m m • •;. •• • . :'/. ' :; l 1 1 " ^HS , '• : -•. :. , ; V | i : ' H • , . . m '':".>.•• • ': ' : : i W.:'--.. :' BACTERIA ix RELATION TO PLANT DISEASES. FERDINAND COHN 2 ROBERT KOCH. 4. EMILE ROUX. 3 LOUIS PASTEUR. 5 EMILE DUCLAUX. BACTERIA IN RELATION TO PLANT DISEASES BY ERWIN F. SMITH, /;/ 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. WILKENS PRINTING CO, WASHINGTON, D 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 continuous 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 tnonography 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 IV PREFACE. together and properly digest all of the literature relating to this subject. This resulted iu the projection of a critical review of the literature, begun in 1896 in the American Xatiiralist 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 even- 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 iu 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 behiud 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 52 Growth in Hydrogen, in Carbon Dioxide, in Vacuo, and in Nitrogen 54 Luminosity 60 Page. 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 iSunlight 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. xrv. 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 and 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 XLIII. 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 LI I. Preparation of Slides, Cultures, etc., for Museums, etc 262 LIII. 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. Pago. PLATE I. Frontispiece. (1) Ferdinand Colin, founder of mod- ern systematic bacteriology. De- ceased. (2) Robert Kocb, 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 metclloidcs eight days after Inoculation with Bacterium solaua- ccariim 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 g. Chamberland Autoclave 84 10. Engine for furnishing Vacuum and Compressed Air 94 Page. PLATE 1 1. Culture-room, i. <-., place for making- Cultures of Bacteria in Still Air.. . . 104 12. Movable Hood of Wood and Glass, under which Bacteriological Trans- fers may be made 106 13. The Reinhold-Giltay Microtome 120 14. Distilled-wator Apparatus 124 15. Zeiss Stand Ha 129 16. Zeiss Photomicrographic Stand Ic.... 129 17. Mounted Camera for Enlarging, Re- ducing, and Natural-size Work 134 iS. Lantern-slide Room 144 19. Black Spot of the Plum 148 20. Bacterial Disease of Broomcorn 150 21. Bacterial Black Spot of Walnut 174 22. Ditto, Late Stage 176 23. Transmission of Wilt of Cucumber by Insects 178 24. Brown Rot of Potato. Natural Infec- tion of Tuber, Artificial Infection of Stems 202 25. Brown Rot of Potato. Shoots wholly destroyed by inoculation 202 26. Tomato-plant inoculated with Bac- terium solaiiaccanun 202 27. Bacterial Wilt-disease of Tobacco. .. . 202 28. Young Pear-shoots blighted by Bacil- lus amylovorus 202 29. Green Pear-fruits eight days after In- oculation with Bacillus amylovorus. 202 30. Quince-shoots and Pear-fruits (cross- section) showing Blight due to Bacillus amylovorus 202 31. Small Green Apples blighted by Ba- cillus amylovorus. . . 202 TEXT FIGURES. Pagt. Fin. I. Cross-section of Sweet-corn Stem para- sitized by Bacterium Stni'arti 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 camf'cs- tre confined to vicinity of Vessels.... 7 5. Bacterium camfcsirc. A small portion of Fig. 4 enlarged 10 Page. Fie. 6. Turnip-root, showing Bundle occupied by Bacterium campestre and the com- mencement of a cavity; a later stage than Fig. 5 n 7. Cauliflower-petiole, showing Bundle de- stroyed by Bacterium campcstre 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 X 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 n. (a) Capsule of Organism plated from Black Spot of Plum; (6) 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 camfestrc when crowded 23 19. Long Form of Bacterium camfcstrc when grown on Sugar-agar 23 20. Hanging-drop Culture 24 21. Involution-forms of Bacillus tracheifhi- his 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 Schiill'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-indium 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 51 1 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 witli Liquid Air 81 68. Petri-dish Poured Plate inoculated with a measured quantity of a Bouillon 'Culture of Bacillus trachcifliilus 82 69. The same as Fig. 68, but poured after Exposure to Liquid Air 83 70. Stomatal Infection by Bacterium priini in Green Fruits 84 71. Stomatal Infection by Bacterium pnuii in Leaf 86 72. Stomatal Infection by Bacterium fruni — a Later Stage in Fruit 88 FIG. 73 74- 75- 76. 77- 7§. 79- 80. Si. 82. 84. 85. 86. 87. 88. 89. oo. 92. 93- 94- 95- 96. 97- 98. 99- 100. 101. Seedling Sweet-corn Plant in Stage when most of Infections occur 89 Stomatal Infection of Sweet-corn Leaf by Bacterium Steward 90 A Detail from Fig. 74, 'highly magnified. 91 Water-pore Infection by Bacterium eam- pestre p2 Bacteria from Fig. 76, enlarged 2,000 93 Single Spiral Vessel occupied by Bac- terium eampestre 93 Water-pore Infection in Cabbage; a later stage than that shown in Fig. 76. 94 Angular Leaf-spot of Cotton, Nearly Natural Size 95 End of Vacuum-pipe on Laboratory- table 96 Portion of Work-table, showing Simple Apparatus for Distilling Water 97 Apparatus for rapidly filling Test-tubes with Measured Portions of Fluid Cul- ture-media 98 Can for holding Culture-media 99 Wrapped Petri Dishes 100 Meyer's Hypodermic Syringe 101 Sections through Tooth of a Cabbage- leaf, showing Entrance of Bacterium campestre 102 Green Cucumbers soft-rotted by Bacillus carotoi'orus 103 Block for holding Test-tube Cultures. .. 104 Constant Burner, with Cut-off for re- ducing Size of Flame 105 Steel Sewing Needle (No. 10) set into Bone-handle and used for Puncture- inoculations 106 Compressed-air Tank and Spray-tube. . . 107 Atomizers for use with 92 108 Hand-sprayer for Distribution of Bac- ILLUSTRATIOXS. Page. teria . 109 103. 104. 105. Inoculation Cage for Herbaceous Plants, no Labels from Test-tube Cultures m Wooden Labels from Inoculated Plants, in Temperature-record Sheets 112 Nitrate-bouillon Records 113 Sample from Card-catalogue, Two-thirds Actual size j j 4 Heading of Large Sheet for Volumi- nous Abstracts i r4 Green-cucumber Skin, Contents rotted out by Bacillus aroideae 115 Pillsbury Slide-boxes 116 Another Form of Pillsbury Slide-box. . . 117 Small Paraffin-oven used by writer 118 XI Page. FIG. 1 06. Infiltrated Tissues embedded in Par- affin in a Watch-glass 1 19 107. Infiltrated Material mounted ready to cut Ilg 108. Drawer with Compartments for hold- 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 eampestre 124 116. 117. Details from Fig. 115 124, 125 nS. Stomatal Infection of Cotton-leaf by Bacterium malvacearum 126 1 10. 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 iso- 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 I4s 135. Top-view of a Small Dark-room shown in Fig. 134 I4Q XII ILLUSTRATIONS. Page. FIG. 136. Case for protecting Squeegee-plates from Dust and Scratches M9 137. Bacterium trilnculare, Ehrenberg's first figure '66 138. Bacterium triloculare, Ehrenberg's sec- ond figure 169 139. Bacterium tcrnio, figured by Cohn.. . 17° 140. Dallinger and Drysdale's conception of Bacterium fernw I7° 141. TVrwo-like Organism obtained by throwing Beans into Water 170 Fie. 142. M3. 144- 145- 146. Page. Iris-rhizome-rot; Crowded Agar-plate after 45 hours at 25° C . 179 Iris-rhizome-rot ; Thin Sowing on Agar at end of 4 days; temperature 25° C.. 180 Bacillus aroideae grown on Agar-plate at 37° to 38° C 182 Bacillus aroideae grown on Agar-plate at 25° C 183 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 nonpathogenic. 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).| It would be still more to the point if they would isolate a dozen bacterial organisms from the soil, air, or water, ami 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, Lehmann & 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 Septcml>i-r, i fFor Bibliography see end of volume. 3 4 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 man}- of the diseases here considered. The writer has used the word "disease" in the common acceptation of the term, meaning thereby Fig. 1* 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 *FiG. I. — Cross-section of the upper part of a sweet-corn stem parasitized by Bacterium Stnvarti (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 token several feet from ithe ground, but the stem in- fection undoubtedly took place Uhrough one or more of the dower nodes. Drawn from photomicro- graph of a section stained with carbol-fuohsin. 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 its cut surface one loop of a fluid culture. The inoculation was made in Che .middle of a cross-section of die 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 below "the inoculated surface. A small portion of it at X is 'shown more highly magnified in fig. 3. This section was stained with carbol-fuchsin and bleached in 50 per cent alcohol. Drawn under Zeiss 16 mm. apochromatic objective with No. 4 compensating ocular and the Abbe camera. 6 BACTERIA IN RELATION TO PLANT DISEASES. Iii the present state of our knowledge (i) and (2) can usually be considered only after a very careful study of (3), (4), and (5), and of the organism itself. They involve a knowledge of modern languages, and a very considerable familiarity with scientific literature. PREVIOUS LITERATURE. One of the first requisites in a student is a knowledge of how to use literature. Previous literature is, however, often of such a fragmentary and uncertain sort, as we shall see, that it is impossible to decide whether a disease is actually new or has been written upon before. Fig. 3 * The literature of plant diseases will not be referred to in this volume, except occasionally and incidentally. The bibliography of this volume deals only with general bacteriology — human and animal diseases, methods of work, etc. *FlG. 3. — A detail from fig. 2. Bacillus carotovorus wedging apart cells of the carrot. Drawn mostly from one plane. In placing the cover-gla^s a few of the bacteria have been crowded out of the intercellular spaces into .parts they did not originally occupy. X 1,000. THE DISEASE. ( iicoo RAPHICAL DISTRIBUTION. Geographical distribution is an exceedingly interesting problem to many naturalists. The writer shares this feeling and has made ever}' effort to determine it, as far as possible, for each disease. There are, however, still many gaps in our knowledge — the whole subject is so new, and information from all parts of the world is desired. The inner temperature of plants conforms nearly or quite to that of the surrounding medium, and we might therefore expect, in some cases at least, to find a rather more sharply restricted distribution than in diseases of the warm-blooded animals. From theoretical con- siderations we should expect the distribution of plant diseases to be more like that of diseases of fish and other cold-blooded animals. Whenever the bacterium is able to endure as wide a range of temper- ature as the host-plant, we should expect to find it as widely distrib- uted. SIGNS OF THE DISEASE. Great care should be exercised in the description of the physical signs and of the lesions due to the parasite, so that the disease may be identified from these alone, if necessary. A great many cases should be examined and the signs must be recorded in detail and with great accuracy. It should be remembered that here is a frequent opportunity for error to creep in, lg' ' since the plant may be affected by two distinct diseases which have been confused. Good figures are always desirable, but are not absolutely essential. If possible, however, photographs, pen or pencil drawings, and good water-color sketches should be secured. *Fic. 4. — Cross-section of a turnip root, showing vessels occupied by Bacterium cainpestre as the result of a pure-culture inoculation hy means of needle-pricks on the leaves. Material fixed in strong alcohol, infiltrated with paraffin, cut on the 'microtome, stained with safranin-picro-nigrosin, and the differential washing stopped at just the right stage. The bacteria are confined to the vessels and their immediate vicinity. They do not occur in the phloem, a small portion of which is shown at the top of the picture. Section made from the same root as fig. 6, but lower, dn the tapering part. Drawn from a photomicrograph. X 85. 8 BACTERIA IN RELATION TO PLANT DISEASES. When all is said, the signs of many plant diseases, it must be admitted, are much alike, and this is particularly true of the bacterial soft rots. This is an added reason for studying them in each case as critically as possible. The captious reader might also remember that while an enormous amount of painstaking labor has been devoted to animal pathology, including twenty centuries in case of human medicine, we are only in the beginning, so to speak, of our knowledge of the minute pathology of plant diseases, and especially of those due to bacteria. PATHOLOGICAL HISTOLOGY. The relation of the parasite to the tissues of the host should be studied both in fresh material and in stained microtome sections made from material properly fixed and infiltrated with paraffin. The organism ma}' be a wound-parasite, or it may be able to enter through uninjured parts, t. ) Isolation of the organism from the diseased tissues and careful study of the same in pure cultures on various media. (c) Production of the characteristic signs and lesions of the disease by inocu- lations from pure cultures into healthy plants. (<{) Discovery of the organism in the inoculated, diseased plants, re-isolation of the same, and growth on various media until it is determined beyond doubt that the bacteria in question are identical with the organism which was inoculated. 10 BACTERIA IN RELATION TO PLANT DISEASES. Under (a) there should be numerous observations on many plants, with very careful microscopic examination of stained and unstained material. The cells of main- plants contain granules which often dance about so actively (pedesis or Brownian movement) as to be very deceptive, and yet they are not bacteria. Living bacteria in plant tissues can always be stained so as to stand out distinctly if the sections are well prepared and sufficiently thin. When bacteria occur in plants as parasites they are usually very abundant in the vascular system, or the parenchyma, or both, and there is, so far as yet known, always a distinct breaking down (solution) of some portion of the tissues (see figs. 6 and 7, and plate 3). If the parenchymatic tissues are sound, if there is no bacterial ooze on making sections, if the vascular Fig. 5.* system is not occupied, and if bacteria can not be demonstrated in the tissues by proper staining, then it is very unsafe to infer their existence from dancing particles, no matter how many may be visible in the unstained sections. Moreover, bacteria may be present in some of the plants and not in others, /. e.} not constantly present, and so not the cause of the disease. It is conceivable that they might also be present *FlG. 5. — Bacterium campcstre parasitic in a turnip-root (inoculated plant No. 53). This figure shows the bacteria crowding out into the cells surrounding the reticulated vessels. The 'lignified pnrtion of each vessel is indicated by fine dots. Material fixed in strong alcohol, infiltrated with paraffin, cut on the microtome, stained with carbol-fuchsin, and the excess of stain removed in dilute alcohol, section then dehydrated and mounted in xylo'l-balsam. Drawn from a photomicro- graph, the contrast here indicated being not greater than that shown in the section. X 500 circa. PLATE 2. Bacterial olive-knots produced on four plants by delicate needle-pricks. Inoculated January 4. 1904. Photographed May 16. 1 904. nearly natural size. The organism came originally from an olive-knol obtained in California, where the disease has been very destructive lor a number of years. A pure culture obtained from one of the California knots was inoculated into young growing olive-shoots and numerous knots resulted. From one of these, after about three months, the organism was plated out and a subculture from one of the colonies was used to produce the knots here shown. METHODS OF ISOLATION. II quite constantly, but merely as followers of something else. When possible, therefore, diseased plants should be examined for the suspected pathogen, in large numbers, in different years, and from widely separated localities. Of course, if fungi are also present they must likewise be examined as to constant occurrence and pathogenic properties. Under (/>) all of the standard nutrient media should be tried, and that repeatedly, until the student is entirely familiar with the appearance and behavior of the organism. It is usually best to isolate the organism for experiment from selected portions of the tissue by means of Esmarch roll-cultures or by the use of poured plates (Petri-dish cultures), generally the latter. Isolations ma}' also be made by inserting a sterile platinum needle or loop into the diseased tissue, obtaining therefrom a little fluid, and drawing this over the Fig. 6.* surface of slant agar, gelatin, or potato a number of times. This is an old method introduced by Koch in 1881. If ten or twelve tubes are used, the final streaks will often consist only of scattering colonies, from one or more of which the subcultures may be made. The plate method has the great advantage of showing just how many kinds of bacteria are present in the tissues (provided they will all grow in the medium used and under the conditions of the experiment), and just how numerous they are. In case of viscid organisms, or those forming compact zooglcese in the *Fic. 6. — Cross-section of root of plant No. 53 (turnip) parasitized by Bacterium campestre, showing an early stage in the formation of a bacterial cavity. The original section was made from material fixed in alcohol, infiltrated with paraffin, stained with carbol-fuohsin, and washed in a mix- ture of alcohol and water. Drawn from a photomicrograph. X 500. 12 BACTERIA IN RELATION TO PLANT DISEASES. tissues, it is sometimes desirable to grow them for a day in bouillon before attempt- ing the plate-cultures ; but one must then be on his guard, since it is quite possible by this method to start with enormous numbers of the right organism and have the bouillon culture filled with something else at the end of the 24 hours. Pure cultures may also sometimes be obtained by cutting out pieces of the tissue and throwing them into tubes of culture media. This method, however, shows little or nothing as to the prevalence of the organism in the tissues, and in $x -'^'•W"^''" -mi .-^IfSSrr pssi 'Av'. ?1r.! ^ -. _-'v- «J.l ^j|€*(||-, ^%K:||^B 7^5^^; / '"^^^ WMK Fig. 7* the hands of beginners is very liable to miscarry. If growth is obtained it may indeed have come from man}' organisms of one sort pervading the tissues and causing the disease, but it is not certain that it did not result entirely from one or *Fic. /. — Bundle in a cauliflower-petiole entirely destroyed by Bacterium camfestrc. The re- sult of a pure-culture inoculation. Plant No. 112 inoculated March 10, 1897, by needle-punctures on the blade of a leaf .without 'hypodermic injection. First signs of disease March 20. Petiole put into alcohol on April 5. Longitudinal section. Tissues surrounding .the bundle entirely free from bac- teria. Section not made from the inoculated leaf, but from the first leaf that showed secondary signs. Drawn from photomicrograph of a paraffin section stained with carbol-fuchsin. X 206. PLATE 3. jfB Cross-section of petiole of muskmelon No. 150 attacked by Bacillus tracheiphilus. The bacteria arc confined to the bundles, in each of which cavities have appeared. This section was taken liom near the point marked X on the inoculated leaf (see fig. 8 1 . The inoculations were made on the blade of the leaf by means of delicate needle-pricks. The material was collected and fixed m strong alcohol on the 6th day after the appearance of the disease. METHODS OF ISOLATION. 13 more bacteria accidentally introduced from the surface of the plant, from one's clothing or body, or from the air ; or it may have resulted from a few non-pathogenic organisms accidentally present in the inner tissues of the plant, particularly in case of roots which have been dug some time. It is therefore much better for the student to begin with plate cultures. Generally speaking, the parasite will be more easily obtained in a state of purity from plants or organs of plants recently attacked and from deep tissues, or from just within the margin of advancing diseased areas, rather than from near the surface, or from parts which have been diseased for a considerable time. Parts long affected almost always contain mixed growths due to the multiplica- tion of saprophytes of various kinds. From such parts it is usually much easier to obtain the saprophyte than the parasite, even if the latter has not been entirely crowded out and destroved. Fig. 8.* Great care must be exercised to avoid introduction of surface organisms which might complicate results, especially if rapid growers. The easiest and most satis- factory way, when the tissues will admit of such treatment, is to sear the surface with a hot knife or spatula so as to burn all surface organisms and then cut or dig through this sterile surface with hot or cold sterile scissors, scalpels, forceps, or needles to a part which has not been affected by the heat, from which some of the diseased fluids and solids may be removed on a sterile platinum loop. I frequently sear upon sound tissues at one side of the spot from which I desire to make cultures *Fic. 8. — Muskmelon plant No. 150. inoculated with a pure culture of Bacillus tracheiphilus. The pricked leaf is on the left side. The section shown in plate 3 was taken from the point marked X, three days after the photograph was made and ten full days after the inoculation. 14 T.ACTERIA IN RELATION TO PLANT DISEASES. and then dig under into the periphery of the diseased portion. If the tissues are rather dry the bacteria may be forced into the cavity by careful squeezing, or some drops (loops) of sterile water or beef-bouillon may be introduced into the cavity and stirred around before the bacteria are removed. If heat is inadmissible, the speci- men may be washed or soaked for a time (15 seconds to 60 minutes) in mercuric chloride water (1:1000) and the surface thus freed from many contaminating organ- isms. Carbolic acid (5 per cent in water) or lysol (5 per cent in water) may also be used for sterilizing surfaces. Of course these substances must be removed as far as possible before the surface is broken. This may be done to some extent by swabbing with sterile absorbent cotton dipped into sterile water or by plunging into sterile water and shaking. The disinfectants will be more certain to touch and sterilize every part of the surface if all adhering particles of air are driven off by first plunging into alcohol for a moment. In case of bacterial leaf-spots the writer generally obtains satisfactory cultures by cutting out the spot and plunging it for a few seconds (15 to 45) into 1:1000 mercuric chloride water, then rinsing in sterile water for a few minutes, crushing and throwing into a tube of bouillon from which the plates may be poured in course of an honr, /'. e., as soon as the bacteria from the interior of the spot have had time to diffuse into the bouillon. I frequently crush with a sterile glass rod, after throwing the material into a tube of bouillon, or else on a small sterile cover-glass which is then thrown into the bouillon. In cases where heat and chemical disinfectants are both inadmissible on account of danger of destroying the organisms within delicate tissues, as iu thin leaves and other soft parts, the bacteria or fungus-spores accidentally lodged on the surface may be greatly reduced in number by gently rubbing all parts of the surface between the thumb and finger xmder distilled water and then washing them in three or four successive beakers of distilled sterile water, the fragments being transferred from one beaker to the other by means of sterile forceps. Of course, the thumb and fingers must be well cleaned iu advance by scrubbing and sometimes by the use of alcohol and corrosive sublimate, followed by sterile distilled water. When dry, these washed specimens may be scraped into, directly for plate cultures, or after the epidermis has been peeled off with cold sterile knives and forceps. Quantitative determinations may be made by grinding up a given quantity of the suspected plant tissue, c. g., a cubic centimeter or a gram, in a sterile mortar with clean sterile sand and 10 or 20 cc. of beef-broth or sterile water, and then making plates from carefullv measured portions of the fluid, e.g., from one 2-mm. loop, from o.i cc., 0.5 cc., etc. A like number of check plates made from equal portions of healthy tissues ground under precisely similar conditions will soon demonstrate about how many colonies are to be expected per plate (and what kind) as the result of surface contamination or air-borne bacteria introduced during the process of grinding. The procedures described under c and d should be repeated a number of times (the more the better) and always with uninoculated plants in abundance for compari- son. 77/i .«? control-plants or check-plants must remain healthy. If they also become BEHAVIOR OF CHECK-PLANTS. diseased, then the experiments must be done over with more care and times enough to remove all possible chance of error. When check-plants become diseased, especially in any number, there is always room for grave suspicion. Either the experimenter has been grossly careless, assuming that he used the right organism in his inoculation-experiment, or else he is working in a locality where the cause of the disease is naturally abundant. In either case, however well convinced he him- self may be, his readers will generally have a lingering suspicion that even his inocu- lated plants succumbed not to what he inserted into them, but to some entirely differ- ent cause naturally present and overlooked by the investigator. The remedy for the first is to learn to use infectious material with more caution, and for the second is to make the in- oculation-experiments in localities or under conditions where the plant shall be less subject to natu- ral infection. If the experiments must be per- formed in localities where the dis- ease is naturally present, then a large number of plants must be selected for inoculation and for control, and such a high percent- age of infections secured in the inoculated plants that the few cases occurring naturally in the control-plants may be neglected as not casting any doubt on the general result. For example, if, in a region subject to the given disease, too plants were reserved for control and 100 similar plants were inoculated, and out of this number 50 of the latter and 40 of the former should contract the Fig. 9.* disease, it is manifest that no deductions of any value could be made from the experiment. All might be the result of some cause totally different from the *Fic. 9. — •Cross-section of a s i largest bacteria are several thousand times as bulky as the smallest. Errera has described a spirillum the largest specimens of which measured 23 to 28 by 3 to 3.4 micra ('02, Errera, Bibliog., X), and Schaudinn has described a bacillus the largest forms of which are 24 to 80 by 3 to 6 micra ('02, Schaudinn, Bibliog., XI). In shape the bacteria vary according to genera and species and sometimes within the limits of the species, from globose cells or very short straight rods, through curved forms or spirals, to filaments which are many times the diameter of the organism. To what ex- tent does form vary under changed conditions ? With the eye-piece micrometer make careful measure- ments of unstained organisms taken from the host- plant and from cultures of various ages and kinds. There is frequently considerable variability in the size of individuals of the same species. Is the breadth more constant than the length? Does the size or Fig. 12.