gram's method 222 Night-blue, capsule-stain 221 stain for flagella 220 Nitrate bacteria, fluid culture-medium for isolation of 199 nutrient agar for isolation of 109 Nitrate bouillon 63 diphenylamin test 267 Nitrates, reduction of, test for 63 Nitrifying organisms, magnesia-gypsum blocks for 200 Nitrite bacteria, fluid medium for isolation of. 199 Nitrites, test for 63 Nitro-bacteria 175 Nitrogen-assimilating soil-bacteria, medium for 199 Nitrogen-bacteria 175 Nitrogen-free media 51, 198 Nitrogen, fixation of, by bacteria 64 growth in, apparatus for testing 58 removal of oxygen from 57 Nitrogen-nutrition, a basis for classification.. 175 Nitrogen salts, in special media 51, 197 Nitromonas 240 Nitrous and nitrate bacteria 175 Nomenclature 154 Non-halation plates, uses of 136 Novy, a new thermoregulator 228 jar for anaerobic cultures 56, 57, 58, 231 Nucleus, existence of, in bacteria. 159,216,217,219 Nutrient material, amount needed by bac- teria 225 Objectives, achromatic, disadvantages of 140 of excellent definition 265 Objectives, apochromatic, Spencer 16 mm 140 Zeiss 130 Occlusion of vascular system due to bacteria . . 8, 12 Oculars 130 for photomicrography 139 Ohlmuller, germicidal action of ozone ^51 Oleomargarine, bacteria in 259 Olive-knot organism, artificial inoculations 10 crystals produced by 66 optimum temperature for 85 Omelianski, blocks for nitrifying organisms 200 isolation of hydrogen-cellulose ferment. 106, 209 sodium-formate medium 50 spore readily stained by anilin dyes 22 Page. Optimum reaction of medium for bacterial growth 69 Optimum temperatures 75, 85 Origin of bacteria 177 Ortol developer 140 Ostwald-Pfeffer water-bath 78 Oven, paraffin no Oversteaming, effect of 98 Oxidases 67 Oxidation, pigments due to 66 Oxygen, compressed, where obtained 81 little, in deep layers of agar 230 pigment- formation, dependent on 238. 230 removal from hydrogen, nitrogen, and car- bon dioxide 57 Oxygen, relation of bacteria to, in, fermentation-tubes 52 surface and deep growths 51 Ozone, action on bacteria 251, 252, 253 Pacinia 172 Pake, ruled counting-plate 36 Paper, blue-print, for pen-and-ink work 150 drawing-board 266 salted silver, for pen-and-ink work 150 Paraffin, infiltration with 118, 226 melting point of that used for embedding. . . 119 recommended for cotton plugs 99, 263 trueing edge of blocks 123 Paraffin-oven 1 19 Parasite, relation of, to tissues of host 8, 10 reaction of host to 8 means of gaining access to tissues of host. 8,92 Parasites, animal, harbored by plants 89 Paratrophic bacteria I7S Parenchyma, intercellular spaces of, occupied by bacteria 8 Park, effect of freezing on B. typhostis 247 Pasteur, culture-fluid 197 definition of ae'robe and anaerobe 230 influence on bacteriology IS 2 Pathogenesis, rules of proof 9 Pathogenicity, developed by association 72, 215 of plant parasites to animals 88 regeneration of 237 Pane, Pneumococcus, origin of capsule 221 Payen, agar-agar, composition of 223 Pear-blight organism, action of light on 7 l blackening of foliage attacked by 65 differentiated from B. coli by KNO 3 51, 113 hosts of 87 natural infection 9 2 result of inoculations with 202 Pedesis i Peirce, root-tubercles of bur clover 64 Pelargonium, natural infection in 92 Pencils for writing on glass m Pepsin 66 INDEX. 28l Page. Peptone, commercial 45 reaction of cholera organism with 229 tests for purity of 229 Peptone-bacteria 175 Peroxides, organic, germicidal action of 253 Petri, nitrates in commercial gelatin 224 Petri-dishes 226 for excluding light 228 for quantitative work and photographing. . . 