* shape as observed in the plant differ from that observed on culture media? How does the living organism differ in size and general appearance from the dead, stained one? CAPSULES. The presence of capsules may be suspected whenever a bacterial growth becomes viscid. They are often difficult to see because their index of refraction is so nearly that of the fluid in which they are usually examined. In ex- • V~v ", ;,', I .«. amining unstained material the field should be illuminated with j a narrow pencil of rays, and the effect of illumination with ob- lique light should be tried. Several methods of contrast staining are in use. By one '^xj. fc-l0v method the capsule remains un- stained or nearly so, while the central portion of the bacterium and the slime lying on the cover between the bacteria stain more or less deeply. By another method which has been spe- i' \ \ •' V"s < Fig. I3.t Fig. I4.{ *Fir,. 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 trachcifhilus 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 ithe threads shown in fig. 13. The 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 «he 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. ii«. This may be compared with fig. 12, in which the same organism is shown without capsules. Fig. nb shows the extreme viscidity of a culture due to the formation and deliquescence of capsules. Fig. 13 shows the tenuous threads into which Bacillus tracheiphiliis 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 volutans. In 1875 Dallinger & Drysdale saw and figured them on Bacterium tcrmo. In 1875 Warming determined their existence on Vibrio rugula and Spi- rillum undiila. 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 tcrmo and also on Spirillum volutans. After 1879 no 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 bod}' 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 l>vt\veen 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 Fig. 15.* lich's aniliu-water gentian violet. Smeary dark lines and 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 LoefHer's stain ; Van Ermengern's nitrate of silver method ; Lowit's copper-sulphate fuchsin mordant, followed by Ehr- (For other methods consult " Formulae " and "Bibliography of General Literature," XII.) In connection with flagella-staiuing 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. 17. This should be attached to a wash-bottle, such as that shown in fig. 16. This will deliver a small stream, very good for washing excess of mordant and staiu 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 auilin stains, and (2) the use of moist heat. If at room temperatures the glistening bodies refuse to take the simple staius even on long exposure and at the same time are very resistant to steam heat or to hot water, /. 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. IS- — Flagella of yellow organism plated from 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 this flask on the 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 (hanging-drop studies. A-bout two-fifths natural size. 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). CELL-UNIONS— ZOOGI.CE/E, 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 zooglceae, or pseitdo- sooglcEa:, if one prefers to retain zooglcese 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 zooglceoe, 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). *Fic. 17. — Double blow-bulb for attachment to drop-bottle shown in fig. 16. By use of this de- Mr, -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. 21). Under what conditions do they occur? Are they living or dead? Isolate in hanging drops of bouillon and determine whether the}- 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 man}- 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 hours. 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 11. 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. I9.t *Fic. 18. — Bacterium camfcstre. 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 camfestre from an old culture on 23 per cent grape-sugar agar, showing long filaments. Cover stained I hour and 20 minutes 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 directly from the slide. X 1,000. 24 BACTERIA IN RELATION TO PLANT DISEASES. Good photomicrographs should be secured 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. 1-5 (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. I ~ *l 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 ol the camera, unless a weak light is used and the exposures are very long, in which case a slight jarring is of no great consequence. Direct sunlight is the best light, but the light of the open sky may be used (with full open diaphragm) if one is willing to make 5 to 20 minute exposures. Electric light is often used by those who live in cloudy regions or who occupy rooms not exposed to F. 2| . the sun, but the writer has had no experience with it. Very good pictures also may be made by gaslight if the Welsbach burner is used. Ordinary lamp light (kerosene) is too yellow and not sufficient!}' intense. Photographs can be made with a kerosene light, but the time and trouble involved make it scarcely worth while to consider this source of light. The writer has obtained the best results by using direct sun- light and slow isochromatic plates behind Zett- now's light filter. Of course, with upright cameras a dry light-filter must be used, such as the yellow one devised by Carbutt or by Ives. i> ---IIiillow-Kfouinl slide with cover-glass bearing hanging drop for examination under the microscope. tFic. 21. — Involution forms of Bacillus tracheiphilus from extremely ropy potato broth. Drawn in i hand, X 1,000 circa. Many as large as 8 by 2 micra and others larger. Nov., 1894. tFif.. 22. — Y -sliapcd (dichotomonsly branched) bodies from the root-tubercles of clover (Tri- folium). From a photomicrograph by tlie author, made from a slide furnished by Dr. Geo. T. Moore. X 1,500. INSPECTION OF COLONIES. For the inspection of colonies and of subcultures in tubes the best hand-lens known to the writer is the Zeiss aplanat magnifying six times (fig. 25). That magni- fying 10 times is also very useful, but will not reach to the center of an ordinary test tube. Those in apple-tree wood cases are in some respects more convenient than those provided with metallic swing covers (fig. 26). The best general work to consult on the morphology of the bacteria is undoubtedly Migula's System (see Bibliog., III). PHYSIOLOGY. In the description of bacteria we are compelled to make large use of physiolog- ical peculiarities, owing to their very simple and monotonous morphology. Within the limits of the genera now recognized the form differences are so very slight F,g. 23* that mail)- bacteria, c. g., Bacillus coli, />'. f lunar, B. siiipeslifcr, H. tv/>/ioxns, B. a»t\loi'onis, etc., are indistinguishable under the microscope. In mixed cultures, or stained preparations, no one could distinguish one from the other with any cer- tainty, and in pure cultures of unknown origin certain identification by means of the microscope would be equally impossible. Nevertheless, these same forms are so widely different in their behavior in culture media, in their pathogenic properties, in their relation to heat, air, antiseptics, etc., that we are certainly warranted in regard- ing them as distinct species, using the word "species" in its common acceptation. These well-ascertained facts should not, however, lead one to neglect slight differ- ences of form, even when they can be expressed only in fractions of a micron. On *Fic. 23.— Zeiss compensating ocular No. 12 with .screw-filar .micrometer. 26 BACTERIA IN RELATION TO PLANT DISEASES. the contrary, as much as possible should be made out of morphology, particularly that of the living organism, and in this connection the recent efforts of Migula and Fischer are especially deserving of commendation. MOTILJTY. If motile, determine kind of motion and rapidity (margin of small hanging drops on thin covers sus- pended over hollow-ground slides).f The cover may be prevented from sliding by touching one edge with a very little vaseline or cedar oil ; if too much is used it runs under, mixes with the hanging drop, and spoils the mount, and possibly in the end the objec- tive is ruined, if the student continues to search for a clear field. The beginner is very apt to mistake Brownian movement for self-motility. It sometimes requires very careful observation to be quite certain. Rods which appear to be motionless will sometimes be seen to dart away quite suddenly if watched. In some species young cultures are much more apt to be motile than old ones ; in others motility appears to be an almost constant characteristic. The move- ments of bacteria are sometimes quite characteristic for particular sorts. They may be slow or rapid tumbling motions centering in the shorter axis, or straight or sinuous slow or rapid darting move- ments in the direction of the longer axis, with rotation on this axis. The media of Hiss ('97, Bib- Hog., XVI) and of Stoddart ('97, Bibliog., XVI) are sometimes useful for distinguishing macroscopically between motile and non-motile forms. The former spread as a thin layer over the whole surface, the latter pile up in restricted areas around the points of inoculation. The student should not remain con- tent with merely determining motility, but when this has been settled he should turn his attention to staining the organs of motion. Fig. 24.* *Fic. 24. — Upright Zeiss camera for photomicrographic work. The cup (a) slips over the end of the microscope and forms a light-tight connection with the bellows without touching it. The microscope rests on the table independent of the camera. The stout rod turns freely in the socket X and is locked in place by a set-screw on the side opposite the observer. The height is about 45 inches. tLehmann and Fried (Arch. f. Hyg., Bd. XL VI, 1903, p. 311) found the swiftest movement of bacteria to be i mm. in 22 seconds; the slowest I mm. in 222 seconds; average: cholera, I mm. in 34!4 seconds; typhoid, I mm. in 56 seconds; B. vulgarc, I mm. in 73 seconds; B. subtilis, I mm. in 40 seconds; B. megatcrium, i mm. in 2 minutes n seconds. PLATE 5. Large horizontal Zeiss photomicrographic outfit ready for use, except that when photographing the curtain is raised and the mirror is placed farther away. i. e.. out of the south window on the triangular extension shown on the front table at the nght. In the newer forms each table top may be raised or lowered at will. There is also a device for raising or lowenng the plate on which the microscope rests. STUDY OF COLONIES. GROWTH. The manner of growth and rapidity of growth at given temperatures in hanging drops and also 011 the margin of young colonies on plates of nutrient gelatin and agar of van-ing density should be determined. Frequently characteristic and interesting ar- rangements of the rods forming the surface layers of the colony, especially when it is voting, may be discovered by means of a direct inspection of the colonies under low powers of the microscope or by means of cover-glass impressions. Covers are carefully placed on the colony, removed, dried, flamed, and stained. There are also often curious Fig. 25.* arrangements of the deeper layers of the surface colony. In direct examination the colonies should be viewed by reflected as well as by trans- mitted light. Drawings or photographs of surface colonies should be made under low or medium magnifications. By a little practice using Lister's dilution method ('78, Bibliog., XVII), hanging- drops containing a single bacterium for study under the microscope may be obtained with Beyerinck's capillary drop-flask ('91, Bibliog., XVII). CHEMOTROPISM. Fig. 26.t On the general subject of chemotropism, see papers by Pfeffer, Miyoshi, Jennings, Buller, Rothert, etc. Jennings maintains that contact irritation inducing motor reflex is responsible for movements which were formerly attributed to chemical stimulus. Consult Jennings, " Contributions to the study of the behavior of lower organisms," Carnegie Institution of Washington, 1904, and especially Jennings and Crosby, "The manner in which bacteria react to stimuli, especially to chemical stimuli," Am. Jour. Physiol., Vol. VI., pp. 31-37, and Jour. Roy. Mic. Soc., 1902, p. 88. Spirillum volutans was used in the tests. REACTION TO STAINS. Proper staining is a very important part of the study of bacteria. Its founda- tion principle is the fact that the bacteria, in a living vegetative condition, all show a great affinity for the basic anilin dyes. Spores ordinarily show no such affinity, but may be made to take up stains by acting on them with strong acids or alkalis, or by heating them very hot. Flagella also show no affinity for stains until acted *Fic. 25. — Hand Jens suitable for examining bacterial cultures. Zeiss aplanat magnifying six times. Three-fourths natural size. tFig. 26. — Zeiss swing-cover aplanat magnifying six times. This is now sent out in a neat little chamois-skin purse. About two-thirds natural size. 28 BACTERIA IN RELATION TO PLANT DISEASES. on by severe reagents, when they may be stained in mordanted solutions or in dyes which have been preceded by a mordant. The outer wall of the bacterium generally reacts to stains in the same way as the flagella, /. e., it usually remains unstained. Staining media may be roughly divided into four groups : (a) Simple stains dissolved in water, e. g., fuchsin (basic), gentian violet, methylene blue ; (b} alcoholic solutions and various complex stains, c. g., saturated alcoholic solutions of anilin dyes, alcohol-iodine, iodine potassium iodide, Russow's cellulose test, Ziehl's carbol- fuchsin, L/oeffler's alkaline methylene blue, Ehrlich's anilin-water gentian violet, Gabbett's stain, Gram's method, Delafield's hsematoxylin, Ehrlich's acid hsema- toxylin, Heidenhain's iron-hsematoxylin, Fleming's triple stain; (r) flagella and capsule stains, c. g., Loeffler's stain, Moore's modification, Fischer's modification, Bunge's stain, Lowit's stain, van Ermengem's nitrate of silver method, Zettnow's gold method, etc.; (g- 27.' the cover-glass preparations or the stains does not itself contain bacteria. It is usually wise first to dry a drop of the water on the cover and stain without addition of the bacteria. Eternal vigi- lance is the price of trustworthy results. It is best to make all mounts on cover- glasses of a known and uniform thickness (o. 15 mm.). Many a good preparation has been spoiled for examination with lenses of a short-working distance by mounting under a thick cover-glass, and sometimes the lens itself has been ruined in the attempt to focus. The thickness of covers often varies greatly from the statements of dealers, and they should not be accepted until tested with a reliable cover-glass measurer (fig. 27). -T- — Zi/iss cover-glass measurer. The cover in place shows a registered thickness of o.lS mm. Fractions of an inch are also registered on this instrument. REACTION TO STAINS. 29 To determine whether bacteria are properly stained examine with the diaphragm of the condenser wide open. If they can not be seen distinctly with this flood of light they are not well stained. The bacteria should be well separated on the cover and deeply stained, while the background should be very free from stain. Dr. Weigert seems to have been the first to use nnilin stains for the demon- stration of bacteria in tissues. This was about 1875. Since that time staining in tissues has been worked up carefully for bacteria causing animal diseases, but very little is known respecting best methods of staining bacteria in vegetable tissues. The difficulty lies in the fact that the tissues of the higher plants often take the basic anilin stains as readily as the bacteria and retain them even more tenaciously. Special remarks may be looked for under particular diseases. CULTURE MEDIA. NUTRIENT GELATIN. («) Plate Cultures. — Colonies, young and old, buried and superficial, crowded and wide apart, should be examined for color, translucency or opaqueness, shape, thickness of the surface growth, and character of the margin. They should also be studied under low powers of the compound microscope for lobes, branches, granulations, wrinkles, flecks, concentric rings, radial filaments, arrangement of the dividing rods on the margin of the colony, iridescence, etc. The microscopic appearance of the surface colony during the first 48 hours is often different from that later on. The rapidity of growth should be compared with that of some common and easily accessible organism, e.g., Bacillus coli\ B. ainylovorus, Bacterium campestre. The comparative rate of growth of buried and surface colonies should also be carefully noted. How is the appearance of the colony changed by increasing the amount of gelatin, or varying the brand of gelatin? Are the surface colonies viscid, or can they be lifted bodily in one mass from the substratum ? (b) Stabs. — The nature of the sxirface growths and of the deeper growths should be carefully examined. Is there any marked tendency of the latter to grow down- ward or outward into the body of the gelatin, either in distinct masses or as a dif- fused cloudiness? Observe effect, if any, on growth when the gelatin is acid or only feebly (litmus ) alkaline. If liquefaction of the gelatin occurs, note its rapidity and whether it is mostly restricted to the surface or is equally rapid along the line of the stab in the depths ; note also whether the liquefied gelatin is clear or cloudy in tubes which have not been shaken, and whether a pellicle has formed on its surface. Liquefaction may be very rapid (taking place within a few hours), may occur after three or four days, may be long-delayed and feeble (only visible after some weeks), or may not occur at all. It is the cases of feeble and long-delayed liquefaction which lead to contradictory statements on the part of different observers, and con- sequently cultures should remain under observation for a considerable time and on a variety of gelatins. Various substances interfere with liquefaction. Determine whether liquefaction can be prevented by the addition of grape-sugar or cane-sugar (10 per cent). Look for gas-bubbles, for crystals, for any fluorescence or staining of the medium (green, brown). Inasmuch as the growth of some bacterial plant 30 BACTERIA IN RELATION TO PLANT DISEASES. parasites is restrained by some nutrient gelatins which are neutral or only feebly alkaline to litmus, it is advisable to add to a part of the stock more caustic soda than is commonly used, /. t\, enough to render it neutral to phenolphthaleiii (strongly alkaline to neutral litmus), especially if gelatin is selected as the first medium for isolation experiments ; otherwise perplexing failures may result. (c) S/rcaks. — Record the character of the streak, whether wet or dry, smooth, wrinkled, or rough, thin or piled up, margin well defined or indistinct. Note also whether the surface is ever iridescent, whether growths are sent down from the under surface into the substratum, whether the streak spreads rapidly and widely over the surface or very slowly. The sur- face behavior depends to some extent on the motility of the organism, on the amount of water in the surface layers, /. e., whether the slants are fresh or old, and on the amount of gelatin in the me- dium,which in temperate climates should usually be 10 per cent, but may be 15 or even 20 per cent. By minimizing heat in prepara- tion and by increasing the quan- tity of gelatin to 20 or 30 per cent a medium may be obtained which will remain solid at 30° C. Growth is less satisfactory, how- ever, 011 such a dense medium, or at least was in the few tests made by the writer. Chester has applied the ordinary botanical terminology to the varying mar- gins of colonies, etc., and has pub- lished some useful figures ('01, Bibliog., III). No substance used in the bac- Fig. 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 "So 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 diie 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 London and well known to the makers of photographic dry-plates, who use it in large quantities. It conies 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 niters with difficulty. Other expensive gelatins, said to be of quite uniform quality, are Fig. 29* L,ichtdruck 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 (Cockaynej. 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 the Japanese as slender " Kan-ten." 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 algae in this group the following, according to Rein (pp. 81-82), deserve special mention : (10.) G. cartilagincum Gail. (ir.) G. rigidiim Grev. ; Jap. Tosaka-nori, i.e., (i.) Chondrus ftinctatus Sur. (2.) Gigartina tenclla Harvey; Jap. Ogo. (3.) G. intermedia Sur. (4.) Gloiopeltis tcna.v Kg. (Sfhaerococcus lena.r Ag.) (5.) 67. capillaris Sur.; Jap. Shiraga-nori. (6.) Gl. coliformis Ha™. ; Jap. Kek'Kai. (/) Gl. iiitricata Sur. ; Jap. Fu-nori. (8.) Gelidium contemn Lamouroux; Jap. Tokoroten-gusa. (a) (7. .-{inansii Lamour. confcrvoidcs A. ; Jap. flabcllifonnis Harv. ; cockscomb algx. (12.) Sfhaerococcus S'hiramo. (13.) Gymnogongrus Jap. IIome-iK>ri. (14.) G. japonicus, Sur.; Jap. Tsuno-mata. (15.) Kallyiiienia dcntata; Jap. Tosaka-nori. (16.) Porfliyra rulgaris 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. *FiG. 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 size of these sticks is about 10*4 by 2!^ by i% .inches. tMetcalf has described a bacillus which slowly softens it, and *he 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. Glyccrin-agar, mattose-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., no°C. 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 /. £•., 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 b Fig. 3 1.* *Fic. 31. — Red sea-weeds from which agar-agar is manufactured, a, Gelidium contemn Lam., one-third natural size; b, Gelidium subcostatum Lam., one-half natural size. Prom 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 enameled-iron dish in a minimum quantity of water or beef-bouillon over a hot Bunsen flame with constant and rapid stirring and Fig. 32.1 *The folded filter papers are the most convenient (fig. 34). These filter papers give the starcli reaction (Wue) with iodine, and reduce Fehling's solution on being boiled in it. tFig. 32. — Unnamed species of red sea-weeds (CTc/irf««m) furnishing agar-agar. From a Japan- ese chart showing "The principal aquatic plants of Japan," supposed to be an official publication. One-half natural size. Original in library of United States Fish Commission. PREPARATION OF NUTRIENT AGAR. 35 ,-.. r>v '"••/;•' I P {/•• *-» ' ^/iv/tt ,'/v?5l ^ii\7C^7IV/" 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 streaming1 steam for two hours, after which, if the first heating was sufficient, it filters 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 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 Lautensch lager or Merck (fig-33). If one has au 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 "Formulae," 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-agaT 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 student 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 ot 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, Mac£, 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. c., 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. SILICATE JELLY. 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 34 * *Fic. 34.— Folded filter papers made by Schleicher & Soliull. PREPARATION OF SILICATE JELLY. 37 H 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 Omelian- 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° Beaume) 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 1 10° C. The thermo-regulator shown in fig. 35 is 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, an(j •£ toQ Jnuc|1 ether they are said to become brittle. After some 35* *Fic. 35. — Toiler's thermo-regulator 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-toth 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 with mercury. 38 BACTERIA IN RELATION TO PLANT DISEASES. experimenting it was found that 5 grains 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 up, 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 uniform 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 12 hours, and sometimes for 24 hours, if ever)' 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° 011 the scale of the hvdrometer 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° Beaume" 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° Beaume" 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, 0.2 ; moiiopotassium 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 oxit, 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 011 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 witli distilled water and steamed 20 minutes at 100° C. on each of three consecutive 4" I of Duclaux. They are chemical substances, the exact / composition of which has not been determined. They Pjg 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). (S.) 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 *Fic. 57. — Crystals formed in cultures of Bacterium syriugac (van Hall). I. From tube II, Aug. 14 i ,-iuar stock 003), fn>m van Hall's II, i. <•., his own isolation corresponding to a, fig. 56. j. Knmi tube I, Aug. 14 (stock '193'), from van Hall's I, which is from Beyerinck's old isolation (see 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. c., 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. c., 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 cam pc sir e 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 Jour. 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-sporifcrnus 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 N 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 TVT XT •4^-' with - — or - - solutions, and to reckon the acidity in cubic centimeters of normal 1 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 fractious. 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 ueutral 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. •(•The plus and minus on Fuller's scale denotes, respectively, acid and alkaline media. The + 10, 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 am ins) produced by bacteria in culture media (see Sutton, 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 tracheiphilns in peptonized beef-bouillons of vary ing 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 -f- 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 clown 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 +15 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 plus 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., 011 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. Lehmami & Neumann ('96, Bibliog., Ill), state that they have found bacteria that will endure 100 cc. of normal acid per liter of fluid culture media, /. e., 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 (}{-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. Organisms believed to be non-sporiferous 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- ipliilus, B. carotavorus, Bad. hyacinthi, etc. Tests may also be made in air dried over sulfuric acid or calcium chloride. Harding & Pmcha have shown recently that Bacterium campcstre 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. 6 1* Fig.62.f 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 omylovorus (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 fhascoliCErw. 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, i. e., the plate was sown too thickly. 72 BACTERIA IN RELATION TO PLANT DISEASES. better, 011 larger Petri dishes rilled 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 am vlozwiis and Bacterium phascoli 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, Bad. Stcwarti in milk, etc. Some organisms are quite resistant to their own decomposition products, e. g., Bacillus coli, Bad. pcricarditidis. 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 campestrc and Bad. 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, /. c., 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). (n.) 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 BacteYiologie. 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 cholerac 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 o( the building and lighted by electricity. Ventilated in the same way as the photographic dark-rooms, i- e-. by an exhaust-fan run by an electric motor. Three of the thermostats were made by Bausch & Lomb. the fourth I felted) is a Rohrbeclc. ENZYMES. 75 depended upon to keep the solutions free from bacteria. This has been the case very frequently, and iu 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 thermo-regulator should be at least i ^ inches in diameter (so as to take a Roux metal-bar thermo-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^°, 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 Lautenschlager's catalogue). The improved Koch safety burner is probably the best. All burners require frequent inspection. 76 BACTERIA IN RELATION TO PLANT DISEASES. The writer lias 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 Ki irdliurf; burm-r 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 ma}- 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 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 (+10 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- bouillon (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 thcrmo-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. •(•The thermal death-point in acid media is considerably higher — at least that of several organ- i-.ms 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 thernio- regulator ma)' 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 Mumll'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, orCentralb. 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 Ansclmtz 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 0.2 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 iu 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 |i i 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 iu 5 cc. portions of distilled water or bouillon in block-tin test-tubes, or preferably iu tubes of resistant glass, for standard periods, e. g., one-half hour, i hour, 6 hours, 1 2 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, /. 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 zoogloeae, 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 uniform 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. 63. — 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. lions 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 moiith 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 ot 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, i. e., 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. 8l the germicidal 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 y> 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-bine 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., XLVI.) 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 Iraclidfliilus. 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. Full}' 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 'or, d'Arsonval (Bibliog., XXXIII) and Fig. 69 * Smith & Swingle, the Effect of Freezing on Bacteria, Proc. Sixth Ann. Meeting Soc. Am. Bacteriologists, December 27, ^04; Science, N. S., Vol. XXI, 1905, pp. 48^483. 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- *Fic. 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. 84 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 110° 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 lo the bottom by means of a double ring of Bunsen burners. No wrench is required for fastening on the top. About one-eighth natural size. THERMAL RELATIONS. 85 pattern designed by Cliainberland 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 miy 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 IHTII 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 hrcad 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 carotovonts, 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 aroidea:, 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 friini. 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 Iffii'est 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 thennophilic 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 trachfiphihts, 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 plios- fhoreitm 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 P.ACTERIA 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 *Fir,. 72.— Bacterium pruni. Vertical section through a green plum fruit (var. Hale) showing li.-icterial cavities and the escape of the organisms through the ruptured stoma. In this case beyond iluubt 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-PAT! IOCKNIC TO ANIMALS? 89 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, z. r., 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. Dot., 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 Ste^'arti 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.* 9o 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 A. 2, 3, and 4. A knowledge of 2 and 3 will frequently lead to some simple and effective means of prevention. B Fig. 74 * LOSSES. 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 *Fic. 74. — Bacterium Steivarti (Erw. Sin.) 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. QI 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 *Fio. 75. — Bacterium Ste-varti 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. *FiG. 76. — Bacterium cainpsslrc. 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 nf 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 .iK-'ihol. 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 *Fio. 77. — Bacterium campcstrc from the cavity shown in fig. 76, illustrating water-pore infec- tion of the cabbage. X. 2,000. tFir,. 78. — Bacterium camfcslrc 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, arc 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, i. c., 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 n;,'. 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 it. 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-S-> general laboratory rooms, chambers for special workers, sterilization -room, thermo- stat-room, cold-storage and stock-ciilture rooms, storage rooms for chemicals, small Fig. 79« 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. *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. m 3 re a- o =- n fl a: 3" a- •* ID T 3 to * o O EQUIPMENT OF THE LABORATORY. 95 There should be hot-water pipes, cold-water pipes, steam pipes, a steam hath, 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 apochroinatic lenses, photographic and photomicrographic appliances, liqitid-air receptacles, cylinders of compressed carbon dioxide and oxygen, microtomes, paraffin baths, glassware, balances, chemicals, and many minor pieces of apparatus. " 0 V 19 -. Fig. 80.* *Fic. So. — 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. c.. 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 inahace- anun suspended in sterile water. 96 l:\CTERIA 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. I'n,. Sr. — End of the vacuum-pipe on laboratory table. The gage serves to show the degree of exhaustion, i. c., 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. 97 PREPARATION AND CARE OF CUT/TURK MKIHA. Even-thing should be carefully weighed or measured. Even-thing should be clean as possible to begin with. I!y water is usually meant distilled water, and this should be free from copper or other germicidal metals (see Bolton, Bibliog., XXX VI 1 1). 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 truc/ieip/iilus 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, /. <•., 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 *FiG. 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 llask 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 011 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 011 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 Fig. 83.* should consult the standard text-books, a dozen or more of which should be kept within easy reach in even' laboratory. Some formuke 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 1 10° C. Milk, gelatin, and media containing sugars should never be sterilized in the autoclave. Sugars FIG. ^.v — Apparatus for rapidly filling U-.t-Ui'n's with 10 cc. portions of agar, bouillon, etc. By of tlriv device an expert assistant can fill 500 tubes an hour. Wade to order by Emil Greiner. Height, Jj 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 phenolphthaleiii 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. IOO 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 iu 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 iu, leaving V - shaped flaps, which are folded down on to the plate. The second cover- 1^—^ ^ ing is folded at right angles to \^ "S^/ ^^ 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, /. c., they should not be crowded together tightly, and the recording thermometer should project well down into their midst. 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 Lautenschlager. The improved form of the IYautenschlager 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. 101 iii 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 consecmently the air between this wrall 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 main- 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. 86.* 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 L,auUjiiM-hl;iger. Desirable on account of perfect workmanship, and because it is easily sterilized without injury. This size holds I cc. Bv 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. Flg. 87* *Fic. 87.— Early stage in the infection of a cabbage leaf by Bacterium campestre; 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-fuchsm. 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 caml>cstrc 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 S°°- ITOW TO AVOID CONTAMINATIONS. 103 THK MAKIM: AND TRANSFERENCE OF 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 2Ol/z 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 1 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. *Fic. 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 9^/2 by 3$^ by 1% inches. PLATE 11. The author's culture-room. At the left hand (back) are nairow ihelves for culture-media, pipette-boxes, etc. At the right is the work-shelf, covered with plate glass. 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 over 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 reqxiires a smart little jeik, 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 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 wha/soei'er, 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). *Fir.. 90.— A constant Btinsen burner with cut-ofl flame. Very useful for the laboratory table ami the culture room. About two-fifths actual size. Fig. 90.* agar, may be held level or inverted for inoculation. 106 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, /. £., 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. g., 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 pastciiriamun 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) mcsenterioidcs by Liesenberg & Zopf and of Bacillus hortitlanits 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 nictal-srri-w 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 transfers may be made under this frame in the open room if windows and doors are kept closed. IHSI'OSAL OF INFECTIOUS MATERIAL. ID/ 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 longtime 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 (1:1000) 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 ma}' 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 u'hole 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 Boeekel, 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 procure 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 n! tbr air-chambiT, which requires more frequent pumpings. Of course the apparatus may be usnl rqu.dly \\rll for the distribution of fluid germicides or insecticides. loS BACTERIA IN RELATION TO PLANT DISEASES. MKTHODS 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 on 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, /. 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 Fig. 93.* 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 fewr 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. tj,3. — Atomizers for use with the air-tank (fig. 92). These are made by the Davidson Rubber Company, Boston, Mass. Aliout 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- i/ed in boiling water without becoming twisted out of shape, it can be attached more easily to large flasks ami to the tube leading from the cornpressed-air tank, and the spray may be directed up, down, i>r straight ahead witlnmt 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. IOQ washed with equal care in sterile distilled water. Wlu-n many inoculations are made with lar^e numbers of check plants and when dne 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., anyone of which may be "room-tempera- ture," depending on the latitude> altitude, and time of year. He should not say, " Organism is killed at temperature of 65° C.," without at the same time stating the age of the culture, condi- Fig. 94.* 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- l;i.,. Q4. — Hand-sprayer which may lie used for distributing bacteria on plants. Some form is usually kept in every pharmacy and sold as a cologne atomizer. 110 r.ACTKKIA 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, c. 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 carefully 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 :Fn.. 95. — Small cage of \vood and glass in which herbaceous plants may l>e placed for inocu- lation by spraying. The inside measurements are 12 by 12 by 30 inches. The large door is a great convenience. 1 lock-fastenings are better than spring catches. KM ( )K I IS. 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 enongh 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 bauds 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 Fi % * be pasted on or the stock number written on with the proper pencil. All plate cultures and all subcultures made on a given day, 110 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. 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 *l"i<:.. cy > —Labels from tcst-tulie culture-;. ;. 97. — Wooden lalcrls 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 ma}' be inoculated, the separate inocula- tions being kept distinct, if necessary, by sub-numbers. Each series begins with 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. FIG 1.18. — Three sheets showing method of keeping maximum and minimum temperature rec- onK One-half actual size. RECORDS. 113 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 particular 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- _. 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; Fi IQQ* 1 6 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 future, heavy sheets (6jg by 8-;H Fig. 10 1. 1 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 M/<' |Fn.. 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, (>"s inches. RF. CORDS. 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). vSteel 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 Fig. 102.* 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 micrometer and throw the image of some portion of the ruled scale on the paper I 102. — Green cucumber soft-rotted by Rat'illits arnidcae. Contents emptied out and skin tilled uitb \\;iicr 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. Il6 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. loj. — 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 TIIK MAKINC, OK 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- 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. Fig. 104.* *FiG. 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. IlS 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. I05.:< 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 <> by 7 by 5 indies. The thermo-regulntor is like that shown in fig. 35, but with chloro- form substituted for glycerin. PARAFFIN-INFILTRATION. 119 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 Flg- 106' " 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 (fi-s. 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. J In this series of jars the gradations are as follows, beginning Fig. I07.f *Fic. 106. — Infiltrated tissues embedded in paraffin in a watch-glass and now ready to cut out and mount on blocks for the machine. f FIG. 107. — Infiltrated material embedded in paraffin and mounted on a pine block ready to cut on the microtome. Actual si/i-. JSections 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. — If 3 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 Fig. 108.* . . on. To retain the stain it is necessary sometimes to omit some of the graded alcohols. The time required for properly staining sections varies from one or t\vo 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. 1 12). /" inS. ( )nr of a M-rie* of drawers divider! into small compartments for holding infiltrated, material, cut and uncut. PLATE 13. I n 1 2. ? o" s s i' e ri o 3 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, /. e., i to 25. The slide- boxes are then filed away on shelves either serially or in groups, according to the parasite. Slides con- taining particularly good fields are marked X, and when the best fields are Fig. llO.t 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 0, 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- 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, Ill.t 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 *FlG. 109. — Coplin's staining jar. About one-half actual size. fFic. no. — Cross-section of Coplin's staining jar. About actual size. |Fic. 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 Lentz & Sons, Philadelphia, with very satisfactory results. Knives suitable for serial sections are shown in fig. 1 14 A and C. In fig. 1 14 B 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 Lentz & Sons at a cost of about $6 each. An end-on view of all these knives is shown in fig. 1 14 a, b,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 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 In such cases thick Fig. 112* X, X X 0 oooo GOOD JB/acK spot of PluTTV. fine the tissues. 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. Fig. Il3.f SECTIONS. I23 moves forward at each stroke a distance governed by the set-screw of the scale (% h 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 an}- 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 trueing 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 b. 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 this method some organisms can be kept alive on agar a year without transfer, and even sensitive organisms will generally live for some months, especially Fig. 115* B 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., LIT). 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. t *Fic. 115. — Cross-section of tooth of cabbage-leaf infected by Bacterium camfestrc. 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 n below the apex of the leaf-tooth. A few micromillimeters further down (370 n ) 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 33IC3 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 camfestre. A detail from fig. 115 at A. The bacteria have not yet entered the vessels. JThat 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 pip; ; (2) wiit--steim pip; ; (3) hydrant-water inflow pip; ; (4) hydrant-water outflow pips (flush) to sewer ; (5) gilvanized-iron b)iler ; (6) water gage : (7) brass top, tinned on the under side ; (8) copper catch basin ; (9) steam safety valve; (10) block-tin steam pipe to condenser; (II) block-tin water pipe from condenser; (12) hydrant-water pip; into condenser tank; (13) hydrant -water pipe from condenser tank; (14) flush pipe (or condenser lank ; ( I 5) reservoir, capacity 80 gallons ; ( 1 6) water gage ; ( I 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 18 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^-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 l/, 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 C" I 1 7 * from a securely riveted tin-lined copper catch basin (8) into a J^-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 ^ -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 i/J-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 (i^-inch) block-tin pipe (i i), which leads the distilled water into (15) the storage tank (34-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 („'(,- 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. e., when some more satisfactory storage tank can be found. The problem of the proper storage of distilled water in quantity is the hardest one, the solvent power of the r ig. 118.* water is so great. From the bottom of this bath-tub several hundred feet of i^-inch block-tin piping lead to various rooms in the building. In addition to the terminal faucets there is a general cut-off just above 18, which is necessary in case of an accident to any faucet or part of the piping. There is also an overflow pipe (17), which does not enter the sewer, but *Fic. nS.—^Early stage of stomatal infection in angular leaf-spot of Rivers cotton. Hothouse infection produced by spraying Bacterium malvacearum upon the surface of the leaves. For a much later stage see fig. 80. DISTILLED WATER. 127 ends free in the laboratory about i foot above a deep sink. The sides and top of the boiler, the copper catch basin, and the ?4' -inch block-tin pipe leading to the con- denser are all coated with 3 inches of best non-conducting magnesia covering. The catch basin, designed to hold back solid particles carried up with the steam, is 9 by 12 inches and is made of --inch copper, securely riveted and soldered with tin solder. It is bolted clown to the flat brass top and a steam-tight connection Fig. 1 19.* is secured by means of a red rubber gasket. The heavy- brass top (7) is tinned on the inner surface and is bolted securely to the iron flange on the top of the boiler by means of 18 screw-bolts. The junction is made steam-tight by means of a corrugated *Fic. 119. — The Reinhold-Giltay microtome arranged for cutting celloidin or very hard paraf- fin sections. The machine is very solidly and accurately constructed out of the best materials, and, in addition, provision is made by means of set-screws for compensating the wear due to long use. The device governing the thickness of the sections is especially ingenious. This particular machine has been in constant use by various persons for over four years, and nothing has been paid out for repairs. With good use it ought to last a lifetime. About one-fifth actual size. 128 BACTERIA IN RELATION TO PLANT DISEASES. tinned-copper gasket. The steam which runs the apparatus is brought to the lab- oratory floor through a ij^-inch pipe, in which (in the engine room) there is a steam gage registering up to 150 pounds, and a reducing valve set at 55 pounds. This very considerably lessens the steam pressure in the copper coil, moderates the violence of the ebullition, and makes the apparatus perfectly safe. The hydrant- water outflow pipe (flush) to the sewer, for occasionally washing out accumulated mud (4) passes from the bottom of the boiler immediately above fig. 19. Gate-valves are used. All brass and copper parts in contact with the steam are tinned; all metal parts in contact with the distilled water are tin, tinned, or nickel-plated. With 60 pounds steam pressure in the engine-room boiler, 40 pounds pressure at the reducing valve, 35 pounds pressure in the pipe at the laboratory floor near where it enters the still, and one-half pound pressure or less in the steam chamber above the coil of copper pipe, the capacity of this still is 60 liters (16 gallons) per hour. The apparatus must be built very substantially in all parts, so as to withstand at least twice as much steam pressure as any part of it will be subjected to, e. g., 160 pounds in the iron pipes and in the copper coil and its attachments, and at least 20 pounds in the catch basin, and other parts subject to steam generated in the still. A steam gage, in addition to the one in the engine-room, shows the pressure in the coils, and another the pressure in the steam chamber above the coils. They are not shown in the plate, as they were put on after that was made. The former is attached to the steam supply pipe near the floor, and the latter to an arm of the safety-valve pipe. The boiler should be taken down and the parts retinued once a a year, or at least once in two years. If a much greater quantity of water is needed the block-tin condensation coil should be lengthened to 60 feet, the diameter of the inflow pipe of the condenser should be increased to 2 inches, and the outflow pipe to 2 y2 inches, and the cubic contents of the condenser tank should be quadrupled. The capacity of the bath-tub (or other receptacle), for a large laboratory should be at least 500 liters, and might well be i ,000 liters. The above apparatus has been in use for two years. It works very smoothly and satisfactorily when the proper amount of steam is let into the coil of copper pipe, which ordinarily should not be nearly the whole amount available. The inflow of steam is governed by the valve a few inches below fig. i in plate 14. When too much steam enters the coil, the pressure in the steam chamber above it rises to five pounds or more, hot water is forced back through the feed pipe (3) into the neighboring pipe which furnishes cold water to the condenser (12), and steam in- stead of distilled water is furnished to the water tank. This is at once obviated by cutting off part of the steam inflow and moderating the force of the boiling. It might also be obviated by reducing the length of the arm of the safety valve (9) which in any event should not be weighted. Sufficient water for small quantities of culture-media and pure enough for most purposes may be obtained from the simple glass still shown in fig. 82 by one dis- tillation. Water of a high degree of purity may be obtained by two distillations, adding 0.5 gram to i gram of potassium permanganate per liter of water before the NON-SOLUBLE GLASS. 