36 of good quality, where obtained 82 sterilization of 100 Petri und Maassen, hydrogen sulphide from bacteria 243 Phenol 74 tests for 63 Phenolphthalein, for use in titration 99 neutral point of 69 Photographic enlargements 147 Photographic plates, for special work 136 Photographic prints, drawings on 151 making of 150 Photographing, of bacteria in tissues 136 of poured-plate colonies 134 of test-tube cultures 135 Photography, focusing planars 132 lenses for 130, 133 Photomicrography 263, 264 Photomicrographic apparatus 23, 129, 136 care of 139 Photomicrographs, making of 138 time of exposure for. 143 Phragmidiothrix 162 Physical conditions, influence of, on mor- phology 177, 222 Physiological changes due to changed environ- ment 222 Physiological peculiarities, for identification of species 25 Picric acid, as a fixing agent 8 germicidal properties of 250 Pierce, work on walnut disease 176 Pigment- formation, conditions of 239 discussion of 65, 223 effect of symbiosis on 223 how regenerated 237 literature on 236 variability of 222, 238 Pipettes, discarded, care of 107 sterilization of 100 Pitfield's flagella stain 191 Planococcus 160 Plant acids, sensitiveness of bacteria to 69 Plant juices used as culture-media 41 Page. Plasmolysis, concentration necessary for 254 Plastids of Hallier 176 Plate-cultures, advantages of, for isolating bacteria n arrangement of rods in colonies on 27 characters to be noted 29 discarded, care of 107 how to avoid contaminations in 103 Koch's, for obtaining pure cultures 226 labeling of in poured, how made 105 Plates, isochromatic, for photographing stained sections 140 isochromatic, time of exposure with 143 isochromatic, uses of 136 non-halation, uses of 136 non-isochromatic, when preferable 136 Platinum black, enzyme-like action of 234 Platinum-iridium, inoculating needles and loops 43 Pleomorphism 176 Pleuro-pneumonia, organism causing, at limit of vision 18 cultivation of, outside animal body 213 Plugs, paraffined 99 Plum, natural infection in 86, 88, 92, 148 organism from 18 subject to pear-blight 88 Pneumococcus, origin of capsule 221 Polyangium 165 Popoff, germicidal action of mercuric chloride. 252 Potassium, chloride, effect on luminous bacteria 60 iodide, effect on luminous bacteria 60 nitrate, effect on luminous bacteria 60 nitrate, for differential purposes 51 sulfate, effect on luminous bacteria 60 Potato, steaming of 98 Potato-broth, preparation of 42 Potato-starch, aseptic, preparation of 50 Prazmowski, arthrospores 218 culture-fluid 197 Precipitate, in culture-fluids, nature of 42 Pregl, method of staining 222 Preparation of nutrient agar 195 Pressure, effect on bacteria 245 Prevention of disease, methods of 93 Proskauer & Beck's culture-fluid 198 Prudden, bacteria in ice 258 Pseudomonas 160, 173 Ps. indigofera, small size of 18 Ps. syringae, reaction to nitrates 64 Pseudo-zoogloeae 22 Pure cultures, how obtained n Pyocyanin, test for 65 Pyoktanin 74 Pyrogallol, with caustic potash as absorbent of oxygen 57 Pyrogallol developer 200, 201 .'82 BACTERIA IN RELATION TO PLANT DISEASES. Page. Quantitative determination, of bacteria in diseased tissues 14 of effect of freezing 79 Quince blight, due to B. amylovorus 202 Rabies-virus, removed by filtration 214 Rabinowitsch, thermophilic bacteria 248 Radium rays 2 45 Raulin's culture-fluid 197 Ravenel, bacteria exposed to liquid air 246 Razors, sharpening of I21 Torrey, Rogers, Lentz 123 Reaction, for maximum growth in liquid media. .. 69,203 of host to parasite 8 Record-books, for culture-media IO 9 for field-notes "O Records, methods of keeping 109 Reducing negatives '42 Reducing powers of bacteria 62 Reichenbach, branching in Spirillum 217 Reichert, thermo-regulator, improved 78 Reimers, bacteria in soil 255 Rein, Florideae, source of agar 224 Reinitzer, on gum-ferment 233 Re-isolations, necessity for 1 6 Relationships of bacteria 177 Rennet 67 Rhabdochromatium 164 Ribbert, method of staining capsules 194 Rice for culture-media 48 Ridgway, color scheme of 263 Roentgen rays 245 Rogers, pathogenicity developed by association. 