129 first distillation, aud 5 grams of c. p. sulphuric acid per liter before the second distillation. The flasks in which such water is collected or stored should be of resistant (non-soluble) glass and absolutely clean to begin with. With use such flasks or bottles become more valuable and should not be employed for other purposes. The solubility of glassware is best tested by determining from time to time the degree of electrical conductivity of pure water stored in it. The specific resist- ance of pure water stored for a week in such tubes, flasks, or bottles should not fall below 250,000 ohms. The specific electrical resistance is determined upon i cubic centimeter of water exposed between electrodes having an area of i square centi- meter, and is read by means of a special Wheatstone bridge. Distilled water redistilled with chromic-acid cleaning mixture, and afterwards with alkaline potas- sium permanganate (method used by the Physical Laboratory in the Bureau of Soils) gives a resistance of 700,000 ohms. The following determinations made by the Physical Laboratory- of the Bureau of Soils show the diverse behavior of two lots of clean test-tubes recently purchased as non-soluble glass by the Laboratory of Plant Pathology. Kind of tube. Time of exposure, in days. Specific resistance, in ohms. Resistant test-tubes, (R) from Greiner Do ad test . . . . Tubes received from the School Sup- ply Co Do , ad test The twice-distilled water used was taken from a Jena flask and its initial specific resistance was 240,000 ohms. MICROSCOPES. Microscopes of a much better grade are required for bacteriological investigations than for ordinary histological work. The writer has for many years employed those made by Carl Zeiss, of Jena, as, on the whole, most serviceable. Good microscopes are also made by E. Leitz, of Wetzlar, and recently by the Spencer Lens Company, of Buffalo, N. Y. The Zeiss stand shown in plate 15 does very well for all ordinary work, but is not well adapted for the making of photomicrographs or for recording the exact location of particular spots in the section. The latter difficulty may, however, be overcome by means of a removable slide-carrier attached to the stage. The stand may also be used with the small upright photomicrographic outfit shown in fig. 24 when the lens does not require a microscope having a wide tube. This microscope has a half-mechanical stage, an excellent fine adjustment, and good substage apparatus. It is thoroughly well made and very durable. One in the writer's laboratory has been in use for twelve years. The lacquer has disappeared in places and it is no longer attractive to look at, but it has required no serious repairs during this time and is still serviceable. For photomicrographic work and also for recording the exact location of desir- able fields in a section, the writer uses the large photomicrographic stand shown in plate 16. This is provided with a specially wide barrel, a fine adjustment of very PLATE 15. Zeiss microscope stand IIa. This form of microscope and lhat represented on plate 16 are (he two patterns used princi- pally in the Laboratory of Plant Pathology, U. S. Department of Agriculture. The objectives are apochromatic, and have proved very serviceable. In carrying do not grasp by any part above the level of the stage, as this brings an undue weight upon the fine adjust- ment. Seize by (he base. PLATE 16 . Zeiss photomicrographic stand I1'. The barrel "T" is of greater diameter than in stand Ha. The fine adjustment Is at " W" and no weight rests on it in lifting the instrument by the handle " H." The set screw " K " locks the upper part of the instiument at any angle. The objective is set in place by means of a very convenient slide carrier- The fine adjustment screw has an extremely slow movement ; and the vernier screws are on the same axis (a great convenience). The stage rotates and may be locked at the desired place by means of a set screw. For the substage arrangement see figure 120- 130 BACTERIA IN RELATION TO PLANT DISEASES. slow movement, a swing-out condenser (fig. 120), two substage iris diaphragms, and various other conveniences. For example, the screw-heads, determining the cross and sidewise movement of the section, are on the same axis and may be reached and moved without changing the position of one's arm. The apochromatic objectives are the only ones recommended for bacteriological work. They cost more than achromatic objectives, but expense is a minor con- sideration. In hot, moist climates the older apochromatic objectives of Zeiss fre- quently became clouded, but those made in recent years have given the writer no trouble in the latitude of Washington. They yield sharp images even with high eye-pieces. Of course, compensating oculars must be used with the apochromatic objectives. It is de- sirable to have the whole series of ob- jectives and eye- pieces, but if one is limited for means, very good work can be done with two objectives and three oculars, viz, object- ives 1 6 mm. and 3 mm. 1.40 n. a., and compensating oculars 4, 6, and 12. The newer forms camera furnished by Zeiss (fig. 121) leave little to be desired in the way of a drawing camera. PHOTOGRAPHY AND PHOTO- MICROGRAPHY. For permanent records nothing equals photography. It constitutes, therefore, a very important special Flg' 120-* part of laboratory work, and every student of pathology should make a knowledge of this subject part of his education. Some of the following suggestions will be useful to beginners. The Zeiss Double-Protar lenses, series Vlltf, are the best all round photographic lenses made by that firm, and are excelled by none made by any firm. The back or front lens is usually as good as the combination. Excellent photographic lenses are also made by Voigtlaender and by Goerz. Zeiss photographic lenses may be *Fic. 120. — Swing-out condenser and other substage arrangements on Zeiss photomicrographic stand, No. ic. There is an iris diaphragm in D, and a second one in S, which is for use when the condenser is thrown out as shown in this figure. D swings under when C is thrown into place. W racks the entire suhstage up or down. PHOTOGRAPHY AND PHOTOMICROGRAPHY. obtained from Bausch & Lomb, who are under contract to manufacture them according to the Zeiss formulae. In buying a photographic outfit it is economy to get one of the high-priced lenses. It is frequently stated, by those who do not know, that "just as good results" can be obtained with cheap lenses, but one may easily satisfy himself that such is not the case by photographing buildings on a Fig. I2I.« street or any object having many vertical parallel lines and other lines crossing at right angles. The pictures made by the cheap lenses generally show serious distor- tions. In buying a lens one should know in advance exactly what he wishes to do with it, otherwise he may be greatly disappointed. If he wishes to photograph only *Fic. 121. — Viewer form of Zeiss-Abbe drawing camera. The camera is clamped at K by means of S. The prism within R is centered over the eye-piece by screw movements of L, and Z. When not in use the prism is swung to the right, as indicated by the dotted lines. The mirror A throws down the prismatic image to the drawing paper. The amount of light is governed by the substage iris-diaphragm and by rotating B and R, which contain smoky glasses of graded densities. P is an extra prism. The image on the paper will also be clearer if it is placed in shadow by means of a screen of some sort. 132 BACTERIA IN RELATION TO PLANT DISEASES. flat surfaces he will select a lens with no great penetration, but with a very clear field, sharp to the edges, /. e., a Planar or some similar lens. If he needs a lens with very little depth of focus (but more than the Planar) and one allowing dark objects to be photographed in a very short time, e. g., luminous bacteria by their own light, he will select a Zeiss Unar or its equivalent, /'. c., an extremely rapid lens. If he desires in one picture as much as possible of a landscape, e.g., a large tree or an interior, he will select an extremely wide-angle lens rather than one distinguished for its rapidity or for the perfection of its definition, e. g., a Zeiss Protar, series V. The Double-Protar, series Vila, combines as wide an angle, as flat a field, as great rapidity, and as sharp a definition as it is possible, apparently, to obtain in a lens and at the same time have great depth of focus. These lenses may also be unscrewed and each half used separately, if one wishes some portion of a picture more highly magnified. They are furnished with front and back lenses of equal or unequal focal distance, as may be desired. In using Planars and all lenses which magnify, it is necessary to secure a very exact focus with the stop -vide open, for, unlike lenses which give pictures less than Fig. 122* actual size, only a very little increased depth of focus can be obtained by stopping down. With many objects — e. g., the surface of a leaf, or of bacterial colonies- there is considerable difficulty in deciding which is the proper focus when a Planar is used, what seemed like a good focus often yielding a poor negative. On this account the writer is in the habit of focusing on a fragment of very fine, sharp print laid 011 the surface of the leaf or of the agar-plate near the colonies to be photographed. A lens magnifying 6 times is used in judging of the image on the ground glass, and when the best possible focus has been secured, the paper is removed, the lens is stopped down two-thirds, and the photograph is made. In case of white colonies the best results are obtained by resting the Petri dish on a piece of black paper while the photograph is being made. The exposure is shortened by illumi- nating the surface of the object with a bright beam from a mirror. The apparatus *Fic. 122. — Zeiss Planar lenses, series la, Nos. I to 5. Nos. i, 2, and 3 may be attached to the funnel-shaped carrier shown in the figure. This screws into the top of the microscope barrel in place of the eye-piece tube. The one attached is No. 3. The condensing lenses necessary for these Planars are also shown in this figure, at right and left. PHOTOGRAPHY AND PHOTOMICROGRAPHY. 133 shown in fig. 24 may be used for this purpose. To avoid shadows the mirror should be held some distance above the object when the surface is not even. The first five of the Zeiss series of Planars are all that are usually required. No. i gives the highest Fig. 123.* *Fic. 123. — Simple apparatus for holding the camera in place when one wishes to photograph down. The camera here shown is a Rochester Optical Company, reversible back 5 by 8, fitted with a Bausch & Lomb rapid universal lens, and has been used very often by the writer for natural-size work and for lantern slides. 134 BACTERIA IN RELATION TO PLANT DISEASES. magnification ; No. 5, the largest field ; No. 3 will give a sharp image of a flat object a centimeter in diameter. Special condensing lenses are required. These fit into the substage in place of the Abbe condenser. One condenser serves for Nos. i, 2, and 3, and another for Nos. 4 and 5 (fig. 122). In photographing ponred-plate colo- nies natural size, there are several ways. It may be done by reflected light, as shown in fig. 123, in which case the colonies sometimes cast deep shadows. Such shadows may be avoided by mounting the camera as shown in fig. 124 and gently twirling it during the exposure. The Petri dish may also be photographed by transmitted light ex- actly as if it were a negative for a lantern slide. The Petri dish is then held in place in the darkened window or in front of the camera box by crowd- ing it into a hole cut in a square of thick leather, paper, or sheet-rubber (1 inch), which is then fastened over the kit or framework by eight thumb- tacks, or, better, it may be held in place by two stout rubber bands, as shown in the photographs (plate 17 and fig. 125). With stop 32 11. s. and Seed's 2J-X plates the right exposure in Washing- ton is usually somewhere between ^ second and ^ second in sunny weather and 3 to 5 seconds in cloudy weather, using a Voigtlaender collinear lens, series III, No. 6, and south light. Atkinson gets very good results by Fig. 124.* *Fic. 124.— Modified Collins-Brown camera swung from the ceiling and set to magnify about X i?4. The four suspending strings, which are of very strong fish-line, end in an S-shaped hook, the upper end of which hooks over a ring attached to a stout cord pendant from the ceiling. The length of bellows in this camera as modified by the writer is 25 inches. The lens used with it is a Zeiss Double-Protar, Series Vila. No. 13, made by Bausch & Lomb, Rochester, N. Y. This is the type of lens known also as the Zeiss Convertible Double Anastigmatic. This lens has a focal dis- tance of pJ4 inches, or, when only the front or back half is used, 165/2 inches (16 according to Zeiss catalogue). It is provided with a Bausch & Lomb No. 2 Volute shutter. A cork support was placed under the object carrier to steady the apparatus while it was being photographed, but in actual use the camera swings free, and if one desires to avoid shadows the apparatus is given a gentle twirl just as the exposure begins. The object carrier is easily removed, and is held in place at any level by two set-screws. PLATE Enlarging and reducing camera, showing method of mounting the apparatus. On the table at the left U a Petri-dish poured plate held in place by two rubber bands and ready for photographing. On the table at the right is a specia camera-back used in making lantern slides. This allows the ground glass to be raised or lowered, pushed to right or left, or rotated at will. PHOTOGRAPHY AND PHOTOMICROGRAPHY. 135 placing a circular black disc centrally some distance behind the plate to be photo- graphed, using for illumination the diffused light which conies in around this disc. The result is a very sharp contrast, /. ~_ II i 1 — CLCK.O. S.O.f)U tI.tp«T ..Tk.0! » KOITIUITl ..T,«, ,u, mlu .,„ ,. ? / 64 «5 32 22 16 II 8 56 / us US JS6 128 6j» 32 16 8 d 2 US i it M r i ii li 4-1 — t-t ii \ i II it 'i i ii • i i ^L ??.? i1 £ ' ' ' II !!-L_L-L.L_L_LJ_J_-L_!. to MO 34 iiTt IT ^^T ^£ c A iy IK ^K ^9 i:nc^. inn 17^ ^nmtnijiflc^ i farair^ "li., ...L j. ~ UIN B^IOHl DUU •.. . WftLU • • 1 L •iiuiu . ' .' J n 1 1 1 1 i \ ii | 1 1 1 1 1 1 i 1 1 ri 1 1 1 1 r i TIT 1 1 1 1 1 1 1 1 1 oi.nco.0 20 52 SO SO (55 300 SO 500 800 Fig. 128.* over and o\-er 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. vFic. 128. — Exposure scale set to show proper time for buildings and average near views at 10 a. m. 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 2;-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, •'. 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, /'. '. triloculare in the earlier work. They show a transversely 2 to 5 septate, rod- shaped organism, with rounded extremities, and bearing one polar flagellum about one-third the length of the body. There are no paired rods, or constrictions at any of the septa, but some of the rods are slightly curved. The shape and septation of the figures is slightly suggestive of some of the drawings of de Bary's Bacillus niegaie- rium (Pilze Mycet. u. Bact., fig. 194). They also look somewhat like some of the involution forms of Bacillus hortulaiius (Phil. Tr. Royal Soc., Series B, vol. 191, pi. 16). Both of these organisms, however, have peritrichiate flagella. The flagel- lum resembles that on species of Vibrio. Cohn's drawings of Bacterium tcr»io are shown in fig. 139, copied from his " Beitrage " (Bd. I, Heft 2, Tafel III). Colin did not consider motility of any generic value, and consequently paid no attention to organs of motion. Dalliiiger & Drys- *FiG. 138. — Ehrenberg's Bacterium triloculare, showing flagella. From Die Infusionsthierchen, Plate V, fig. I, i, 2. 170 BACTERIA IN RELATION TO PLANT DISEASES. dale's conception of this organism, at a time when the air was full of talk of Cohn's researches, is shown in fig. 140. Dallinger & Drysdale's drawings were made from unstained material, and there is no doubt that these expert microscopists actually saw what they figured, viz, a schizomycetous organism provided with one polar flagellum and belonging to the family Bacteriaceae. Dallinger afterwards care- fully measured the diameter of the flagellum many times over in unstained material, grown in Cohn's fluid. As bearing on the question whether Ehrenberg could see the flagellum of an unstained bacterium with the microscopes at his disposal, it is inter- esting to note Dallinger's statement that Koch could not see the unstained flagellum of Bacterium termo because he used "low-angled glasses, which are incompetent to that demonstration." Another remark of Dallinger is also pertinent. "I have learned," he says, "from experience that there is as great a diversity in different individuals in the sensitiveness of the retina as there is in sensitiveness of the olfactory or auditory nerves." The writer's own conception of Bacterium termo is shown in fig. 141. These organisms are green-fluorescent species cultivated in Cohn's solution, from water into which beans had been thrown in the manner described by Cohn. The very distinct flagella were stained by Lowit's method. The particular species from which this was obtained did not / liquefy gelatin. * 600 ca To the writer, then, the genus Bacterium is Bacterium lg' '* (Cohn emend.), and is based on the morphology of the green-flnorescent organisms, i i capable of growing in Cohn's nutrient solution and called by him Bacterium tcrnioS. It corresponds ) j to Migula's genus Pseudonumas, for which name it ( should be substituted as a proper generic name for ^-~, V "P * i ^^ , straight or slightly curved Bacteriacese, motile by ^-^^ of one to several polar flagella. It ~ m m most of the yellow bacteria and all of the green-fluor- pi |4] j esceut bacteria (vide Migula's system, Bd. II, p. 875). *Fic. 139. — Bacterium tcnno: a. motile form; h, xoogkeae. After Cohn. Untersuchungen tiber Bakterien. Beitrage z. Biol. d. Pflanzen. Bd. I, Heft 2, Plate TIL tFir,. 140. — Dallinger and Drysdale's conception of Bacterium tcnno. See Dallinger and Drys- dale "On the Existence of Flagella in B. tcrmn." Monthly Micros. Jour., Sept. I, 1875. Plate CXI 1 1, p. 105, figs. 6 and ;. tFir.. 141. — The writer's conception of Cohn's Kactcrium Icnun. Organism obtained by throw- ing beans into water and then making a transfer from the green-fluorescent liquid to Cohn's solu- tn'ii Stained by Lowit's method. X 2000. jThese organisms have no necessary connection with Bacterium termo Ehrenberg or with Monas termo Miiller. We shall never know what these were. NOMENCLATURE AND CLASSIFICATIONS. \Ye have therefore the following : Bacterium (Cohn emend.). Type : The one-flagellate, green-fluorescent schizoniycetes, capable of growing in Cohn's nutrient solution. To these should be added all the morphologically sim- ilar, non -fluorescent and yellow species. Synonym : Pseudomonas Migula. Among others the following plant parasites belong here: Bacterium campestre (Pammel), B. pruni (Erw. Sin.), B. hyacinthi Wakker, B. vasailantm (Cobb), B. phascoli (Erw. Sin.), B. juglandis (Pierce), B. Steivarti (Erw. Sin.), B. mak'accarnw (Erw. Sm.). These changes leave no generic name for the anthrax organism and other nou- motile forms. The writer would like to name the anthrax organism and related forms in honor of the distinguished man who first pointed out the generic significance of non-motility in this organism, but who unfortunately selected for it the preoccupied name of Bacteridium. There is, however, already a genus Davainea in helminth- ology, and it does not seem wise to make another, even in botany. Bacteria are now classed as plants, but we do not know what may finally be done with them. It remains, therefore, to adopt some old name, if an unobjectionable one can be found, and if not, to devise some entirely new name for the non-motile bacteria. There are several old names not now in use, e. g., Aletalactcr and ^fclanella, but so far as I have been able to determine, none of them were given to organisms at all resembling the anthrax organism, and for one reason or another all must be rejected. I therefore propose the name Aplanobactcr (from Greek words meaning ivithoitt motion and a rod), and shall use it as the generic name for the anthrax organism called Bacteridium by Davaine, Bacillus by Colin and Fischer, and Bacterium by Migula. Under Aplanobacter I include all non-motile forms morphologically similar to the anthrax organism (Bacillus anthracis Cohn), the latter, however, being taken as the type of the genus : Aplanobacter nov. gen. nom. An unattached, non -motile, rod-shaped organism, destitute of chlorophyll and multiplying by fission, sometimes forming threads of considerable length. The type of the genus, in the family Bacteriacete, is that organism causing anthrax and most commonly known in literature as Bacillus anthracis Cohu. For the present iion-sporiferous forms, resembling Aplanobacler anthracis, are also included under this genus, but if it shall be decided, later on, that the difference between sporiferous and non-sporiferous forms is of generic significance, then the latter may be excluded. This genus, as now understood, includes Aplanobacter anihracis (Cohn) and many other non-motile species called Bacillus in most books, but Bacto iiim by Migula. For a list of the species see Bacterium (p. 279) in Bd. II of Migula's "System." A few species there given are now known to be motile. Forms related to Bacillus tuberculosis Koch and Bacillus leprae Hansen do not seem to belong with the anthrax organism, and some name must be found for these. 172 BACTERIA IN RELATION TO PLANT DISEASES. Lehmann & Neumann have suggested Mycobacterium, and we ma}' use this name without in any way committing ourselves as to the significance of the branching forms. I would include also under it Bacillus diphtheria Loeffler (Corynebac- terium L. & N.). The writer has not inquired critically as to whether this is the earliest available name for this group, but that of Sclerothrix, given by Metchnikoff in 1888, is twice preoccupied, and that of Cocothrix, given by Lutz in 1886, is too near the earlier Cocothrichium Link. In 1889, in Saccardo's Sylloge Fungorum, De-Toni and Trevisaii included these organisms under the genus Pacinia Trevisan, but Trevisan's original draft of this genus included only vibrios, his type being the organism causing Asiatic cholera. Another difficulty is to decide what name shall be used for the cause of Asiatic cholera and its relatives. The majority, perhaps, of pathologists and bacteriologists use the genus name I 'ibrio. They understand by it small spirally bent organisms common in water and possessed of one polar flagellum or rarely of several such organs, the / 'ibrio cholera" being taken as the type. Others call most of these organisms Vibrio, but speak of Spirillum cholera:. Others use the two names Vibrio and Spirillum interchangeably. Others try to escape the difficulty by avoiding Latin names altogether, speaking in the same article indifferently of "the cholera vibrio," "the cholera bacterium," and "the cholera bacillus." This is the case frequently in the recent big monograph by Kolle & Wassermann. A few per- sons, following Migula, have used Schroeter's name, Microspira, given in 1886. Microspira is inadmissible, according to strict rules of priority, because Trevisan's name Pacinia is one year earlier (1885). Trevisan's genus, although badly defined, following Zopf 's ideas of pleomorphism, is tied hard and fast to the cholera organ- ism. Apparently this name was given without any personal acquaintance with the organism named, but according to current rules of nomenclature this makes no differ- ence. The choice, therefore, is between Pacinia and Vibrio, the one tied fast to a known species, but not used by working pathologists or bacteriologists since it was coined, so far as my reading goes, the other in common use, but a floating name — that is, one which can not be used for bacteria, and at the same time tied to any definite species or group of species included in the original draft of the genus. Miiller's genus Vibrio was published in 1773 in his "Vermium terrestrium et fluviatilium." It contained 15 species — bacteria, eel-worms, etc. Other things were also afterward put into it by Miiller, e.g., diatoms. We will be content with the first draft of the genus. It is described as follows: "Vibrio. Most simple, inconspicuous, terete, elongate worms." The first species is described as follows: Vibrio lineola. Vibrio linear, hard to see. Danish, Strseg-strsekkeren . A most minute animal, almost exceeding in smallness Afonas termo and 30 times less than Vibrio bacillus and entirely different. A trembling motion of myriads of oblong and obscure points is seen in a single drop, or with the highest magnification undulatory movements. In infusions of vegetables it almost fills the substance of the water after many days. The second species, I7, bacillus, first obtained from hay infusions, is described at a little greater length, but not any better. The third species is a mixture of nematodes. The first two species are bacteria. One other species of schizomycete is described, viz, Vibrio undula. This last, or what was supposed to be it, together NOMENCLATURE AND CLASSIFICATIONS. 173 with Vibrio spirillum, a subsequent addition by Miiller, was removed by Ehrenberg to form his genus Spirillum, which we still retain. The eel- worms were removed to form the genus Angnillula, and the other infusoria were variously distributed. Only UK- first two species of the original genus remained in Cohn's time, and neither one was used by him. Cohn used Vibrio ntgitla, one of Miiller's additions, for the first species under his emended genus Vibrio, but this has now been put by Migula into Spirillum. The only other member of Cohn's genus Vibrio (emend.), V. set-pens, is still less like the cholera organism. Ehrenberg's figure of I 'ibrio lineola Miiller (Infusionsth.) shows crooked little organisms not unlike what we now call vibrios. As a general proposition the writer believes that if a genus name is to be retained one should be able to tie it to some definite type-species, and it ought to be a species put into a genus when it was first published, and not one put in after the genus has been emended out of all recognition. Of course, nothing can be done with Miiller's, or Cohn's, genus description of Vibrio. If the name is to be retained for any organisms whatsoever, the description must be made over and the name anchored to a known species. Ordinarily such a name should be discarded. Under the circumstances, we may perhaps strain a point, make over the genus description in toto, and use the name Vibrio, as many pathologists have done, for Koch's comma bacillus and related forms. Logically, perhaps, we should adopt the strange Pacinia; for convenience sake we may continue to use the familiar Vibrio. The name Vibrio is not used by helminthologists or algologists, and, if we connect it to the first species described by Miiller under the genus, we may anchor the name to any small motile species, withoiit fear that subsequent researches will require changes to be made. This may be done, because the description of Miiller's Vibrio lineola, the first species, is so imperfect that identification is out of question ; the name can never be attached to any morphologically definite organism or group of organisms different from the cholera vibrio, even the gelatinization of the water after many days being probably enough due to other bacteria. The writer follows Lafar (ist ed.), Alfred Fischer, Lehmaiin & Neumann, ct al., and would write : Vibrio (Miiller, Cohn, emend.).* Type of the genus, Koch's comma bacillus. Synonyms. — Spirillum choleric-asialiar Koch; Microspira comma Schroeter; Pacinia cholcra-asiaticce Trevisan . Kendall has criticized Migula's use of the word Pseudomonas on the ground that he has combined under it two distinct groups of the family Bacteriacese, the monotrichiate and the lophotrichiate forms, and because the name implies, he says, a relation to " pseudomonads." The second criticism implies that to be tenable a name must conform etymologically to all the facts in the case. This is a miscon- ception. No one is warranted in setting aside a generic or specific name simply because it seems inappropriate. It is not inappropriate, however, since the first species in Miiller's genus Fionas was undoubtedly founded on small bacteria of some sort. As to the first criticism, that lies also against my use of Bacterium and requires a word. This criticism appears to me not well taken, since in the BacteriaceEE, as Migula first pointed out, there is no such sharp distinction *According to Fischer, 1903, and Lehmann & Neumann, 1896, this emendation \vasmade by Loeffler. BACTERIA IN RELATION TO PLANT DISEASES. between the moiiotrichiate and the lophotrichiate forms as there seems to be among the Spirillaceae. Within the limits of the same species, and on the same cover-slip, forms may occur with one flagellum and others with two or more (see fig. 15, which is not the only one I might offer). The name Psendomonas is of earlier date than Fischer's or Kendall's equivalents, has priority, and can not be set aside 011 the grounds named. If the reader is not satisfied with the reasons I have given for substituting the earlier name Bacterium, then he should continue to use the name Pseudomonas. For the present, therefore, I follow Migula's classification, except in so far as relates to his use of the words Microspira, Pseudomonas, and Bacterium. The following names should be rejected : Acromatiura. Actinobacter. Actinomyces. Aerobacter. Aethylbacillus. Amylobacter. Arthrobacter. Arthrobactridium Arthrobactrillum. Arthrobactrium . Ascobacillus. Ascobacterium. Ascococcus. Astasia. Astrobacter. Azotobacter. Babesia. Bacteridium. Bacteriopsis. Bactrillum. Bactrinium. Bollingera. Botryomyces. Cenouiesia. Chromatium. Clathrocystis. Clostridium. Clostrillnm. Clostrinium. Coccos. Cocobacillus. Cocobacteria. Cocothrix. Cohnia. Cornilia. Corynebacteriuni . Cryptococcus. Cystobacter. Dicoccia. Diplectridium. Diplobacteria. Diplococcus. Discomyces. Dispora. Ereboneuia. Ery throbacillus . Erythroconis. Feiiobacter. Gaffkya. Gallionella. Gliabacteria. Gliacoccus. Glischrobacterium . Gonococcus. Granulobacter. Guruniibacillus. Hrematococcus. Halibacterium. Heliconionas. Helobacteria. Hyalococcus. lodococcus. Klebsiella. Kurthia. Lactobacter. Lampropedia. Leptothrix. Leucocystis. Leuconostoc. Lineola. Macrococcus. Megabacterium. Megacoccus. Melanella. Meningococcus. Merisniopedia. Mesobacterium. Mesococcus. Metallacter. Microbacterium . Microhaloa. Microphyta. Microsphsera. Microspora. Microsporon. Microzoa. Microzynia. Monas. Monobacteria. Monococcus. Mycoderrna. Myconostoc. Mycothece. Mycothrix. Neisseria. Newskia. Nitrobacter. Nitrosococcus. Nitrosomonas. Nocardia. Nosema. Octopsis. Ophidomonas. Pacinia. Paracloster. Paraplectrum. Pasteurella. Pasteuria. Pediococcus. Perroncitoa. Petalococcus. Photobacillus. Photobacterium . Photospirillum. Plectridium. Pleurococcus. Pneumobacillus. Pneumococcus. Polleudra. Proteobacter. Proteus. Rhabdomonas. Rhizobium. Saccharobacillus. Saccharobacter. Schinzia. Schuetzia. Sclerothrix. Sphaerococcus. Sphoerotilus. Spirobacillus. Spirodiscus. Spiromonas. Spirulina. Sporonema. Streblothrichia. Streptobacillus. Streptobacteria. Streptothrix. Tetracoccus. Thermoactinomyces. Thermobacillus. Thermobacteriuni . Thioderma. Thiosphtera. Thiosphcerion. Torula. Tyrothrix. Ulvina. Urobacillus. Urobacter. Urocephalum. Urococcus. Urosarcina. Zooglcea. Zopfiella. PLATE 21. Walnut disease. Bacterial black spol of the Persian walnut (Juglans regia). more commonly known as the English walnut. Hall -developed green fruits from an orchard in California, snowing the badly spotted epicarp ; spots due to Bactenum juglandis (Pierce) . Leaves and shoots are also subject to this disease, which has become serious in Southern California, where large quantities of these nuts are grown for market. The attacked parts are conspicuously blackened as if charred. The numerous small white spots show the location of groups of stomata. Infection takes place readily through unbroken tissues. NOMENCLATURE AND CLASSIFICATIONS. 175 A very few of the preceding may perhaps some time make good their claim to be considered as independent genera. Many of these names are preoccupied in this group or in other groups ; some represent mixtures ; others, purely physiological genera ; but some of them may be used within the limits of genera to designate special physiological groups whenever such use leads to clearness of understanding. Naegeli, Beyerinck, and Winogradsky have studied especially the food require- ments of bacteria. Many others have, of course, contributed. Alfred Fischer has given a good summary in the second edition (p. 96) of his " Vorlesungen." Follow- ing this, and considering them especially with reference to their nitrogen-nutrition, the bacteria may be classified into seven groups : 1 . Paratrophic bacteria. The obligate parasites, capable of growing only on substrata similar in composition to the fluids of the host. 2. Peptone-bacteria. Organisms requiring peptones or albumoses. 3. Amido-bacteria. Organisms which also grow well when their nitrogen food is restricted to amido-bodies — asparagin, leucin, etc. — but not able to use ammonia. 4. Ammonia-bacteria. Able to take nitrogen from ammonia compounds. 5. Nitrobacteria. The denitrifying organisms. They require organic carbon com- pounds. 6. Nitrous and nitrate bacteria. The saltpeter-bacteria. Nitrates, nitrites, or ammo- nia-compounds furnish the necessary nitrogen. The carbon dioxide of the air serves as their carbon-food. 7. Nitrogen -bacteria. Organisms able to assimilate free nitrogen, but only in the presence of organic carbon compounds. In 1895 Wyatt Johnston suggested that all the important characteristics of a species might be recorded by numbers arranged in a definite order. Gage & Phelps and Kendall afterward made use of the Dewey numeral system. By this means the leading features of a hundred or of five hundred organisms might be recorded on a single page, so as to be very easily compared. Chester has modified this system for application within the genus as follows: ioo. Endospores produced. 0.002 Acid without gas from saccharose. 200. Endospores not produced. .003 No acid from saccharose. 10. Aerobic and facultative anaerobic. .0001 Nitrates reduced. 20. Anaerobic. .0002 Nitrates not reduced. 1. Gelatin liquefied. .00001 Fluorescent. 2. Gelatin not liquefied. .00002 Violet chromogens. o.i Acid and gas from dextrose. .00003 Blue chromogens. .2 Acid without gas from dextrose. .00004 Green chromogens. .3 No acid from dextrose. .00005 Yellow chromogens. .01 Acid and gas from lactose. .00006 Orange chromogens. .02 Acid without gas from lactose. .00007 Red chromogens. .03 No acid from lactose. .00008 Brown chromogens. .001 Acid and gas from saccharose. .00000 Non-chromogenic. According to this scheme the formula for Bacillus coli and Bacterium campcutrc would be respectively B. 212.11110 and Bact. 211.33315. Such a system admits of indefinite extension, and the reader can see at a glance that, if well worked out so as to include all the more important facts, it would be invaluable for unification of methods and for quick, easy reference. Each group of digits should include as 176 BACTERIA IN RELATION TO PLANT DISEASES. many facts as possible. Kendall, for instance, under the gelatin group has also included action on dextrose as follows : Liquefaction of gelatin. Fermentation of dextrose, gas production. Acid production. I. Negative. Negative. Negative. 2. Negative. Positive. Positive. 3- Negative. Negative. Positive. 4- Positive. Negative. Negative. 5- Positive. Positive. Positive. 6. Positive. Negative. Positive. 7- Unknown. Negative. Negative. 8. Unknown. Positive. Positive. 9- Unknown. Negative. Positive. The subject is now in the hands of a committee of the Society of American Bacteriologists for consideration and recommendation, and criticisms are desired. They may be sent to Prof. F. D. Chester, Wilmington, Del. ; Prof. F. P. Gorhain, Providence, R. I., or to Erwin F. Smith, Washington, D. C. VALUE OF MORPHOLOGICAL CHARACTERS. Ebb and flow, growth and change, this is the order of the world. Living things conform to a certain set of conditions and we say they are constant in structure and function because the conditions are fairly constant ; change the environment too much and they are destroyed ; change it essentially, ever so little, and the animal or plant begins at once to respond to it. This is especially true of simple uni- cellular forms. We can not, then, expect more than a moderate amount of constancy in these low forms of life. If under slight changes of environment they are fairly constant morphologically, it is all that we can expect, and in interpreting all descriptions we must make due allowance for these slight changes which an author may not have observed. There have been two extreme views respecting the morphology of the bacteria. Bechamp, Hallier, Billroth, and Zopf stand for one extreme ; Koch's earlier views for the other. To Hallier bacteria were only the developed plastids (protoplasmic granules) of fungi, and under widely different forms we might have the same organism functioning at one time as a harmless mold and at another as a micrococcus, causing the dreaded cholera or some other human or animal disease. Bechamp's microzymas were granules or fundamental elements more minute than the plant or animal cell, granules out of which all life developed and which persisted in other forms after the death of the cells. To Billroth all ordinary forms of bacteria, however dis- similar they might appear, were but stages of one unique species, viz, his Coccobacteria septica. Zopf did not carry his doctrine so far, but taught pleomorphism as a funda- mental characteristic of the bacteria. To-day an organism might be a Micrococcus, tomorrow a Bacterium or a Bacillus. Koch, on the other hand, insisted on the fixity of forms. To him a bacillus was always the same thing, and the views of the polymorphists were explained as the result of errors in technique, the confounding of entirely different things. Koch's own methods were exact and his views had PLATE 22. Bacterial black spot of the walnut. A late stage of the disease on the nuts. Photograph by Pierce. Mr. Pierce, who discovered the cause of this disease, has demonstrated 50 per cent of the losses preventable by spraying, and is now endeavoring to obtain resistant varieties by hybridizing and selection. The sum of $20.000 was offered by the waJnut growers of California some time ago for a satisfactory remedy, and recently the legislature of California has appropriated a considerable sum for its investigation. NOMENCLATURE AND CLASSIFICATIONS. 177 enormous weight, since he depended not on mere assertion, but pointed out many errors of fact and many flaws in the reasoning of his antagonists. To-day the majority of bacteriologists hold a sort of middle ground. Very few are willing to accept the views of the old polymorphists, but there is a spirit of rational inquiry abroad. We know that bacteria are much more responsive to changed environment than was supposed by Koch and his followers in the eighties, and we are prepared to believe anything respecting their origin and their poly- morphism which can gain the suffrage of the great body of critical workers who now cultivate this field, and who at once begin to investigate from all sides any new and strange statement. Duplication of work, so called, is not waste of time.* If sharp criticism abound, so much the better. In this way we shall gradually reach a clearer understanding of these organisms. Meanwhile, let each one cultivate his own little field as best he may, and, above all, let him be very sure of his facts before he publishes. There can be no doubt that the same organism sometimes exists as a long fila- ment in which no septa are visible and at other times as a short or nearly isodia- metric rod, but we are not thereby compelled to consider the short form as a Micro- coccus, i. e., as something very different from the long form. Physical conditions probably have much to do with bringing about these differences. Respecting the meaning of the branched forms, described by so many writers, the author is in doubt and can only wait for more light. Several hypotheses are open : (i) The bacteria, as now understood, are not a homogeneous group, but consist of many organisms of dissimilar origin and differing morphologically, which will be gradually separated out and put into their proper places, just as the Oosporas (Streptothrices) have already been removed, leaving as /Tw-bacteria a genuine residuum of morphologically similar forms ; (2) the bacteria do not any of them represent a natural group, but are stages of various higher forms, just as certain cells, multiplying indefinitely in yeast form, are now known to be conidial stages of the higher fungi (smuts, mncors) ; (3) the branched forms, which come mostly in old cultures, or in other crowded conditions where the organisms are subject to the injurious action of their own by-products (root-tubercles of L/eguminosse, lung-tubercles, etc.), are to be regarded simply as involution or degeneration forms, and not higher stages of development ; (4) the branchings are incomplete longitudinal fissions favored by special chemical or physical conditions. Time will show where the tnith lies. No harm will come to any one if all of these perplexing questions are not settled definitely within his own generation. So far as can be judged from structure the bacteria appeared in early geologic ages (in coprolites, decaying bones, tree-trunks, etc.) in forms closely resembling those now existing, but we have very little definite information as to their origin. Probably they are related to the lower algce and of as ancient origin. On rela- tionship of the bacteria to Algae, Fungi, Flagellata, and Myxomycetes, see Migula's remarks on the systematic position of the bacteria, in his "System," part I, page 237. *Karl Pearson has recently stated that 50 per cent of the scientific work of the igth century will have to be junked as worthless. In bacteriology 75 per cent would be nearer the truth. 178 BACTERIA IN RELATION TO PLANT DISEASES. VALUE OF CULTURAL CHARACTERS. Of what worth are the cultural characters commonly mentioned in descriptive bacteriology ? Much depends on the proper answer to this question. There are undoubtedly two extreme views, neither of which is correct. One investigator would maintain that no dependence can be placed on them ; another seems to have no suspicion of any source of uncertainty. The truth undoubtedly lies somewhere between the two. That great progress in bacteriology has come from their use must be admitted by all. To cast doubt on everything already done is only to bring chaos back again. It is wise to make haste slowly. No necessity exists for making a rubbish heap of the past before beginning one's own work. Old methods should be tried repeatedly, scrutinized from every standpoint, and only abandoned when they have yielded all that can be obtained from them, or when there is some- thing distinctly better to take their place. New methods should be hailed with enthusiasm only in so far as they have actually made good their claim to be genuine improvements. A great deal of writing on bacteriology is worthless because not based on well-considered and properly conducted experiments. Hypotheses ad libitum, the more the better ; but let us not forget to test each one in the crucible of experiment, and generally before publishing, rather than after. In other words, give to the world only the well-established facts. As a means toward arriving at the truth, let each person not only experiment as carefully as possible, but let him set down all the steps in his procedure, so that others may repeat his experiments. Many misapprehensions and supposed contradictions arise from the fact that workers are led to believe they have exactly duplicated another man's work when they have done nothing of the kind. The temperature at which they have worked has been dif- ferent, or some other physical or chemical condition, important but not recognized or not recorded by the first writer, has been unlike, and the results are not the same. Bacteria are not so simple as they appear. While monotonous morphol- ogically they are complex in their multitudinous physiological activities, and are extremely apt to vary under a slightly changed environment. When we repeat an experiment we must know, therefore, whether we have preserved substantially the former environment. If we have not, then it should not surprise us if the results are somewhat different from those we anticipated. A very frequent source of error in interpreting descriptions consists in not making sufficient allowance for changes due to slight variations in the culture-media. I can perhaps make my meaning plainer in the following way : Let the curve A B represent all the variations in color and appearance of a given organism on a given medium, c. g., steamed potato. Now, if a worker describes his organism from a PLATE 23. Bacterial wilt of the cucumber. (Introduced to illustrate transmission of the disease by insects). The central plant (variety Long green) was inoculated on June 17 with Bacillus tracheiphilus by the striped cucumber-beetle (Diabrotica vittata). As a result the gnawed leaves first wilted and then the whole upper part of the plant, the vascular bundles being occluded by the sticky white slime of this bacillus. Photographed July I, 1905. About 1 - 14 natura size. The entire plant was dead about two weeLs later. VALUE OF CULTURAL CHARACTERS. 179 few cultures, he will then in all probability have covered only a fraction of the curve A B, let us say between C and D, and not the whole curve of growth. If, now, another worker should happen to experiment with potatoes capable of giving rise in the organism to phenomena represented by that part of the curve lying between A and A', he would get somewhat different results and yet this would not prove steamed potato to be a worthless culture-medium. The only real facts in the sup- posed case are that neither person has experimented sufficiently to draw up a proper description of the characteristics of the given organism on potato. Let us suppose we have to do with a yellow organism, e. g., Bacterium phaseoli and that A to A' represents a pale yellow growth, with no graying of the potato, while D to B repre- Fig. 142.* sents a very deep yellow growth, with very decided graying of the potato. The cultures look like different organisms, but they are not. The descriptions would differ. Neither account alone would form a proper description of the behavior of this organism on potato, but there should be rather a combination of the two and of all intermediate stages, viz — Potato : Color van-ing from pale to deep yellow, flesh of the potato usually grayed, but sometimes remaining unchanged, etc. The same remarks apply to other non-synthetic media. *Fic. 142. — Iris-rhizome rot. A dense sowing of the organism in an agar-plate culture after 45 hours at 25° C. The buried colonies small. Not van Hall's organism, which, as received from Krai of Prague, is non-pathogenic in my hands. i8o BACTERIA IN RELATION TO PLANT DISEASES. Iii case of agar and gelatin there are numerous variations due to inadvertent changes in the culture-medium, especially if this is made by students. The media should be made by competent, experienced persons, and then the descriptions of the behavior of the organism on it should be broad enough to include slight differences in the aspect of the colonies, streaks, and stabs, which often depend 011 chemical and physical conditions within the control of the experimenter, c. g., on the water-content, 011 age of the medium, amount of moisture in surface-layers, kind of peptone, kind of gelatin, length of exposure and degree of heat during sterilization, etc. The dense or thin sowing of the plate may sometimes make a very decided difference in the aspect of the colonies. Fig. 142 shows a densely- Fig. 143* sown plate, the colonies round or roundish. Fig. 143 shows the same organism, and from the same set of plates, but thinly sown and two days older. Here the colonies are radiate. In case of Racillns aroidecc when grown on agar-plates, near the maximum and minimum temperature limits, the surface-colonies are round even after many days, but they are promptly and strongly radiate when grown at or near the optimum temperature (see figs. 144, 145). When very thin sowings of this organism were exposed to the high temperature, the colonies were also round. It occurred to the writer that the round colonies obtained on the agar-plates exposed in the thermostat at 37° C. might be due to physical changes in the surface *l''jc. 143. — Iris-rhizome rot. The same as 142, but sown thinly and kept for 4 days at 25° C. VALUE OF CULTURAL CHARACTERS. l8l layers of the agar, /. e., to rapid loss of water, and experiments have shown this to be the case. Two sets of Petri-dish poured plates were made, inoculating from the same culture. One set was exposed in the open thermostat at 37° C., and these developed round colonies, similar to those shown in fig. 144. The other set was inclosed in the same thermostat, but inside of a closed glass vessel containing water. The colonies on these grew in radiating form, the same as in a third set of plates exposed at 30° C. This does not account, however, for the appearance of circular colonies at low temperatures. After twelve days' exposure in an ice-box the writer obtained the same result as Townsend ; the colonies were not radiate, but looked like those shown in fig. 144. UNDERGRADUATE WORK. As a rule, the results of this kind of investigation are to be distrusted. The fresh ambition of students and their delightful eagerness to take up hard problems are sources of great pleasure to every good teacher. At the same time such students must be held back rather than uiged on, since for the most part they are still unfitted to do independent work, especially that which involves the drawing of general conclusions from a variety of experiments. The ordinary training of botanical and zoological laboratories will not fit the student for specialization in pathology and bacteriology. Skill in this sort of work must be obtained from consorting with the professional pathologist and bacteriologist. In general, at the present time a well-equipped modern laboratory devoted to animal pathology is a much better place for the plant bacteriologist to learn methods than even our best-equipped botanical laboratories. One of two alternatives is open to the ambitious student. Either he must submit to a long and rigorous course of elementary study in a bacteriological laboratory, under a competent and critical teacher, or else he must be content to pick up the general principles of the science out of books and journals, with much blundering and stumbling in the first years of his study. During this nursery period, if he is jealous of his own reputation, he will not publish much. My experience has led me to discount very liberally the conclusions of student investigators, and I consider those students very unfortunate whose teachers urge them into precocious publication. In many cases nothing could be more damaging to their own reputation as scientific inquirers, or more injurious to the progress of science. Bad papers also react upon the teachers of such students, who can not by any shift evade responsibility. My advice to teachers is to discourage all students who do not show marked aptitude, and to give to those who do show signal ability the best possible training in methods of work, but to discourage them from undertaking difficult pieces of original investigation. The only alterna- tive is for the teacher to follow their work step by step and assume joint responsi- bility for it in the end. Even this latter course is sometimes risky, as the history of science shows very conclusively. After a year or two of careful work on methods, under the watchful supervision of a good teacher, the bright student will have learned how to avoid many of the pitfalls which beset his way, and, if he has acquired a proper training in other directions, such as general botany, modern physics, chemistry, the modern languages, 182 BACTERIA IN RELATION TO PLANT DISEASES. etc., he ma}- be trusted to undertake some original research. Even when once on his feet as an investigator, my advice to him would be : Try every conclusion repeatedly and make haste slowly. When he becomes uneasy at delays, let him reflect that one really good paper does much to set an unknown worker on his feet among scientific men, whereas one or two hastily written, poor papers will injure his reputation as an investigator more than half a dozen good papers subsequently published will suffice to repair. Moreover, in this age of enormously multiplied publication it is impossible to read everything, and consequently if a writer wishes to attract attention he must have a commanding grasp of his subject ; must present Fig. 144.* its leading features in a clear, interesting style; must be as brief as the importance of his subject will warrant, otherwise his words are certain to be overwhelmed and lost; and, finally, must publish in a proper place, /. c., not in some obscure "Transac- tions" or in some local journal with a small circulation. II 'hen ready to publish, stop and do your work all aver again it'it/i more care. This is my advice to begin- ners. In the course of such general revision the chances are that many statements will require correction or modification, and some may have to be omitted altogether. *FiG. 144. — Colonies of Bacillus aroideae, circular when grown on an agar plate at 37° to 38° C., i. c., at ;\ temperature near the maximum. Photograph hy Townseml. CONSTANCY OF CHARACTERS. In any event, the student must have a considerable body of knowledge, gained by actual experiment, before his judgment is worth much. In the beginning he is apt to depend too much on the constancy of organisms and is certain to be misled by names. To illustrate : To him all agar is agar and all gelatin is gelatin. Not so, perhaps, to the organism with which he is experimenting. Slight differences in the composition of a culture-medium sometimes make considerable difference in the growth and general appearance of the bacteria, and this must be taken into account. After the student has passed this stage of development he can interpret his results much better. If, then, on some culture-medium he obtains results slightly Fig. 145.