215 Rohrbeck thermostat 75 Roll-cultures, Esmarch's 226 Root-tubercle bacteria 64, 240, 241 Roth, contact-irritation 253 Rothberger, differential diagnosis with anilin dyes 230 toluidin red for differentiation 230 Rothert, effect of ether and chloroform 254 Roux filter, for culture-fluids, etc 44 Roux thermo-regulator 78 Rubber, caps for tubes of media 99 note on best solvents for 207 Rulers for measuring colonies, glass, steel. ... 115 Rules of proof 9, 10 Russell & Babcock, fermentation of silage. . . . 257 Ruzicka, inner structure of bacteria 217 Saccardo, color scheme of 263 Sachs, reduction of cane-sugar in plants 66 Safranin-picro-nigrosin 189 Salicylic acid 74 Salt-bouillon 51 Salt-water bacteria, minimum temperature for 87, 255 Sander, growth of tubercle bacteria on vege- table media 222 Sand-fleas, bacterium of luminous 60, 242 Page. Saponification of casein in milk-cultures 46 Saprophytes, behavior of, when injected into plants 89 presence in diseased parts long affected .... 13 Sarcina 160 Sauerkraut, fermentation of 257, 258 Scales for photographic exposures 143 Schaffner's safranin-picro-nigrosin 189 Schaudinn, bacillus of large size 19 two endospores in a cell 22 Scheffler, neutral red for differentiating 230 Schild, formalin for detection of B. typhosus. . 229 Schill, preparation of cultures for museums. . 262 Schilow, germicidal value of hydrogen pe- roxide 251 Schneider, studies on pigment- formation 237 Schottelius, B. prodigiosus, non-pigmented races of 222 preparation of nutrient agar 223 Schliiter, effect of acids on bacteria 249 Schultz, structural changes due to antiseptics. 252 Schumburg, bromine, water treated with 252 Schu'tz, method of making nutrient agar 34 Schwartz, on antiseptics 250 Sclavo's flagella-stain 192 Sclerothrix 172 Sea-weeds furnishing agar 33 Sections, microtome, balsam for mounting 117 boxes for preservation of 117 cutting and care of 119 for photographing 140 keeping of material for 117 mounting of 119 preparation of 1 18 staining of 119 Sedgwick & Winslow, influence of cold on B. typhosus 247 Setchell, thermophilic bacteria 248 Signs of disease 7 Silage-fermentation, bacteria active in 256 bacteria not active in 257 Silicate-jelly, for differential purposes 39 method of preparing 37, 206 value, as a culture-medium 36 Winogradsky^Sleskin 198 Sjoebring, nucleus in bacteria 216 Skschivan, branched forms 217 Slater, branched forms 237 Slide-boxes 117 Slides, care of discarded 107 numbering of T2I Smith & Swingle, effect of freezing on bac- teria 83 Smith, Theobald, discovers cause of Texas fever 153 method of filtering 226 tubercle bacteria difficult to destroy by heat. 247 Soaps, germicidal action of certain 253 INDEX. 283 Pago. Sodium chloride, effect on luminous bacteria 60 restraining influence of 70, 252 Sodium fluoride, germicidal action of 253 Sodium hydrate, method of increasing tolera- tion of 70 Sodium nitrite, used for indol test 62 Softening hard tissues, fluid for 200 Soft-rot bacteria, many plants attacked by. ... 87 Soil, bacteria in upper layers of 255 parasites carried in 91 sterilization of 85 Solid vegetable media, behavior of organisms on 40 preparation of 40 substances recommended 40 Solio paper, toning bath for 201 Solubility of glassware 129, 223 Solution of tissues occupied by bacteria 10 Solution persulphate of iron, preparation of. . 188 Sorghum, natural infection in 92 Soy-bean, natural infection in 92 Sphaerotilus 162 Species, morphology not sufficient for differentiation . 25 Specimens, method of substituting alcohol for water in 226 Spectrum, part possessing germicidal action. . 