* different from those already published by some author, he is not immediately driven to suppose (i) that he has a new species, or (2) that the earlier writer was in manifest error. Other hypotheses now lie open to him. He is dealing with a living and variable organism, and perhaps the conditions in his experiment are not precisely like those to which it was subjected by the previous experimenter. It may also be an organism which has already varied into many races having slightly different peculiarities. Only when full weight has been given to these possibilities is he entitled to fall back on the others. On the other hand, however, he must not FIG. 145. — Colonies of Bacillus aroideae, radiate-fimbriate when grown on an agar plate at 25° C. Photograph by Townsend. .84 IIACTKKIA IN RELATION TO PLANT DISEASKS. escape Scylla only to fall into Charybdis. It may be that his organism varies in all sorts of ways, but he is by no means to assume this. Every hypothesis must be tried in the reducing fire of exact experiment. Probably the best acquirement a student can get from his years of training is a spirit of self-distrust leading to habitual caution in the drawing of conclusions and the making of general statements. Such a spirit will preserve him from many foolish statements and will enable him to serve his generation to the best of his ability. He will not go far, however, without a tremendous earnestness, an indomitable energy, directed in proper channels. I^et him concentrate this energy, the most priceless of all human attributes, and attack specific problems, one after another or a few at a time; not all at once. Honesty, industry, and self-reliance, tempered with the self-distrust already mentioned, will then carry him very far on the road he desires to go. Filially, the student should remember that the ideal man of science, and to a large extent also the actual man of science, is a modest man, always inclined to be cautious, always willing to revise his conclusions in the light of fresh evidence, generally plain-spoken, always an enemy of shams, and never offended by frank and honest criticism, preferring the white light of truth to the plaudits of the multitude. A FINAL CAUTION. Probably more mistakes arise from failure to carefully check up the work behind one than from any other source. What is meant by this can be explained in a few words, by means of a series of examples. ( i ) I make subcultures from a poured-plate colony. The first subculture is on slant agar, the second is from the agar into beef-broth, the third is from the beef-broth into potato - broth, and from the latter I propose to inoculate a plant. The in- Fig. 146.' FlG, 14(1. — Apparatus fur removing writer fnitii tissues with a minimum of injury. The speci- men is plaeed mi die wire carrier at X in water. The tube at the right also contains water. Alco- hol (i)5 per cent) is then poured into the funnel and allowed to pass into the apparatus drop by drop, ll perfect diffusion through the water is obtained by making the basal ends of the carrying tubes llarin.u or funnel-shaped. I'.y yat:ini; the time between drops the alcohol may be substituted JAT the owly or rapidly, in any desired time. About one-third actual size, MKTIIOOS OF WORK. 185 ference is that this tube of potato-broth, which is only the third remove from the colony, contains a pure culture of the organism with which I started, but simple observations of the tube, even when coupled with a very firm persuasion, do not assure me that such is the fact. I check the inference by making plate-cultures and find in the tube either (a) only the original organism ; ($) a mixture of two or more organisms; (c) a pure culture of some wholly different organism, which entered during one of the transfers as an accidental contamination and has crowded out the original organism. (2) A plant is inoculated from a solid culture or fluid culture of a supposed para- site, and becomes diseased. The inference is that the inoculated organism has caused the disease. I check this inference by making plate-cultures from the interior of the diseased tissues and find (a) great numbers of the inoculated organism in pure culture and capable of again producing the disease, which I determine by actual experiment ; (fy a mixture of organisms ; (c) some wholly different organism ; (rf) no bacteria whatever. (3) Fermentation-tubes of cane-sugar bouillon inoculated with a supposedly pure culture soou show clouding in the closed end, with an abundant production of gas and acid. The inference is that these phenomena are due to the presence of this particular organism. I check this inference by making plate-cultures and find (a) a pure culture of the original organism; (//) only an intruder; (c) a mixture of two organisms, in wrhich case both ma}- break up the sugar in the manner described, or only one of them. (4) Drops of fluid containing a supposedly pure culture are dried on sterile cover-glasses and subsequently put into sterile beef-broth, which becomes clouded. The inference is that the organism in question has resisted the drying. I check thi* inference by making plate-cultures from the fluid and find (a) a pure culture of the right organism ; (/;) a pure culture of some intruder. (5) The thermal death-point of an organism is tested by inoculating tubes of beef-bouillon and exposing them to a given temperature in the manner already described. Subsequently the bouillon clouds or does not cloud, as the case may be. The inferences are that the organism is killed or is not killed by the exposure. The first inference is checked by having at the same time inoculated other tubes of the same bouillon, which have been kept at room-temperatures, and which (a) do not cloud, showing either that the bouillon itself inhibits growth or that only dead organisms were inserted, i. e., those from too old a culture ; or (b} which cloud readily, showing that failure to grow in the exposed tubes is actually, as it was presump- tively, attributable to the temperature of the water-bath. I check the second infer- ence by making poured plates from the clouded tubes and find (a) pure cultures of the right organism ; (b) pure cultures of some intruder. (6) A plant, which we will designate as A, is subject in the field to a certain disease, and this disease is readily reproduced under experimental conditions, using pure cultures of a given microorganism. A related plant, which we designate as B, is subject in the field to a similar disease. A microscopic examination shows sim- ilar lesions associated with a morphologically .similar organism, and Petri-dish l86 BACTERIA IN RELATION TO PLANT DISEASES. poured plates indicate the presence of a physiologically similar organism in both plants. The first inference is that the two diseases are caused by the same organism. A test-experiment is now instituted, viz, one or two varieties of B are inoculated with the organism obtained from A, but these do not contract the disease. An easy second inference now is that we are dealing with two distinct diseases. This may be perfectly correct, but it is not established by the experiment. Owing to an oversight, plants of A were not inoculated at the same time and in the same man- ner as B, to serve as checks, and consequently we are not assured as to the virulent nature of our culture — it may have been dead, or non-virulent, or the wrong organ- ism. Check-plants should have been inoculated. Assuming, however, that this was done, and that A promptly contracted the disease while B remained unaffected, it is not yet certain that the disease in the two plants is due to different organisms. The question of individual and varietal resistance to disease may have entered to com- plicate results. To eliminate this possible source of error a greater number of varieties of B should be tested with a larger number of individuals in each variety. Cross-inoculations should also be made, /. t\, numerous varieties and individuals of A should be inoculated with the organism isolated from B. Enough has been said to show the ordinary method of work. All inferences should be carefully confirmed by frequent poured-plate cultures in Petri dishes, by cultivations on the media which have been found to give most characteristic results, and, finally, by frequent inoculations into the host-plants. In case of unexpected or striking results it is always safe to determine whether they can not be obtained in the absence of the assumed cause. These methods involve an almost endless amount of drudgery, but they are fundamental to any large success in the domain of pathology, and those who are desirous of winning a shining reputation without much labor are advised to culti- vate some easier science. For those who are really in earnest, who do not mind hard work, and who have acquired the requisite training, no field affords greater opportunity for brilliant and useful work than that of plant pathology. FORMUL/E. When not stated the solids are reckoned in grains and the fluids in cubic cen- timeters. \Yater is understood when no particular solvent is mentioned. STAINS. GENERAL AND MISCELLANEOUS. Alcoholic Solutions of Anilin Stains. These should be saturated solutions, made preferably with Griibler's stains and absolute al- cohol. In well-stoppered bottles they keep in- definitely. Watery Solutions of Anilin Dyes. These do not keep long and must be made up fresh each time. If made directly from the dry powder or crystals, rather than from the alcoholic solution, the resulting fluid should be passed through filter paper before using. Watery solu- tions are usually made by adding the alcoholic solution to distilled water in any strength de- sired. Usually a few drops of the alcoholic solution to 5 or 10 cc. of water is sufficient. Anilin Water. Anilin water is made by shaking thoroughly one part of anilin in 20 parts of distilled water and filtering it clear by passing one or more times through filter paper moistened with water. It should be prepared fresh each time. Anilin, known also as anilin oil, is a colorless, oily- looking fluid. It oxidizes to a brown color if exposed to the air, and it should therefore be kept in a close-stoppered bottle in the dark. The brown fluid is still usable, at least for some purposes. Ziehl's Carbol-Fuclisin. Puchsin (basic) I Absolute alcohol 10 Carbolic acid (5 per cent sol. in water) 100 The fuchsin should first be dissolved in tin- alcohol and then the two fluids mixed. A pow- erful and much-used stain. Ehrlich's Anilin-Watcr Gentian Violet. Alcoholic solutii'ii "i" gentian violet (saturated ) 5 Anilin water too This should be used as soon as prepared. It does not keep well. Flexner's Anilin Gentian Violet. Anilin oil 2 Alcohol, 95 per cent 5 Saturated alcoholic (absolute) solu- tion of gentian violet 8 Distilled water 80 Mix well and filter. Hhrlich-Weigert Anilin Methyl Violet. Alcoholic solution of methyl violet (saturated) n Absolute alcohol 10 Anilin water 100 Does not keep well. Anilin Fuchsin. Prepared in the same way as Ehrlich's anilin gentian violet. Ziehl-Nielson's Stain. Used chiefly as a means for identifying tuber- culosis. The cover-glass bearing the specimen is floated for 3 to 7 minutes on carbol-fuchsin which is heated until steam begins to appear. It is then washed in distilled water, plunged into 10 per cent nitric or sulphuric acid long enough to decolorize (a very short time). It is then passed through 60 per cent alcohol for a few seconds (just long enough to remove the stain from the background), washed thoroughly in water, dried, and mounted in balsam. The cover-glass preparation may be obtained also by dropping some of the stain upon it and holding it over the flame. This method is more eco- nomical of stain and time and less mussy than the preceding. Fricdlaender's Stain. This has been used so far mostly for identify- ing the tubercle organism in sputum. It is made as follows: A few drops of carbol-fuchsin are placed on the prepared cover (which has been gently flamed) and heated over a flame until the fluid steams. The cover is then washed in distilled water, and plunged for a half 187 1 88 BACTERIA IN RELATION TO PLANT DISEASES. minute or so into acid alcohol (c. p. nitric acid So per cent alcohol 100 cc.). It is then \va>hed in water, stained about 5 minutes (for contrast) in an aqueous solution of methylene 1)1 lie, dried, and mounted in cedar oil or balsam. L.ncfflci-'s Alkaline Mcthylaic Blue. Alcoholic solution of methylene blue (saturated) 30 Caustic potash I Distilled water 10,000 This fluid retains its valuable properties for a considerable time and is an excellent stain. Kiihuc's Carbol-Mclliylciic Blue. ( i ) Methylene blue 1-5 Absolute alcohol 10.0 (2) After triturating the above in an agate or pon ' lain mortar, or in a watch glass, add grad- ually 100 cc. of water containing 5 per cent car- bolic acid. Methylene blue is not the same as methyl blue. (See Pregl, Bibliog., XIV. ) Grain's Stain. This is a method of differential bleaching after a stain. The cover-glass preparations or sections are passed from absolute alcohol into Ehrlich's anilin gentian violet or into a watery solution of methyl violet, where they remain I to 3 minutes, except tubercle bacilli prepara- tions, which remain commonly T2 to 24 hours i Cram). They are then placed for I to 3 inin- i occasionally 5 minutes) in iodine potas- sium iodide water (iodine crystals I, potassic iodide 2, water 300), with or wilhont first wa-h- ing lightly in alcohol. In this they remain I to 3 minutes. They are then placed in absolute alco- hol until sufficiently bleached, after which they arc1 cleared in clove oil and mounted in Canada balsam. By this method the stain is removed from some kinds of bacteria and not from others. Too much confidence must not be placed in this method, since in some cases the removal or non-removal of the stain from the organism de- pends on the length of exposure to the iodine water. It would be belter, therefore, to expose all for the same period, c. g., 2 minutes. Gabbctt's Stain. Used mo-tly for tubercle bacteria in sputum. Slain first with carbol-fuchsin, then place the lass for i to 2 minutes in acid methylene blue (methylene blue 2 grams, 25 per cent sul- phuric acid water 100 cc.). When washed in water and dried it may be mounted in cedar oil or in balsam. The ordinary bacteria of sputum are decolorized ; the tubercle organism retains the red stain. The Bkrlich-Weigert Stain. Used for detecting the tubercle organism in sputum. The prepared cover is floated face down on anilin methyl violet, which is heated until steam rises. After 2 to 5 minutes on this hot stain plunge for a few seconds into acidu- lated water (i part nitric acid. 3 parts distilled water ). then wash for a few seconds in 60 per cent alcohol, and afterward thoroughly in water. For a contrast stain the cover may be placed for 5 minutes in a saturated aqueous so- lution of vesuvin. It is then washed in water, dried, and mounted in balsam. Bacteria which hold the stain after such treat- ment are sometimes called " acid-fast " bacteria. Flciiuning's Triple Stain. The slide is first placed in (i). (i) Safranin O (saturated alcoholic solution) 50 Distilled water 50 Anilin water 5 After washing in water, it then goes into (2). ( j) Saturated aqueous solution of gentian violet 50 It is then washed in water and passed into (3). ( 3 ) Aqueous solution of orange G. strong or weak (generally about one-half saturated). The slide is then washed quickly in 95 per cent alcohol, dehydrated, cleared, and mounted. Prcgl's Method. (See '91 Pregl, Bibliog., XIV.) Nieoltc's Mcth,n1s. (See '95 Nicolle, Bibliog., XIV.) Bcnda's Iran Haematoxylin. .Mordant the sections for several hours in I part of the following ferric solution* diluted with 2 parts of water : Ferrous sulphate So Water 40 Sulphuric acid 15 Xitric acid iS This solution, known to the German Pharmacopoeia as Liquor ferri sulphiirici oxydati and to the U. S. P. as Liq. f. tersulphatis or sol. persulphate of iron, keeps indefinitely. It is made as follows : Heat in a flask ou a water-bath until fluid is brown and clear, and a drop diluted with water is no longer colored blue by potassium ferricyanide, orate in a tared porcelain capsule to 100 parts, add a little water and evaporate again. Repeat the dilution and evaporation until the hot fluid is free from the odor of nitric acid. Finally dilute to a weight of 160 parts. STAINS. 189 \V;ish tin- sections in distilled water and then in tap-water. Stain (until very black) in water containing i per cent li:u-mal<>\ylin. Differen- tiate in 30 |ii-r cent acetic acid water with care- ful watching, or in more dilute acid, or in ver\ dilute ( I : jo) liquor ferri. if it is to he followed h\ acid fuch-in a-, a contrast stain. (Verhandl. d. Anat. ('.e^elkch., iX<>3, Jena. Onstav Fischer.) Ilculi-iiliain's Iron lliiciiiiitn.\-ylin. Mordant the sections from one-half hour to 12 hours in a 2.5 per cent watery solution of iron alum (ammonio-fcrric sulphate (NH,)_. Fc, ( Si >,>, dissolved cold). This salt comes in violet crystals. Yellow or green crystals should ho rejected. Rinse well in water. Stain in water containing 0.5 per cent of haematoxylin. Rinse, expose again to the iron-alum solution, watching the differentiation under the micro- -> ope, the examination being made in tap-water. When properly differentiated wash 15 to 45 min- utes in running water. Dehydrate, clear only 'with xylol, and mount in xylol balsam. The mordant does not keep indefinitely, but is said to retain its properties for some time (Dodge). Schaffncr's Sutruiiin Picro-nigrosin. The slides are stained for a few minutes in anilin-safranin made as follows: ( i I . \nilin water 50 Saturated alcoholic (95 per cent) solution of safranin.... 50 Rinse quickly in water. They are then stained in (2). (2) Distilled water 100 Picric acid thoroughly dissolved in the above i Then add nigrosin i Rinse in water, wash rapidly in 95 per cent al- cohol, dehydrate, and mount in balsam. Malachite Gi-ccn. For plant tissues, as well as animal tissues, this may be used as a contrast stain, follow- ing carbol-fuchsin. It is dissolved in auilin (i : 1000) and used fresh; generally the ex- posure to it should be for only a very brief period, i. c., I to 3 minutes. If not fresh, much longer exposures are required. CLEANING COYER-GLASSES FOR FLAGELLA STAINS. Van Ermengem recommends boiling in a mixture of water 100 cc., concen- trated sulphuric acid 60 cc., potassium bichromate 60 grams. The covers are afterward thoroughly washed, first in water and then in absolute alcohol. They are set on edge and dried under a bell-jar. Loeffler recommends heating covers in concentrated sulphuric acid. They are then washed in distilled water and put into alcohol-ammonia, from which they are wiped with a very clean linen cloth. Covers cleaned in the ordinary way may be freed from fat by passing them through a Bunseri flame immediately before using. FLAGELLA STAINING. There is no easv road to success. Some of the common stones of stumblin°- * o are (i) oily or otherwise dirty covers ; (2) cultures uusuited either by age or by composition of the medium ; (3) the casting off of flagella on dilution or during the slow drying of the fluid on the cover ; (4) an uneven or too copious distribution of the organisms on the cover ; (5) imperfect mordanting ; (6) excessive mor- danting ; (7) uuderstaining ; (8) overstating ; (9) precipitates on the cover-glass during some stage in the process. If clean covers are used, if the bacteria are derived from young moist agar cult- ures, if a very small quantity of such culture is put into a large drop of well aerated water, or, better, into a test-tube or watch-glass containing 5 or 10 cc. of water, and a tiu\- quantity of this dilution is taken on a platinum needle and deftly swept over the whole cover ; or if the needle is touched to the bacterial fluid and then touched 190 BACTERIA IN RELATION TO PLANT DISEASES. to one or more parts of a large drop of water on the cover, which is then put into the thermostat, so that it shall dry quickly and yet allow time for various rods to swim free from the tangle of their fellows ; if the mordanting is thorough, but not excessive, and, finally, if there is no unforeseen mishap in the subsequent staining, good preparations of many organisms are easily secured. Others have given the writer much trouble (Bacillus amylovorns is one of the worst), and the only con- clusion he has been able to reach is that the bacteria vary greatly in their response to flagella stains. Sometimes well-cleaned covers give trouble and then the surface of the glass itself is at fault (V. A. Moore), and covers of another origin should be tried. Young agar cultures are usually preferred, i. e., those not over twenty-four hours old, but good results may occasionally be obtained from much older cultures and also from other media. In case flagella are to be stained from fluid cultures by ordinaiy methods, the medium in which the organism is grown must be very dilute, and there must usually be an additional extensive dilution on the cover, or before putting thereon, to avoid a dense ground stain. Bouillon contains too many fine particles, but Zettnow has found a way to stain from bouillon (Bibliog., XII). Recently the writer has obtained very good results with several organisms by growing them for some days in 10 cc. of distilled water to which 2 or 3 drops of Uschinsky's solution was added. Covers were prepared directly from this solution. Others have reported good success by transfer of agar-grown organisms to consider- able water in a watch-glass or test-tube and incubating this in the thermostat for some hours before preparation of the covers. Others have recommended the use of filtered, sterile, hydrant water instead of distilled water. Nearly every worker has some favorite stain. The writer prefers Van Ermengem's. Loeffler's Flagella Stain. 1 i ) Mordant : Solution of tannin (20 per cent in water) .... 10 Saturated (cold) aqueous solution of ferrous sulphate* 5 Saturated alcoholic solution of basic fuchsin. i (2) Stain : Carbol-fuchsin. (3) Corrective solutions: (a) i per cent solu- tion of caustic soda; (6) a sulphuric-acid solu- tion of equivalent strength. V. A. Moore's Flagella Stain. This is a slight modification of Loeffler's. (1) Mordant: Tannic acid (20 per cent in water) 10 Ferrous sulphate (cold saturated water solu- tion) 5 Basic fuchsin (saturated alcoholic solution) . i (2) Stain : Ziehl's carbol fuchsin. Use the mordant fresh, and filter each time before using. Fix the film on the cover-glass by passing it quickly face up twice through the open flame, or by exposing for 5 or 10 minutes to a temperature of 120° to 140° C., the better way. A few cubic centimeters of the mordant are now placed in a wide test-tube (i inch), the cover is lowered into it, and the mordant is heated over a flame until steam rises. It is then removed from the flame and the cover is allowed to remain in the hot fluid 5 or 10 minutes, with occasional gentle shaking, after which it is drawn to the mouth of the tilted tube by means of a hooked platinum wire set into a glass rod. After thoroughly rinsing under the tap or in a fine stream of water the cover is lowered into the stain (held in another wide test-tube), where it is heated until steam rises (i to 3 minutes). One should know from be- ginning to end which side of cover bears the bacterial film. The cover is drawn to mouth of tube by means of a hooked platinum wire. Some- times i per cent sol. NaOH (1A cc.) may be added to mordant with advantage. *The iron oxide may be removed from the solution of ferrous sulphate by passing it through a filter paper, ected crystals should be used. FLAGELLA STAIN'S. 191 Fischer's Flagella Stain. This is a slight modification of Dr. Corner's, which is itself a modification of Loeffler's. (1) Mordant: Dry tannin 2 Water 2O Solution ferrous sulphate (1:2).... 4 Concentrated alcoholic solution basic fuchsin i Filter. This mordant will keep for several weeks. Drop it on the cover and heat over a gentle flame until steam rises ; then continue one- half minute longer without boiling. Wash. Treatment with alcohol is unnecessary. (2) Stain : Place on the cover a few drops of concentrated watery solution of basic fuchsin. Heat slowly, so that steam rises after about I minute. Then expose for one-half minute longer, so that the stain boils up once or twice. Wash, dry, and mount. Fischer says tannin ab- sorbs moisture readily, and advises keeping it in a desiccator. Bungc's Flagella Stain. (1) Mordant: Tannin 20, water 100 30 Liquor ferri sesquichlorati I Water 20 Saturated watery solution basic fuchsin 5 This must ripen some weeks by exposure to the air in a flask loosely plugged with cotton. (2) Stain: Carbol-fuchsin. Expose to the filtered mordant 5 minutes, using heat if necessary. Wash and stain. Van Ermengem's Flagella Stain. This is made as follows : (1) Mordant: Osmic acid (2 per cent water solu- tion) 50 Tannin (10 to 25 per cent in water) .. 100 Four drops of glacial acetic acid may be added to this. (2) Silver bath: 0.25 to 0.5 per cent nitrate of silver dissolved in distilled water in a very clean bottle. (3) Reducing and strengthening bath : Gallic acid 5 Tannin 3 Fused sodium acetate (some books say fused potassium acetate) 10 Distilled water 35° The flamed cover-glass (it may be unflamed) is first covered with the mordant for one-half hour, or if in a thermostat at 50° C. for 5 or 10 minutes. The mordant is then carefully re- moved by thorough washing in water, alcohol (some say absolute alcohol), and water. The cover (film side up) is now put into the silver bath (a few cubic centimeters in a small, per- fectly clean beaker or watch-glass) for a few seconds, during which it is gently agitated. Without rinsing, it is put next into a few cubic centimeters of the reducing solution and gently agitated until the fluid begins to blacken. It is then washed in water and examined. If not stained deeply enough the cover is returned to the silver bath, then once more passed through the reducing bath. It is finally dried and mounted in balsam. All the dishes must be scrupulously clean. The fluids must not be con- taminated by the fingers nor by dipping iron or steel instruments into them. Kuntze has suggested some improvements. (See Centralb. f. Bakt., I Abt., Bd. XXXII, 1902, pp. 555-500.) PMeld's Flagella Stain. (1) Mordant: Tannic acid, 10 per cent aq. sol 10 Corrosive sublimate, sat. aq. sol 5 Alum, sat. aq. sol 5 Carbol-fuchsin 5 Heat on cover until steam rises; keep at this temperature I minute ; then wash, dry, and stain. (2) Stain: Alum, sat. (cold) aq. sol 10 Gentian violet, sat. ale. sol 2 Kendall, in Journ. Applied Micr., Vol. V, 1902, p. 1836, says this has proved a very satis- factory stain to himself and his associates. Another formula is given as follows : (1) Saturated (cold) aqueous solution of alum 10 Saturated alcoholic solution of gentian violet i (2) Tannic acid (tannin) i Distilled water 10 A mixture of these two fluids is put on the flamed cover, which is held over the flame and gently heated until nearly ready to boil. The cover is then put aside for i minute, after which it is washed in water, dried, and mounted in bal- sam. If the mixed mordant-stain is filtered be- fore using, it is best to stain a second time for a moment in anilin-water gentian violet. 192 BACTERIA IN RELATION TO PLANT DISEASES. Lowit's I'lagclla Slain. Lowit's modification of Loeffler's flagella stain consists in substituting a copper-tannin mordant for ...... i iMMi-lannin. It is made as follows : ( i I .Mordant: Distilled water .................... 10 Tannin .......................... 2.5 Dissolve and filter through two thicknesses of filter paper, then add: Saturated solution copper sulphate... 5 Saturated ale. sol. basic fucbsin ...... I Filter as before. Covers are exposed to this mordant 20 seconds to 3 minutes without heating. Wash thor- oughly. (2) Stain: Expose cover to Elirlich's anilin u.iter gentian violet i to 5 minutes. Wash thor- oughly in water, and it this is not sufficient plunge for a moment into 50 per cent alcohol or into acid alcohol ( i drop of 0.3 per cent HC1 alcohol in 3 to 4 cc. of 60 per cent alcohol). The mordant and stain must be made up each time. If the mordant has been in use for some hours so that an oxidation film has formed on its sur- face, it is well I" stop and remove this by filtra- tion. ,S'i /irro'.v r/u^ella Stain. Sclavo exposes some minutes in the mordant (tannin i, water 50, alcohol 50) ; washes in uq dest. ; exposes some minutes in 50 per cent phospho-tungstic acid: washes carefully in aq. dest. ; stains 3 to 5 minutes in gently warmed anilin-water fuchsin ; washes, dries, and mounts in Canada balsam Some kinds are not stained by tins method. I^i-.chiU's Flagella Slain. This author used two solutions made as fol- lows : ( i ) Orcein ........................ I \bsolute alcohol ................ 50 Distilled water .................. 40 (2) Tannic acid (tannin) ........... 8 Distilled water (hot) ........... 40 Equal parts of ( D and (2) are mixed and filtered. Bacteria from a fresh agar culture are suspended in boiled distilled water. This sus- pension is allowed to stand 5 minutes. Drops are taken and spread and dried on clean covers, which are then taken in the fingers and fixed over the llame. They are now floated, film down. on the mordant (gently warmed) for from 10 to 15 minutes, then washed in water and dried. Elirlich's anilin-water gentian violet is now dropped on the cover, and this is heated over the flame until steam appears. The preparation is then washed, dried, and mounted in xylol balsam. Subsequently Bowhill modified the above as follows, using the orccin itself as a stain : (1) 'Saturated solution of orcein (al- lowed to ripen about 10 days). (2) 20 per cent solution of tannin dis- solved in hot water. The stain is prepared by taking of No. I, 15 cc. : No. 2, 10 cc. : and distilled water. 30 cc. After mixing, the fluid should be filtered. Sub- M'i|ncnt bleaching of the preparation should be avoided. (Hyg. Rundsch., VIII Jahrg., 1898, pp. II and 105.) llintcrbcrgcr's Method for Flagel/a. ('See 'oo Hinterberger, Bibliog., XII.) Ni.^lil Blue Stain fur Flagclla. ('See '(jo. Morton. Bibliog., XII.) w's Method for Flagella. Zettnow fixes with formalin, mordants with tartrate of antimony and tannin, and stains with gold or silver. The fixing is done by taking bacteria from a fresh agar or bouillon culture and adding them to water. They are then killed by the addition of 4 per cent formalin. The fixed bacteria set- tle after a day or two. when the sediment con- taining them is pipetted out and washed, first in i per cent formalin water and finally in pure water. The cloudy water is spread and dried on clean covers, and when these have been fixed by gentle heat they are ready for the mordant. The mordant is made as folows : ( i ) Tannin ........................ 5 Distilled water .................. too Flask and heat to 35° or 40° C. in the water- bath. (2) A solution of tartrate of antimony (i gram dissolved in water in a test-tube) is added drop by drop to solution ( i ). with shaking until the precipitate which forms is not redissolved. It is then filtered. The filtered mordant should be strongly opalescent, but not cloudy or opaque to transmitted light. This is said to be a perma- nent universal mordant, and one which does not cause precipitates on the cover. It is used hot (70° to 80° C.) for 5 or 10 minutes. The cover s then washed and gilded or silvered. After- ward the image may be intensified if desired. FLAGELLA STAINS. 193 The gold method consists of placing on the mordanted cover 4 or 5 drops of an aqueous solution ill nentr.-il gold cliloride (I :2OOO). This is then heated until steam is given off freely. If the mordanting has been sufficient there will be a deposit of metallic gold on the bacteria. Hugh U'illia ins' Method. (Copied from Mallory & Wright's Patholog- ical Technique.) This is a modification of van Ermengem's method along the lines of the modification of Hinterberger and others. It has he-en adopted by Dr. Hugh Williams after a large experience \\itli various methods in the laboratory of the Ma-.-aclm*ctts General Hospital. The method is capable of giving black bac- teria and flagella, with little or no precipitate. The method is as follows : (i) Cover the cover-glass with a mordant con- sisting of — • Alumnol,* I per cent solution... I part Osmic acid, 2 per cent solution. . I part Tannin, 20 per cent solution 3 parts Shake the mixture add three drops of glacial acetic acid, and again shake. i _• i Apply the mordant less than one minute without heating. Wash thoroughly in water. (3) Cover the preparation, during about t minute, with a t per cent solution of silver ni- trate to which sufficient ammonium hydroxid has been added to keep the silver in solution. | l 4 ) Wash in water. ( 5 ) Wa^li with 0.6 per cent solution of sodium chlorid. (6) Flood the preparation with a 30 per cent solution of ammonium hydroxid, and imme- diately wash in water. (/) Apply a few drops of Ortol photographic developer. (The directions for making tip this developer come with the Ortol,) (8) Wash in water. (9) Cover with a I per cent solution of gold chlorid during a few seconds. (10) Wash in water and apply Ortol developer for a few seconds. (n) Wash in water and cover with a I per cent solution of mercuric chlorid for a lew sec- onds. (12) Wash in water. (13) Apply Ortol developer ;or a few seconds. ( 14) Wash in water and repeat the application of chlorid of gold, the washing, and the applica- tion of the developer two or more times. Be- iween the various applications of the chlorid of gold the preparation should be inspected with a high, dry lens to determine the progress of the staining. This is readily done by placing the cover-glass, charged side upward, on a slide. In this way the process of impregnation with gold may be controlled; for the flagella, if stained, may be easily seen with the high-power dry lens. The preparation is very conveniently held dur- ing the process in cover-glass forceps. The washing is best done in a small stream of water from a faucet. The various solutions are con- veniently applied from dropping-bottles. It will be seen that the process consists essen- tially in the impregnation of the flagella with silver, followed by intensification, in the photo- graphic sense, with mercury and gold. The ob- ject of the application of the sodium chlorid and ammonia is to remove the excess of silver com- pounds which adhere to the surface of the cover- glass in spite of washing. This excess of silver compounds is chiefly responsible for the precipi- tates which appear on the preparation after the intensification. In spite of the application of the sodium chlorid and ammonia solutions, some precipitate will occur if the intensification is pushed too far. On this acount it is advisable to observe the progress of the intensification under the microscope, as above indicated. Although this method may appear compli- cated, in practice it requires but a few minutes to stain a preparation. IhickisuU's Method. Streaks are made on 2 per cent agar in Petri dishes from young growths in bouillon. Sus- pensions are made in water according to nature of organism, the motile bacteria being divided into six groups for staining purposes. Pigments and slime are removed by shaking with chloro- form. Cover-glasses must be absolutely clean. Mordant miM be ii'-ed only when fresh; dye must be fre^h and used warm (LoerHer's stain) ; streaks on cover-;:. ass should not be confluent. Fix without injury to flagella; stain without overheating; wash in alcohol and water without breaking film ; clear in xylol ; mount in xylol balsam without previous examination. For de- tails respecting method of making suspensions see The Canner and Dried Fruit Packer. Vol. XX. Feb. 16, 1005, p. 23. *Farbwerke vorm. Meister Lucius u. Briining, Hochst a. M., Germany. fWorkers iu the Bureau of Animal Industry U. S. Department of Agriculture state that too great an excess of ammonia in the silver nitrate may interfere with the working of the method. 194 BACTERIA IN RELATION TO PLANT DISEASES. CAPSULE STAINS. Ribbert's Method of Staining Capsules. Water i°° Alcohol 50 Glacial acetic acid 12.5 Warm and add dahlia to saturation. The covers are barely touched to this stain, and are then washed in water. The cover may then be mounted in glycerin or balsam. The stain keeps well. If the cover is left on the stain too long the capsule becomes deep blue and can not be distinguished from the body of the organism. Friedlaender's Capsule Stain. (See '85 Friedlacnder, Bibliog., XIII.) Richard Mnir's Capsule Stain. (1) 'Mordant: Mercuric chloride (sat. aq. sol.) 2 Tannin (20 per cent in water) 2 Potash alum (sat. aq. sol.) 5 The dried films are mordanted 2 minutes. They are then washed in water, in alcohol, and again in water. (2) Stain : Carbol-fuchsin 2 to 3 minutes, with gentle heat. Wash with water ; re-mordant 2 to 3 minutes ; wash again with water. (3) Counter-stain : Methylene blue (sat. aq. sol.) 2 minutes. Bleach in methyl alcohol, clear in xylol. Welch's Capsule Stain. Fix in glacial acetic acid. After a few sec- onds pour off the acid and flood with anilin- water gentian violet; repeat this operation until all acid is removed ; wash and examine in salt solution (0.85 to 2 per cent). Kaufmann's Method. Stain 2 hours at 35° C. in Loeffler's methylene blue; wash in water containing caustic potash or soda (i : 1500), dry; expose 2 minutes in l/2 per cent silver nitrate ; wash again in the alka- line water; counterstain 30 seconds in basic fuchsin water (i cc. sat. ale. sol. in 20 cc. aq. dest.) ; expose again to the alkaline water (sec- onds) ; dry and mount. Best adapted to demon- stration of capsules in fresh tissues. The bac- terial body is blue and the capsule red. Moore's Night-blue Capsule Stain. (See '99 Moore, Bibliog., XIII.) Buni's Method. Mix white of I egg, glycerin 50 cc., formalin 2 drops; shake well and filter. The bacteria are placed in a drop of this fluid, spread, and heated until the glycerin has entirely evaporated. -Stain 20 to 30 seconds in carbol-fuchsin, wash in water, dry and counterstain 4 to 6 minutes in Loeffler's methylene blue, wash in water, dry, and mount in Canada balsam. The background is red, body of organism blue, and periphery colorless. (See 'oo Boni, Bibliog., XIII.) SPORE STAINS. Hauser's Spore Stain. Pass cover-glass quickly three times through flame. Drop on carbol-fuchsin and heat for 5 minutes over flame, renewing the stain as it boils away. Nearly decolorize in dilute sulphuric or acetic acid (5 per cent). Wash very thoroughly. Counterstain with a dilute watery solution of methylene blue or with Loeffler's methylene blue (Festschrift fiir Zenker). Mailer's Spore Stain. (See '91 Moeller, Bibliog., XI.) Neisser's Spore Stain. The cover is floated on hot anilin-fuchsin for an hour. The temperature should be near the boiling point. The cover-glass is then washed in water and decolorized in acid alcohol (i part hydrochloric acid, 3 parts alcohol). Care must be taken not to expose to the acid alcohol too long; otherwise the color will be removed from the spores also. The cover is now stained for contrast in a saturated aqueous solution of methylene blue. Fiocca's Spore Stain. The prepared cover is placed in a watch-glass or test-tube containing 20 cc. of 10 per cent am- monia (water solution) and 10 to 20 drops of alkalin methylene blue or other alkalin solu- tion of anilin color. Then the fluid is heated to the giving off of steam and left for 3 to 15 minutes. It is now passed for a moment (?) through 20 per cent nitric or sulphuric acid, then thoroughly washed in water and stained for con- trast, if desired, in an aqueous solution of vesu- vin, malachite green, or safranin (saturated?). (See '93, Fiocca, Bibliog., XI.) (For other methods, e. g., Foth's, Klein's, Aujeszky's, see Bibliog., XI.) Ki iK 195 NON-SYNTHETIC CULTURE MEDIA. .Standard Peptonized Beef-Bouillon. Standard peptonized beef-bouillon is made as follows: To 500 grams of finely minced lean beef add 1,000 cc. of distilled water. The solu- ble parts may be removed from the meat by al- lowing the water to stand on it for 24 hours in the ice-chest or for I hour in the water bath at 55° C. The writer prefers the second method. Then boil for 60 minutes either in the steamer or in a covered dish. Filter through a clean cloth, using pressure (meat-press), cool, and remove fat by filtering through S. & S. filter paper ; make up to 1,000 cc. by addition of more water; then add I per cent Witte's peptonum siccum and 0.5 per cent c. p. sodium chloride. Steam one-half hour, filter, cool, titrate, add re- quired alkali, steam again for one-half hour, filter, pipette into tubes or flasks, and autoclave or heat for a minimum time in the steamer. Plugs should be well made and fit tightly; glassware should be scrupulously clean. For some purposes both the peptone and the salt may be omitted. A greenish bouillon indicates insufficient boiling, and will usually throw down some additional vexatious precipitate when heated in the test-tubes. Other meats may be substituted for beef, and other peptones for Witte's. Meat-extracts are not recommended. Such extracts usually contain resistant spores. Media which have been steamed, or boiled in an open dish, are better for many bacteria than those which have been sterilized in the autoclave. (For additional observations on proper steril- ization see Culture Media, p. 29.) Dunham's Solution. Distilled water 1,000 Witte's peptonum siccum 10 C. P. sodium chloride First recommended by Dr. Ed. K. Dunham, of New York. Standard Nutrient Agar. To 1,000 cc. of standard beef-bouillon add 10 grams of agar-flour, steam one-half hour, cool to 58° C. ; add whites of two eggs (beaten thor- oughly and neutralized to litmus by dilute hy- drochloric acid) and thoroughly mix with the bouillon ; steam I hour, filter hot through S. & S. paper which has been thoroughly warmed with boiling distilled water. Use two or three fun- nels. That which remains unfiltered after ; reasonable time must be reheated and put through a fresh filter paper. Sometimes all can be got through a second filter paper without re- heating. Some advise filtering in the autoclave or in the steamer, but the writer has not found that necessary, and in recent years has also abandoned the hot-water funnel. Clear agar may be obtained also by filtering through absorbent cotton, and some prefer this to filter paper. In preparing agar from the " slender kanten " or the " square kanten," snip fine, soak in the bouillon 15 minutes, and then heat on the sand bath I hour at 110° C. or in the autoclave 45 minutes at 105° C. From this point proceed as before. Long heatings in the autoclave at 110° C., or shorter heatings at higher temperatures, are apt to brown the agar, and should be avoided care- fully, as this renders the medium less service- able for the growth of bacteria. Agar which has been properly superheated filters readily. One per cent agar made from the agar-flour does not require to be heated on the sand-bath or in the autoclave, but filters satisfactorily after steam- ing for an hour at 100° C. After the agar has been tubed it may be ster- ilized, if it does not contain sugars, by one steaming in the autoclave for 10 minutes at 110° C., or by short steamings in the steam sterilizer at 100° C. on three successive days. To those who are dependent on the agar-strips and do not have access to an autoclave, Schutz's method may be recommended as very good. The writer formerly made large use of this. It consists in heating the agar very hot in a mini- mum quantity of water or bouillon before add- ing the bulk of the fluid. (See p. 34 and Bib- liog., XVI.) Agar which has been soaked in 5 per cent acetic acid for an hour or two before adding it to the bouillon also enters into solution thor- oughly and filters well after a short boiling. The acid must first be removed completely by wash- ing in running water for some hours under a mosquito-net or a piece of gauze. Unfiltered agar does well enough for certain fungi, and for lazy people, but the agar used for delicate work in bacteriology should be as clear as the bouillon from which it is made., i. c., per- fectly free from cloudiness and precipitates. Sufficient caustic soda is usually added to the agar to render it + IS of Fuller's scale. Other degrees are useful, e. g., + 10, o, — 10, etc. BACTERIA IN RELATION TO PLANT DISEASES. Litmus Lactose .-/«ar. To 1,000 cc. of ordinary agar, preferably that made up with bouillon free from muscle-sugar, add 10 grams of c. p. lactose and 20 cc. of a sat- urated (water) solution of c. p. (lime-free) hlue litmus. Hesse anil Xiei/uer's Nutrient Agar fur H'atcr Bacteria. Distilled water 980 Xahrstoff Heyden (an alhumose) 7.5 Agar-agar 1.2.5 This agar is said to he the most suitable me- dium for the bacteriological examination of water. It gives a much larger number of colo- nies than ordinary agar. It requires no neu- tralizing. The poured plates are counted, ac- cording to Dr. Robin, on the gth or loth day. Chromogcnic species are brilliantly colored. (Zeitschr. f. Hygiene. Bd. XXIX, pp. 454-462. See also Am. Jour. Pliarm., Vol. LXXV1, p. [12.) (,7y, erin . l^ar. To each 1,000 cc. of ordinary agar add 50 cc. of Schering's c. p. twice-distilled glycerin. Standard Xulrient Gelatin. To 1,000 cc. of sterile standard pcptonized href-bouillon add 100 grams of best quality gela- tin. Soak 2 hours at room-temperature. Then strain 5 minutes, cool, titrate, add the necessary alkali, steam 30 minutes, lilter through S. & S. paper washed with sterile boiling hot water, tube at once, and heat in the steamer on three suc- cessive days 15 minutes, 10 minutes, and 5 min- utes, respectively, at 100° C. Do not autoclave, and carefully avoid long heatings in the steamer. Have all the glassware sterile and the fluids sterile ami sufficiently boiled to begin with. Very acid gelatin should be avoided. The very best English, French, and German gelatins should be used. + 10 or + 15 is a good degree of alkalinity for many purposes. Zero of Ful- ler's scale is also useful. See remarks on gela- tin (p. 30). Blond Serum. (See p. 48.) I'lii ut Juices. (See p. 41.) .Vi//i(/ I'e^etal'le Su/'s/aiices. i See page jg > Milk. See i. 4'' ) Litmus Milk. Litmus milk is prepared from fresh milk which has been passed through a separator (cen- trifuge), or from milk which has stood 18 or 20 hours at 20° C. and has had the cream removed by skimming and filtration. To each too cc. of this milk is added 2 cc. of a saturated solution of high-grade, lime-free, blue litmus (litmus I gram, water 15 cc. ). This gives a lavender color of just the right degree, which reddens distinctly under the action of acids, and blues with the development of alkalies. The milk selected should not titrate more than + 16 with phe- nolphthalein and caustic soda. A good quality often gives + 10 to + 14. High readings de- note the excessive multiplication of lactic acid bacteria. .Such milks frequently coagulate on steaming, and are not suitable for culture-media. After adding the litmus water the milk should be pipetted in 10 cc. portions into cotton-plugged test-tubes and heated in streaming steam (100° C.) for 15 minutes on'each of 4 successive days. This is a very useful culture medium. Every organism should be tested in it. All milk used for culture media should be ceutrifuged, if pos- sible, immediately after milking, and secured a I once for the laboratory. Three steamings are- then sufficient. Milk offered for sale in cities is frequently more than 48 hours old and often con- tains from 3,000.0000 to 6,000.000 bacteria per cubic centimeter. Such milk is not fit for labo- ratory use. X'litrient Starch Jelly. The writer makes this as follows: To 10 cc. portions of modified Uschinsky's solution, or of the ordinary solution (glycerin omitted or not, as desired), is added I gram of clean aseptic potato starch. This is rubbed up in the slanted Hind. The test-tubes are then very tightly plugged to avoid loss of water and placed carefully in a blood-serum oven or in the top of an Arnold steam sterilizer with the vents open, where they are heated for 2 hours on each of 5 successive da\s at 85° C. to 93° C. If water is lost during the heating it must be made up, using a sterile pipette. Potato starch is prepared in the labora- tory (p. 50) with care in the washing and dry- ing, so as to avoid retention of other substances than starch and the multiplication of resistant ( spore-bearing) bacteria, which interfere with the sterilization. (See Pruc. Am. Asso. Adv. Sci.. 1898. Vol. XLVII, p. 411.) HiRMUL.F.. 197 SYNTHETIC CULTURE MEDIA. I'aslcttijs Culture /'liiiil. Ammonuiin tarlrate 10 Ashes iif vast 10 Rock candy 100 Distilled water 1,000 1 >isso]\ e cold. Naegeli's Nutrient Solution. Calcium chloride O.I Magnesium sulphate .2 Dipotassium phosphate.. i.o Ammonium tartrate 10.0 Distilled water 1,000.0 ( \>lin's .\'nti'ic:il Solution. Distilled water 1,000.0 Acid potassium phosphate 5.0 Magnesium sulphate 5.0 Neutral ammonium tartrate IO.O Potassium chloride 0.5 (De Bary, p. 86, Vorles. ii. Buet, 2 Auflage.) Ra itlin's Culture-Flu id. Distilled water 1,500.00 Granulated cane sugar 70.00 Tartaric acid 4.00 Ammonium nitrate 4.00 Ammonium phosphate .60 Potassium carbonate .60 Magnesium carbonate .40 Ammonium sulphate .25 Zinc sulphate .07 Ferrous sulphate .07 Potassium silicate .07 Prazmowski's Culture-Fluid. Dipotassium phosphate 5.0 Magnesium sulphate 5.0 Ammonium carbonate 5-O Calcium chloride -5 Distilled water 1,000.0 Dissolve cold. Any desired sugar may be added for the carbon food. .!lnti>n. Distilled water 1,000 Glycerin 30 to 40 Sodium chloride 5 to 7 Calcium chloride o.i Magnesium sulphate 0.3100.4 Dipotassium phosphate 2 to 2.5 Ammonium lactate 6 to 7 Sodium asparaginate 3 to 4 Mudifici/ Uschinsky's Sulniinn. The modified Uscliinsky recommended by the writer for use with starch jelly is made as fol- lows : Distilled water I.OOO.OOO Ammonium lactate 5.000 Sodium asparaginate 2.500 Sodium sulphate 2.500 Sodium chloride 2.500 Dipotassium phosphate 2.500 Calcium chloride .010 Magnesium sulphate .010 Fraenkel tunl fuses' S iln/imi. (Hygienische Rundschau, Bd. IV, 1894, p. 769.) Water i.ooo Sodium chloride 5 Dipotassium phosphate* 2 Ammonium lactate 6 Sodium asparaginate 4 This paper also discusses I'sdiinsky's solution. Fermi's Culture-Fluid. Distilled water i.ooo.o Magnesium sulphate .2 Acid potassium phosphate I.o Ammonium phosphate IO.O Glycerin 45.0 This may be added to agar in place of pepton- ized beef-broth (De Schweinitz) or to silicate jelly, in which case the volume of water must be reduced (see Silicate Jelly, p. 36). Mimre's Culture-Medium fur Leguminous Root- tnhcrclc I-iiicilli. (For field use.) The dried culture (on cotton) is thrown into clean water containing : Cane-sugar, I ; c. p. monopotassium phosphate', o.i ; c. p. magnesium sulphate, o.oi per cent. After 24 hours add Merck's pure dibasic ammonium phosphate to amount of 0.5 per cent. Seeds are drenched with this fluid at end of another day, dried in shade, and planted. *I,e!imanu aud Neumann recommend neutral commercial sodium phosphate ('. c. p. 29.) 198 BACTERIA IN RELATION TO PLANT DISEASES. Maasscn's Culture-Fluid. Malic acid 7 Distilled water 100 Neutralize to litmus exactly with ^ per cent potassium hydrate. 'Make up to 1,000 cc. with distilled water and add : Asparagin 10.0 Secondary sodium phosphate 5.0 Magnesium sulphate 2.5 Sodium hydrate 2.5 When dissolved add o.oi gram of calcium chloride. To this may then be added grape-sugar or any other carbon food desired. Proskauer and Beck's Culture-Fluid. Distilled water 1,000.00 Commercial ammonium car- bonate 3-5° Primary potassium phosphate.. 1.50 Magnesium sulphate 2.50 Glycerin i5-°o Mackcnsie's Culture-Fluid. Acid ammonium tartrate. Bipotassium phosphate... Potassium sulphate Sodium chloride Glucose Lactose Glycerin Water . 1.5- 2.5 i.5 •5 5-0 5-0 iS-O 1,000.0 This is rendered alkalin to phenolphthalein with normal soda solution. Culture-Medium for Luminous Bacteria. (Molisch., /. c. p. 87.) Water 1,000.000 Gelatin 100.000 Sugar 20.000 Peptone 10.000 Dipotassium phosphate .250 Magnesium sulphate .250 Enough sodium hydroxid to render the me- dium feebly alkalin. On this substratum the bacteria grow feebly and are not luminous until sodium chloride or some equivalent substance is added (usually 3 per cent). Then they grow well and become luminous. Winogradsky-Sleskin Silicate Jelly. Ammonium sulphate 0.40 Magnesium sulphate 05 Potassium phosphate .10 Sodium carbonate 0.60- .90 Calcium chloride Trace Silicate jelly 100.00 The salts are dissolved separately in the least possible water, and added to the dialyzed acid. (For further observations on silicate jelly see p. 36.) Nitrogen-free Medium for Bacteria. Triple-distilled water 1,000.000 Cane-sugar 5.000 Monopotassium phosphate. . . . 2.000 Magnesium sulphate .100 Sodium chloride .500 All chemically pure, in scrupulously clean flasks. The water should be freshly distilled, kept in glass-stoppered bottles, and tested fre- quently with Nessler's solution for presence of ammonia. Giltay &• Aberson's Culture-Medium for Deni- trifying, Organisms. Distilled water, t 1,000.0 Potassium nitrate 2.0 Asparagin i.o Magnesium sulphate 2.0 Citric acid 5.0 Monopotassium phosphate 2.0 'Calcium chloride .2 Ferric chli >riik> 2 gtts. The acid should be neutralized by the addition of potassium hydrate. This medium is a modification of that of Gayon and Dupetit, less nitrate being used and the neutralization being made with potash in- stead of ammonia. In preparing this fluid the asparagin and the nitrate of potash are dissolved in 250 cc. of water ; the other substances are dis- solved in 500 cc. of water, and after the citric acid has been neutralized the two fluids arc mixed, cooled to 15° C., and sufficient water added to make I liter. When the nitrate of potash and the asparagin are dissolved along with the other salts a decomposition occurs, and the liquid is browned from the presence of nitrous acid, which should be avoided. Some carbonate of lime is also added to the culture fluid. Instead of asparagin, 2 grams of dextrose SYNTHETIC CULTURE MEDIA. 199 may be added. If the latter is used the fluid must not contain the least excess of potassa; otherwise when it is sterilized there will be more or less humification. (Recherches sur un mode de denitrific.it ion, etc. Archives neerlandaises des Sci. Ex. et Nat, Tome XXV, 1892, pp 341- 361.) Winogradsky's Culture-Medium for Nitrogen- Assimilating Soil-Bacteria. Twice-distilled water 1,000.00 Potassium phosphate i.oo Magnesium sulphate 0.50 Sodium chloride 01 to 0.02 Iron sulphate 01 to .02 Manganese sulphate 01 to .02 Dextrose 2o 1040 To this should be added a small quantity of pure calcium carbonate, 30 or 40 grams per liter is sufficient. The carbonate is freshly washed in boiling water and added in paste or dried rapidly and preserved in flasks with ground- glass stoppers. It is recommended that the sec- ond distillation of the water be made with car- bonate of soda and that pure salts be obtained by repeated crystallizations. It is probable that monopotassium phosphate is meant by fliosphat de fotasse. (Recherches sur 1'assimilation de 1'azote libre de I'atmosphere par les microbes, Archives des Science Biologiques, Tome III, p. 304, St. Petersburg, 1895.) Bcycrinck's Agar for Cultivation of the Nitrite Bacteria. Ordinary agar is added to distilled water, heated until it passes into solution, and poured into Erlenmeyer's flasks, where it is left to solidify. When cold the flasks are filled with distilled water (not necessarily sterile) and set away. After several changes of water and the lapse of one or two weeks the soluble organic substances will have been absorbed out of the agar, and to it may now be added the inorganic nutrient substances desired, after which it is sterilized. Along with the nutrient substances some pure precipitated calcium carbonate should be added. The sterile agar may then be solidi- fied in Petri dishes, test-tubes, etc. Beyerinck considers this medium better for isolation of the nitrite ferment than the silicate jelly. Hydro- gen ammonium sodium phosphate (NH, Na- HPCX-KH-O) is recommended as the best one nf the ammonium salts for addition to the agar, because, upon cooking, the agar is not attacked, and consequently additional soluble substances are not liberated from it. (Beyerinck: Kultur- versuche mit Amoben auf festem Substrate. Centralb. f. Bakt., i Abt, Bd. XIX, 1896, pp 257-267.) Winogradsky's Nutrient Agar for Isolation of Nitrate Bacteria. Tap-water T,ooo Agar (thoroughly washed) 15 Di-potassium phosphate 0.05 Fused sodium carbonate i Sodium nitrite (Merck) 2 (Centralb. f. Bakt, 2 Abt, Bd. V, 1899, pp. 537-549-) Winogradsky & Omclianski's Fluid Culture- Medium for Isolating the Nitrate Bacteria from Soils. Distilled water 1,000.0 Magnesium sulphate .3 Ferrous sulphate .4 Sodium chloride .5 Di-potassium phosphate .5 Fused sodium carbonate i.o Sodium nitrite (Merck) i.o Transfers through a series of flasks are neces- sary in order to isolate the organism. (Cen- tralb. f. Bakt, 2 Abt, Bd. V, 1899, pp. 537-549.) Winogradsky & Omclianski's Fluid Culture- Medium for Isolating the Nitrite Bacteria from Soils. Distilled water 1,000.0 Ferrous sulphate .4 Magnesium sulphate .5 Di-potassium phosphate i.o Sodium chloride 2.0 Ammonium sulphate 2.0 The cultures are made in broad-bottomed flasks in 50 cubic centimeters of the fluid, to each of which about Y2 gram of magnesium car- bonate is added. It is necessary to transfer through a series of flasks in order to obtain pure cultures. If the organism does not grow- well on the start, additional ammonium sul- phate may be added, viz, I cubic centimeter of a 10 per cent solution to each flask. (Centralb. f. Bakt, 2 Abt, Bd. V, 1899, pp. 537-549.) Dubois' Fluid Medium for Luminous Bacteria. (See '93 Dubois, Bibliog, XXVII.) Kuntze's Medium for Bacillus Prodigiosus. (See 'oo Kuntze, Bibliog, XXIII.) 