244 Spencer microscopes 129 Spina, studies of reduction processes 239 Spirillum 161 branching in 217 double staining of 217 Ehrenberg's genus 173 Spirillum undula, action of ether on 254 flagella of 20 Spirillum volutans, flagella of 20 reaction to stimuli 27 Spirochaeta lot Spirodiscus 162 Spiromonas 162 Spirosoma 161 Spores, action of heat on 84 Aujeszky's stain 219 bacilli bearing green 218 cause of resistance to dry heat 218 classification based on 157 common in stringy bread 261 effect of steam on 246 Fiocca's stain 218 Foth's stain 218 germination of 21 influence of environment on formation Of 219, 222 Klein's stain 219 means for identification of 21 Moeller's stain 218 reaction to stains 27 Page. Spores continued, resistance to high temperatures and steam heat 21, 84, 246 resistant, present in meat-extracts 260 resistant, present in milk 46 stains for. 194 Spraying, inoculation by 108 Stadler, effect of sodium chloride on bacteria. 252 Stage-micrometer 115 Staining, bacteria in tissues 29, 222 double 217,218 microtome-sections 119 Staining-media, groups of 28 Staining methods 221 bacteria, vegetative forms 27,187 capsules 19, 194 flagella 20, 21, 27, 189 spores 27, 194 tissues 187 Staining produced by bacteria, in host-plant 65 in nutrient substrata 65, 211, 214, 237 Stains, anilin, alcoholic solutions of 187 general and miscellaneous 187 Starch from potato, preparation of 50 Starch-jelly, nutrient 50, 196 Starch, test for slime derived from 221 Starrett cross-level ^9 Starvation, resistance to 223 Statistics, need of, on plant diseases 90 Steam, spores resistant to 84 sterilization by, possible source of error in. 47 vegetative bacteria sensitive to 83 Steaming, media in tubes 48 milk for culture-media 46, 47 Steam-sterilizer, Arnold 47 Stephens, flagella-stain 220 Sterilization, cold, of culture-fluids 43, 52 cotton IOI culture-media 85 dry, of pipettes, scalpels, etc 41 glassware, instruments 100, 107 infectious material ' IO 6 milk 46 oven for loo silicate-jelly JQ soils \] 8s solid vegetable media 40 starch-jelly g O surface of plants before making cultures from j-j surface of plants to be inoculated 41, 108 syringes IO2 with metallic copper 97 2 r? Still, for water, in small quantities 128 for water, on a large scale 124 284 BACTERIA IN RELATION TO PLANT DISEASES. Page. Stock-cultures, how best kept 72, 12 3 Stoddart, media for differentiation of motile bacteria 2( > Stolz, peculiar growths in Pneumococcus, etc. 217 Stomata, artificial infections by way of 108, 126 natural infection through 9, 92 Stored media, effect of loss of water on 99 Streak-cultures, characters to be noted 3 Streblothrichia l62 Streptococcus Io Streptococcus mesenterioides, isolation of by heat Io6 Streptothrix Io2 Succession of organisms in diseased tissues.. 73 Sugar-cane, red stain in bundles of 66 Sugars, effect on liquefaction of gelatin 29 growth retarded by 223, 248 proper sterilization of media containing 99 reagents for 208, 234 Sulphur-bacteria 162, 261 Sunlight, effect of 71 in photomicrography 24 Surface organisms, partial removal by washing . 14 Surface sterilization !3 Synthetic culture-media, kinds of 49, 197 value of Si Syringes, hypodermic I 02 Systematic position of bacteria 177 Szyszylowicz, corallin as a microchemical re- agent 221 Tannin, method for detecting in cells 207 Temperature, of liquid air 83, 247 optimum 75 maximum 75 minimum 76 range of, suited to growth 86 Temperature, influence on, geographical distribution 7 involution-forms 22 Teratological growths 23 Test-tube cultures, inoculation of 105 labeling of m photographing of 135 Test-tubes, device for filling rapidly 98, 227 holder for 104 Jena glass 81 resistant glass 82 tests of solubility 129 Tetanus poison, lethal dose of 213 Thaxter, Myxobacteria 164 Thermal death-point, apparatus for 78 method of determining 77 range of 87 Page. Thermal death-point experiments, methods of checking 185 Thermal limits, effect of, on pigment produc- tion, pathogenicity, sporulation 87 Thermal relations, of bacteria 75 of enzymes 67 Thermometers, Anschiitz normal 78 Thermophilic bacteria, common in digestive-tract 248 literature on 247 maximum temperature for 87 minimum temperature for 87 often spore-bearing 248 Thermo-regulator, Novy 228 Reichert 78 Roux 78 Tollens 37 Thermostat-room 75 Thermostats 75 Thermotaxis 254 Thiocapsa 163 Thiocystis 163 Thiodictyon 164 Thiopedia 163 Thiopolycoccus 164 Thiosarcina 163 Thiospirillum 164 Thiothece 164 Thiothrix 162 Thomann, bacterium of stringy bread 261 Thumm, green-fluorescent bacteria 238 Thymol 74 Tischutkin, preparation of nutrient agar 224 Tissues, solution of, by bacteria 10 Titration of culture-media 69, 99 Tobacco, fermentation of 257 Tollens, thermo-regulator 37 Toluidin red, for differentiation 230 Toning bath for solio paper 201 Transfers, how to avoid contaminations in making 103 Trenkmann, flagella-stain 219 Trevisan, genus Pacinia 172 Trikresol 74 Trypsin 66 Tsiklinsky, thermophilic bacteria 248 Tubes, care of discarded 107 rapid filling, with fluid media 99 Tumors produced by bacteria 8 Typhoid bacillus, effect of copper on. . 74,250,253 Tyrosin, qualitative test for 63 Ultramicroscopical organisms 18, 211, 213 Unguentum resinae 228 Urocephalum 159, 264 Uschinsky's solution, cultures in dilute, for flagella staining 20 formula 197 in silicate-jelly 39 modified 197 INDEX. 28 5 Page. U-tubes, for testing growth in nitrogen 58 Vacuo, concentration of fluids in 68 growth in 54 Vallin, cleaning filters 227 Van Ermengem, flagella-stain rgi germicidal effect of ozone 251 van't Hoff, modification of plate-method 228 van Tieghem, thermophilic bacteria 247 Varietal resistance to disease 93, 186 Vascular system occupied by bacteria 8 Velox-prints 151 Ventilation, of dark-room 149 Vernhout, bacterial fermentation of tobacco. . 257 Vibrio, use of, as a genus name 173 Vibrio cholerae, action of copper on 74, 250 classification 172 Vibrio, Muller's genus 172 Viscidity 42 of bacterial growths 19 of milk 46 of Uschinsky's solution 20 Vitality on various media 72 Voges, blue water-bacteria 237 cultivates Cholera vibrio on potato 249 Voigtlaender, physical properties of agar 224 von Esmarch, roll-cultures of 226 von Freudenreich, filtration of agar 224 von Rozsahegyi, pigmented-media for differ- entiation 229 V-shaped forms 23, 217 Wager exposure scale 143 Waldo & Walsh, bread not fully sterilized by baking 261 Wall-charts 206 Wall of cell, outer, reaction to stains 28 Walliczek, effect of dry air on bacteria 249 effect of tannin on bacteria 251 Ward, A. R., ropy milk 259, 260 Ward, H. M., bacteria from Thames water.. . 258 Warming 20 Water, apparatus for distilling 124 bacteria in, literature on 258 Water-bacteria, blue and violet 237 Hesse & Niedner's nutrient agar for 196 Page. Water-bacteria continued. how best kept 263 media for 258 Water, examination of. English methods for. 220 Water-bath, Ostwald-Pfeffer 78 Water-pores, infection through 92, 102, 124 Wax-mixture, Darwin's 200 Wehmer, fermentation of sauerkraut 2=;8 Weigert, early use of anilin stains 29 Weiss, bacteria from soured foods 235 ruled counting-plate 36 Welch, capsule-stain 20, 194 influence of 152 Welcke, flagella-stain 220 Wescner, eggs as a culture-medium 225 Weyl, ozone as a germicide 252 Wilhelmy, bacteria in meat-extracts 260 Williams, flagella-stain 193 Winogradsky, agar for isolation of nitrate bacteria 199 elective cultures 106 medium for nitrogen-assimilating bacteria. 199 nitrifying bacteria 240 retting of flax 257 silicate-jelly 36.-24O red sulphur-bacteria 263 Winogradsky & Omelianski, fluid-media for isolating nitrate and nitrite bacteria. . . 199 Winogradsky-Sleskin silicate-jelly 108 Winslow & Rogers, Coccaceae revised by 265 Wood-vinegar, an energetic disinfectant 252 Wounds, disinfection of 107 natural infection through 92 Wright, anaerobes, simple methods of culti- vating 228 Wynne exposure meter 143 Yendo, source of commercial agar 225 Yersen, pest carried by rats 215 Yokote, filtration of agar 225 Y-shaped forms 23 Zeiss lenses for photographic work, Double-Protar 130 Planar 132 Unar 130 Zeiss microscopes 129, 130 Zettnow, cleaning cover-glasses 227 flagella-stain 192 light-filter 137, 201 M