2OO BACTERIA IN RELATION TO PLANT DISEASES. Omelianski's Magnesia-Gypsum Blocks fur Hit- Cultivation of Nitrifying Organisms. One per cent carbonate of magnesia is uni- formly mixed wiih gypsum and water added to it, stirring until it becomes of the consistency of sour cream, when it is poured upon plate- glass and spread out. As soon as the mass be- comes of a doughy consistency and is ready to harden, it is cut into circular blocks for Petri dishes and into strips for test-tubes. The cir- cular pieces may be cut with a Petri dish of a size a little smaller than the dishes it is intended to use. As soon as the gypsum has hardened thoroughly the blocks are pried loose from the plate glass, placed bottom up in the dishes (so as to give a smooth surface), and enough of the above (nitrite) culture-medium added to half cover the block. This is then autoclaved and additional sterile culture-media added from time to time as necessary, being careful not to wet the inoculated surface of the block. The sowings are made on the smooth surface of the block and the dishes are kept in a thermostat at 25° to 30° C. Colonies begin to be visible in 4 to 5 days. In 10 to 14 days many colonies are 0.25 to 0.50 millimeter in diameter. (Cen- tralb. f. Bakt., 2 Abt., Bd. V, 1899, p. 652.) MISCELLANEOUS. Distilled Water. (See page 124.) Chromic Acid Cleaning Mixture. This is made by pouring I gallon or more of concentrated crude sulphuric acid into an equal volume of a saturated aqueous solution of po- tassium bichromate. It should be done in a large enameled iron kettle, the acid being added slowly at intervals, with frequent stirring, so as to keep the mixture below the boiling point. An excess of the sulphuric acid should be avoided. Pure water should be used for dissolving the potassium bichromate, and under no circum- stances should this solution be poured into the acid, since steam might be generated and dan- gerous splutterings occur. The resulting chromic acid is very injurious to the skin and should be used with care. At 15° C. each 10 parts by weight of water will dissolve about i part of the potassium salt. The chromic acid mixture is said to explode violently when brought into con- tact with certain substances, c. g., alcohol, gly- cerin. rinid for Softening Hard Tissues. Frequently grains of cereals and other hard tissues may be softened for cutting on the microtome with slant stroke by soaking from .1 to 6 months in equal parts of alcohol and glycerin. Unguentum rcsinai-. (See Bibliog., XVII, 'oo, Bulloch.) Danvin's N'a.r-Mixlurc. This consists of vast-line 50 parts, beeswax 35 parts, melted together. Then stir in of pow- dered resin 15 parts. If a stiffer mixture is de- sired, add more wax up to 50 parts (see Darwin & Acton, Plant Physiology, p. 3, foot note). Pencils for Writing on Glass. (See page in.) Pyrogallol Developer. (Much used in Laboratory of Plant Pathol- ogy.) (1) Alkali: Carbonate of potash (cry st.)* I Bounces, or 46.5 grams Carbonate of soda (cryst.)* 2 ounces, or 62 grams Distilled water I2'< ounces, or 375 cc. (2) Pyro : Sulphite of soda (cryst.)*. ... 4 ounces, or 124 grams Citric acid 60 grains, or 3.9 grams Bromide of potash.. 40 grains, or 2.6 grams Distilled water i2l/2 ounces, or 375 cc. Pyrogallic acid i ounce, or 31 grams The pyrogallol should be added last of all, and the nearly filled bottle closed at once. For a normal developer take 2 drams of No. I, add 2 drams of No. 2, and make up to 4 ounces with distilled water. Reduce the amount of al- kali to one-fourth dram or less in case of much overexposed plates. In case of exposures likely to exhibit too great contrasts i educe the pyro. Always begin development with one-fourth of the alkali, unless the exposure is known to be correct. Both solutions should be kept in glass- stoppered bottles. Bottles containing alkali should have the in- side of the neck and the ground surface of the stopper wiped dry before replacing; then the latter will not stick. 'In cas. anhy.hnn.. salts are employed, use oiie-lmlf as much. SYNTHETIC CULTURE MEDIA. 2O I Ortol Developer. (See pp. 140-141.) Pyro Developer for Dry Plates. (Recommended by S. G. Lofft.) (1) Water xooz., or 300 cc. Citric acid 10 grains, or 6.46 grams Pyrogallic acid... i oz., or 31 grams (2) Sodium sulphite (crystals) 4oz., or 124 grams Water 16 oz., or 480 cc. Or Seed's sulphite. ...I'/^oz., or 46.5 grams Water i6oz., or 480 cc. (3) Sodium carbonate (crystals) 4 oz., or 124 grams Water 16 oz., or 480 cc. Or Seed's carbonate.. 202., or 62 grams Water i6oz., or 480 cc. To develop take — Water 4 oz., or 120 cc. No. i 2 drams, or 7.5 cc. No. 2 i^ oz., or 15 cc. No. 3 i^ oz., or 15 cc. For underexposures dilute and change fre- quently to fresh developer. For overexposures use old developer or re- strain with a few drops of 10 per cent potassium bromide. Lantern-slide Developer. (Used in Laboratory of Plant Pathology.) Distilled water cc. . goo Carbonate of soda (cryst.) grams.. 39 Sulphite of soda (cryst.) . ..grams. . 39 Hydrochinon grams.. 13 Add the hydrochinon after solution of the soda salts, and put at once into a glass-stoppered bottle. For use take 3 ounces of above and 3 ounces of distilled water, to which add 5 drops of a 10 per cent solution of bromide of potas- sium. If properly exposed the image should appear in 30 to 60 seconds, and the development should be over in 3 or 4 minutes. Good for a dozen or more properly exposed slides. Zettiwvj's Copper-clirom-filter. Dry, pure, copper nitrate 160 Pure chromic acid 14 Distilled water 250 This may be diluted further with water if desired. In case there is difficulty in preparing the above, a solution, stated by Zettnow to be nearly as useful, consists of — Sulphate of copper 175 Bichromate of potash 17 Water 1,000 (Centralb. f. Bakt, IV Bd, 1888, p. 51.) Neuhaus says, water 500 to 1,000, and also 2 cubic centimeters of sulphuric acid. This so- lution extinguishes all the blue and violet rays. Toning Bath for Solio Paper. (A) Hyposulphite of soda... 8 ounces Potash alum (crystals).. 6 ounces Cane sugar 2 ounces Water So ounces Dissolve cold, then add 2 ounces of borax which has been dissolved in 8 oz. of hot water. Let stand 12 hours, and then decant. (B) Pure chloride of gold. . 7.5 grains Acetate of lead 64 grains Distilled water 8 ounces This solution must not be filtered and must be shaken thoroughly each time before using. Solio paper should be printed about one-third darker than it is desired to have the pictures. When the prints are ready they are placed face down in a toning mixture made of stock A, 8 ounces, and stock B, i ounce, taking care that the entire surface of each print is uniformly wetted. They are allowed to remain in this bath, with constant movement by means of the fingers, until the desired color is obtained (usu- ally about 5 minutes). The prints are now im- mersed in salt water (i 132) for 5 minutes. They are then exposed for 15 minutes to the fixing bath, consisting of — • Hyposulphite of soda I ounce Sulphite of soda (crystals) 60 grains Borax 54 ounce Water 20 ounces The prints are finally washed for from I to 2 hours in running cold water. The toning bath should not be cooler than 40° or warmer than 60° F. A New Test for Indol. Herter & Foster have recently described what is stated to be a rapid and accurate method of determining indol, adapted either for colorimet- ric or gravimetric determinations. To slightly alkaline solutions naphthoquinon sodium mono- sulfonate is added. A blue crystalline com- pound, di-indyl naphtho-ketone mono-sulfonate is produced. This is slightly soluble in water, but is readily soluble in chloroform, its solution being red. (Science, n. s., Vol. XXI, 1905, p. 987.) 2O2 BACTERIA IN RELATION TO PLANT DISEASES. FIXING FLUIDS. Absolute Alcohol. Expose 24 hours or more. Very useful for fixing bacteria in tissues, as it prevents their diffusion. It causes, however, considerable shrinkage of the tissues, and the nuclei are often difficult to stain, and are usually distorted. Bacteria fixed in this way stain well in Ziehl's carbol-fuchsin. Picric Acid in Hot Absolute Alcohol. (See p. 8.) Mercuric Chloride in Hot Absolute Alcohol. (See p. 8.) Acetic Alcohol ivith Mercuric Chloride. Absolute alcohol i Glacial acetic acid i Chloroform I Add mercuric chloride until saturated. Wash with alcohol or with alcohol containing tincture of iodine. One of the most rapid fixatives known. Acetic Alcohol (Camay's Fluid.} Glacial acetic acid i Absolute alcohol 3 Expose 24 hours or more and wash in alcohol. This solution preserves the chromatic and cyto- plasmic structures better than alcohol alone, and shrinks the tissues much less. Tissues fixed in this solution take most of the coal-tar stains better also. It has very great penetrating power, which makes it very useful in fixing large pieces of tissue. Chromo-aceto-osmic Acid (Flciiuiiing's 1'Iitids}. A. Stronger solution — 1 per cent chromic acid 15 2 per cent osmic acid 4 Glacial acetic acid i B. Weaker solution — • I per cent chromic acid 25 i per cent osmic acid 10 i per cent acetic acid 10 Water 55 Expose 12 to 48 hours and wash in running \vaU-r. This is probably the most valuable of all the fixing fluids for preserving exactly all the cell structures ; and material fixed in it takes the coal-tar stains unusually well. It has very slight penetration, and therefore the tissues should be cut into pieces not more than 2 milli- meters thick. It should be kept in glass-stop- pered bottles, and should be made fresh every 6 months or less. For additional formula; consult the various standard text-books, especially Eyre's Bacteriological Technique," where may be found, among others, the following: Kitasato's glucose-formate-bouillon . Iron-bouillon. Lead-bouillon. Parietti's bouillon. Carbolized bouillon. Kitasato's glucose-formate-agar. Guarniari's agar-gelatin. Carbolized agar. Glycerine blood-serum. Heimau's serum-agar. Washbourn's blood-agar. Urine-gelatin. Whey-agar. Fish-gelatin. Urine-agar. Fish-bouillon. Fish -agar. Glycerinated potato. Glycerine-potato-broth . Eisner's potato-gelatin. Goadby's potato-gelatin. Beer-wort. Wort-gelatin. Wort-agar. Wine-must. Gasperini's wheat-broth. Bread-paste. Milk-rice. Fakes' iron-peptone-solution. MacConkey's bile-salt-broth. MacConkey's bile-salt-agar. Sabouraud's French proof agar. Blaxall's English proof agar. PLATE 24. Brown rot of the potato. Potato-tuber from Hastings, Florida, crop of 1905, sound externally, but brown-rotted in the vascular system, the bacteria having entered through the vessels of the underground stem. Bacterium solanacearum was plated out and subcultures were used to infect potted potato- plants. The plant shown was inoculated on June 27, by delicate needle-pricks. Disease more advanced on one inoculated choot than on the other; later this also shriveled. Tuber natural size; photographed May 29. Plant about one-fifth natural size; photographed July 15. 1905. PLATE 25. Early Rose potatoes destroyed by Bacterium solanacearum. Plants inoculated at same time, in same way, and from same source as plant shown in plate 24. About one-fourth natural size. Progress of disease rather slow ; no tubers were formed. All of the shoots dead or dying. Photographed August 3, 1 905, i. e. at end of fifth week. PLATE 26. Effect of Bacterium solanacearum on a tomato-plant. The left-hand plant was inoculated in the stem on July 5, by means of a few needle -pric Its. The right-hand plant was pricked with a sterile needle. The infectious material was a subculture from a poured-plate colony. This was obtained from the interior of a potato-stem, naturally infected, in District of Columbia. Photographed July 1 1 . 1904. About one-third natural size. PLATE 27. The Granville tobacco-wilt, a bacterial disease. The Left-hand plant milled naturally in the field, was pruned, transplanted to the hothouse, recovered for a few weeks, developed the leaves shown, and then wilted again. Photographed June 28; one-third natural size. In this disease the vascular ring is browned and cavities are formed in the stem. The bactenal slime is gray-white and abundant. Fungi were nol present in the plants examined by me. Poured plates were made from the interior of several such stems, and these yielded practically pure cultures of one organism. A subculture from one of these colonies was used to inoculate the right-hand plant. The needle-pricks were made on July 13, 1905. Photographed August 3; one-half natural size. Twelve plants were inoculated at ihis time and all contracted the disease. The signs and lesions were the same as in the plants obtained from the field. The organism causing this disease is closely related to Bact. solanacearum. PLATE 28. Pear-shoots blighted by Bacillus amylovorus. Inoculations by needle-pnckson rapidly growing stems. The infectious material came from a green apple. The poured plates yielded practically pure cultures and the inoculations were made directly from colonies tn these plales. Of 12 inoculations (8 shoots. 4 fruits) 1 I were successful. Needle-pricks made July 10. 1905. Photographed July 20. The lower leaves were still green ; the tops (A and A) had shriveled and browned ; the bacteria had passed downward in the bark to points below B and B, and they had also run out into the petioles (P and P) and had browned them, but the blades of these leaves were still green. PLATE 29. Green pears inoculated with Bacillus amylovorus. These were inoculated from the same poured plate and at the same time as the shoots shown in plate 28, but the photographs were made two days earlier. The inoculations were by means of a few needle -punctures. The inoculated parts browned, softened, shriveled slightly, and wereextruding bacterial slime from small cracks and from many stomata. Beyond the browned area there was a water- soaked area. The internal injury was extensive — in one fruit nearly the whole interior had softened and was occupied by the bacteria. These tissues were filled with grayish bacterial slime to such an enormous extent that on handling them the fingers dripped with it. Inoculated July 10. 1903, Photographed July 18; about natural size. Poured plates made from the interior of these fruits yielded pure cultures of B. amylovorus. PLATE 30. Blighted quince-shoots and pear-fruits. Twelve inoculations were made and all were successful — 8 quince-shoots and 4 pear-fruits. None of ihe checks contracted the disease. The organism was introduced by delicate needle-pncks. The subculture used was derived from a poured-plate colony obtained from the interior of one of the pears shown in plate 29. Owing, no doubt, to the riper and therefore less susceptible condition of the tissues, and possibly also to somewhat cooler weather, the disintegration of the deeper tissues of the fruits had not proceeded as far as in the pears inoculated on the same tree I 0 days earlier ; in those fruits first inoculated the decay was from 5 to 10 rimes as extensive on the 8th day as in the second inoculations on the Nth day. Inoculated July 20, 1905. Photographed July 31. PLATE 31. Small green apples blighted by Bacillus amylovorus. One of these fruits furnished the bactena used to inoculate the shoots and fruits shown in plates 28 and 29. The interior of these fruits was gorged with a gray-white bacterial slime. The surface was browned and shriveling in many places and water-soaked in others. Bacteria were oozing from the darker parts. Every one of many fruits examined had been wounded by the curculio, and probably infection occurred in this manner. Arlington Farm. Virginia. Photographed June 30. 1905. BIBLIOGRAPHY. General Literature. As far as possible the references in this bibliography have been arranged chronologically so that the reader may see at a glance the historic development of the subject. In several cases logic has been sacrificed to convenience, e. g., in keeping the nitrifying and denitrifying bacteria separate from the group devoted to oxidation and reduction, the average reader, it was thought, being more likely to search for references to the nitrogen bacteria under the former than under the latter heading. I. Journals. Archiv fur Hygiene. Munich and Leipsic. Verlag von R. Oldenbourg. Begun in 1883. 51 vols. to date. Zeitschrift fur wissenschaftliche Mikroskopie und fur mikroskopische Technik. Braunschweig. Harald Bruhn. Begun in 18 21 vols. to date. Zeitschrift fiir Hygiene und Infectionskrankheiten. Leipsic. Veil und Comp. Begun in 1886. 48 vols. to date. Annales de I'lnstitut Pasteur. Paris, G. Masson et Cie. Begun in i887. 18 vols. to date. Centralblatt fiir Bakteriologie, Parasitenkunde und Infektionskrankheiten. I Abt. Medicinisch-'hygi- enische Bakteriologie und tierische Parasitenkunde. Gustav Fischer, Jena. Begun in 1887 under the title, Ceutralb. f. Bakt., und Parasiteukunde. Title changed and scope limited when the following publication was begun. 37 vols. to date. Centralblatt fiir Bakteriologie, Parasitenkunde und Infektionskrankheiten. 2 Abt. Allgemeine land- wirtschaftlich-technologische Bakteriologie, Ga- rungsphysiologie, Pflanzenpathologie und Pflan- zenschutz. Gustav Fischer, Jena. Begun in 1895. 12 vols. to date. Annales de Micrographie, specialement consacrees a la Bacteriologie, etc. Paris, Geo. Carre et C. Naud. Begun in 1888. Zeitschrift fiir Pflanzenkrankheiten. Paul Sorauer, editor. Verlag von Eugen Ulmer, Stuttgart. Begun in iSgi. 11 vols. to date. Occasional articles only on bacteria. The Journal of Pathology and Bacteriology. Edin- liurgh and London. Young J. Pentland. Begun in 1892. 9 vols. to date. Journal of the American Public Health Association. The number for October, 1895, contains numerous valu- able papers on bacteriology. The number for October, 1807, contains report of a committee on the pollution of watersupplies; also report of committee on disinfectants. Report of meeting held October 3i-November 3, 1899. con- tains notes on the reaction at which the maximum growth of bacteria occurs in liquid media ; a paper on the making of nutrient agar-agar ; a paper on the classi- fication of water bacteria, etc. Tijdschrift over Plantenziekten. Onder redactie van Ritzema Bos en G. Staes. Gent. J. Vuylsteke. Begun in 1895. 7 parts have appeared. Only occasional papers on bacteria. The Journal of Experimental Medicine. New York, D. Appleton & Co. Begun in 1896. Edited by Wm. H. Welch, of Johns Hopkins University. 6 vols. to date. Contains many papers of interest to persons who are not physicians Recently transferred to the Rockefeller Institute under editorship of Simon Flexuer. Archives de Parasitologie. Paris, Geo. Carre et C. Naud. Begun in 1898. 8 vols. to date. Published since vol. 4 by Societe d'editious scientifiques. Journal of Applied Microscopy. Rochester, N. Y., Bausch and Lomb. Begun in 1898. 6 vols. Discontinued. Archives des Sciences Biologiques. Publiees par 1'Institut imperial de medecine experimentale a St. Petersbourg. Valuable. Begun in 1892. 10 volumes to date. The Botanical Gazette. University of Chicago Press. Begun privately in 1875. 38 vols. to date. Papers on bacteria only in recent volumes. Journal of Hygiene, Cambridge, England. Edited by Nuttall. Begun in 1901. Published quarterly by Cambridge Uni- versity Press, Warehouse Ave., Maria I.ane, London, E. C. 4 vols. to date. Journal of Medical Research. Edited by Harold C. Ernst, Boston, Mass. 7 vols. to date. This is a continuation of the Journal of the Boston Society of Medical Sciences. Zeitschrift fiir Physikalische Chemie, Stochiometrie und Verwandtschaftslehre. Herausgegeben von Wilh. Ostwald und J. H. Van't Hoff, Leipzig. Verlag von Wilhelm Engelmann. About 45 vols. to date. The Journal of Infectious Diseases. Founded by the Memorial Institute for Infectious diseases, Chi- cago. Begun in 1904. 203 204 BACTERIA IN RELATION TO PLANT DISEASES. Bulletin de 1'Institut Pasteur. Revues et analyses des travaux de microbiologie, medicine, biologic generale, physiologic, chimie biologique dans leurs rapports avec la bacteriologie. Comite de redac- tion : G. Bertrand, A. Besredka, A. Borrel, C. Dele- zenne. A. Marie, F. Mesnil, dp 1'Institut Pasteur de Paris. Begun iu 1903. II. Transactions, Beitrage, Jahresberichten, Festschriften, Etc. C/o-'92). Beitrage zur Biologie der Pflanzen. Edited by Ferdinand Cohn. 15 Heften. (5 vols.) Breslau, 1870-1892. ('86). Jahresbericbt uber die Fortschritte in der Lehre von den Pathogenen Mikroorganis- men umfassend Bakterien, Pilze und Pro- tozoen. Von P. Baumgarten. Braun- schweig, Harald Bruhn, and more recently Leipzig, S. Hirzel. Begun in 1886. First volume covers the year 1885. The last volume (iSJahrgang) brings the literature refer- ences down to the close of 1902- Contains nothing on plant parasites, and the title is therefore misleading. ("86). Arbeiten aus dem Kaiserlichen Gesundheits- amte. Berlin, Verlag von Julius Springer. Extremely valuable on account of the high quality of the papers, which deal mostly with animal diseases. The first two volumes were published as Mitteilungen, etc. Begun in 1886. ('92-'95). Beitrage zur Physiologie und Morphologic niederer Organismen. W. Zopf. 5 Heften. Leipsic, Arthur Felix, 1892-1895. ('93). The Wilder Quarter-Century Book. Ithaca, N. Y., Comstock Pub. Co., 1893. Contains several vahiable papers. Publications of the Division of Vegetable Physiology and Pathology, Bureau of Plant Industry, U. S. Department of Agriculture, Washing- ton, D. C. Frequently when editions are exhausted, copies may lie procured at actual cost of printing by addressing the JIm-caa of Doru rnents. Union Building, Washinilt'm, D. C. The publications of the Department of Agricul- ture are listed each month, and this list may be obtained gratis on application to the Secretary of Agriculture. ('94). KLEIN, L., UND MIGULA, W. Arbeiten aus dem bacteriologischen Institut der teclmischen Hochschule zu Karlsruhe. Bd. I, n, in, Wiesbaden, O. Nemnich, 1894-1903. ('03). Contributions to medical research dedicated to Victor Clarence Vaughan by colleagues and former students of the department of medi- cine and surgery of the University of Michi- gan on the twenty-fifth anniversary of his doctorate. Ann Arbor, George Wahr, 1903, pp. vi, 620, with numerous plates and figures. III. Manuals. ('80). WINTER, GEORG. Bacteria. In Die Pilze of Rabenhorst's Kryptogamenflora Deutsch- lands. i Abt., 1884. The volume was published in parts, the one on bacteria coming out at an early date ( 1880). Transl. by T. J. Burrill , as part of a paper entitled, " The Bacteria," Springfield, 111., 1882. nth Rep. 111. State Industrial Univ. ('83). DUCLAUX, E. Chimie biologique— Micro- biologie. Paris, 1883, pp. vn, 908. This forms Tome ix, first section, of Frem/s En- cyclopedic Chimique. ('84). DE BARY, ANTON. Vergleichende Morphologic und Biologie der Pilze Mycetozoen und Bac- terien. Leipzig, Engelmann, 1884, pp. xvi, 558. English translation by Garusey revised by Balfour in 1887 under title of " Comparative inorpholo'gy and biol- ogy of the fungi mycetozoa and bacteria." Oxford, Clar- endon Press. ('85). DE BARY, ANTON. Vorlesungen tiber Bacte- rk-n. Leipzig, Engelmann, 1885, pp. vi, 146. There is an English translation by Garnsey, revised by Balfour, Oxford, 1887. The second German edition was published in 1887. pp. vi, 158, 20 figs. A third edition revised by Migula, containing 186 pages and 41 figures, appeared in 1900. ('86?). SCHROETER, J. Schizomycetes. In Die Pilze Schlesiens, pp. 136-174. Breslau, J. U. Kern's Verlag. The volume as a whole bears date of 1889. ('87). LOEFFLER, FRIEDRICH. Vorlesungen uber die geschichtliche Entwickelung der Lehre von den Baoterien. Erster Theil bis zum Jahre, 1878, pp. xn, 252, with 3 plates and 37 text figures. Leipsic, F. C. W. Vogel, 1887. ('90). BAUMGARTEN, P. Lehrbuch der pathologischen Mykologie, pp. ix, 973, 24. Braunschweig, Harald Bruhn, 1890. ('90). CORNIL, A. V., ET BABES, V. Les bacteries et leur role dans 1'etiologie, 1'anatomie et 1'his- tologie pathologique des maladies infec- tieuses. 3d ed. 2 vols. Tome i, pp. vn, 582. Tome n, pp. 608. Paris, Felix Alcan, 1890. ('90). FRAENKEL, CARL. Grundriss der Bakterien- kunde. 3d ed. pp. vin, 515. Berlin, August Hirsclrwald, 1890. An excellent book, but now requires revision. There is an English translation by Linsley. New York : Wm. Wood & Co., 1891. ('90). KRAMER, ERNST. Die Bakteriologie in ihren Beziehungen zur Landwirtschaft und den landwirtschaftlich - teclmischen Gewerben. Theil i. Die in der Landwirtschaft durch Bakterien bewirkten Vorgange. Mil 36 Abbild. Wien, Verlag von Karl Gerolds Sohn, 1890. 8vo. 171 pp. it Theil. Die Bakterien in ihren Verhaltnisse zu den land- wirtschaftlichen-technischen Gewerben. 8vo. pp. vi, 178, with 79 figures. Vienna, Gerolds Sohn, 1891. ('91). EISENBERG, JAMES. Bakteriologische Diagnos- tik. 3d ed. pp. xxxi, 509. Hamburg and Leipsic, Leopold Voss, 1891. ('91). HUEPPE, FERDINAND. Die Methoden der Bakterien-Forschung. Handbuch der ge- sammten Methoden der Mikrobiologie. 5 verbesserte Aufl. Svo. pp. vin, 495, with 2 lithographic plates and 68 wood cuts. Wies- baden, C. W. Kreidel's Verlag, 1891. A French translation of an earlier edition, by Van Ermengem, entitled, Manuel technique de microbiolo- gie, was published at Paris in 1887. ('91). WOODHEAD, GERMAN SIMS. Bacteria and their products. London, Walter Scott, Ltd., 1891. I2mo. pp. xni, 459, with 20 photomicro- graphs. ('92). STERNBERG, GEO. M. Manual of Bacteriology, pp. xn, 886. 8 plates and 268 figures in the text. 8vo. New York, Wm. Wood & Co.. 1892. MANUALS. 205 ('94) FRANKLAND, PERCY, AND FRANKLAND, MRS. G. C. Micro-organisms in water. London, Longmans, Green & Co. pp. XI, 532, with figures, 1894. ('95). ABBOTT, A. C. The principles of bacteriology. A practical manual for students and physi- cians. 3d ed. pp. xn, 493, with 98 figures. 17 of which are colored. Lea Brothers & Co., Philadelphia, 1895. Last (6th) ed. in 1902, pp. xi, 641. ("95). ITZEROTT UND NIEMANN. Mikrophotographi- scher Atlas der Bakterienkunde. Mit 126 mikrophotographischen Abbildungen in Lichtdruck auf 21 Tafeln. Leipzig, Johann Ambrosius Earth (Arthur Meiner), 1895. ('95)- KANTHACK AND DRYSDAI.E. Elementary prac- tical bacteriology, pp. xxn, 181, with 16 figures. I2mo. London and New York, Macmillan & Co., 1895. ('9S). FRAENKEL, CARL UND PFEIFFER, RICHARD. Mikrophotographischer Atlas der Bakterien- kunde. 2d ed. Berlin, 1895. Verlag von Aug. Hirschwald. 76 plates (156 figures) with descriptive text. 8vo. ('96). HUEPPE, FERDINAND. Naturwissenschaftliche Einfiihrung in die Bakteriologie. pp. vin, 268, with 28 wood cuts. Wiesbaden, C. W. Kreidel's Verlag, 1896. English translation by Edwin O. Jordan, entitled, The Principles of Bacteriology. The Open Court Pub. Co , Chicago, 1899. ('96). LEHMANN, K. B., AND NEUMANN, RUDOLPH. Bakteriologische Diagnostik. 2 vols. I2mo. Miinohen, J. F. Lehmann, 1896. Theil I. Atlas of 63 colored plates. Theil n. Text, which also includes some figures, pp. vii, 448, and a folded sheet, giving a synopsis of some of the characters of 62 species. There is an English edition in one volume, issued by Wni. Wood & Co. in 1897. The German edition is a very useful book. The English edition does not give the translator's name. It also omits the extended classifica- tion and description of species and the folded sheet. ('96). FLUEGGE, C. Die Mikroorganismen. Mit be- sonderer Berucksichtigung der Aetiologie der Infektionskrankheiten. 3 vollig um- gearbeitete Aufl. 8vo. 2 vols. Bd. i. pp. xvi, 596, with 57 text figures. Bd. n. pp. xxn, 751, with 153 text figures. Leipsic, F. C. W. Vogel, 1896. Very useful. ('96). STERNBERG, GEO. M. Text-book of bacteriol- ogy- PP- xi, 693, with 9 heliotype and chro- molithograph plates and 200 engravings. 8vo. New York, Wm. Wood & Co., 1896. Iu the second revised edition (1901) there is a chapter on plant parasites, but the saprophytes are omitted alto- gether. ('96). CROOKSHANK, EDGAR M. Bacteriology and in- fective diseases. 4th ed. pp. xxx, 715, with 22 plates and 273 wood engravings and pho- tographs. Small 8vo. London, H. K. Lewis, 1896. ('97). MACE, E. Traite pratique debacteriologie. 36 ed. pp. vin, 1144, with figures. Paris, J. B. Bailliere et fils., 1897. 4th ed., Paris, 1901, pp. vin, 1196, 338 figs. Much new matter added. A very useful book. ('97). MALLORY, FRANK BURR, AND WRIGHT, JAMES HOMER. Pathological technique. A prac- tical manual for the pathological laboratory. pp. 397, with 105 illustrations. Philadelphia, W. B. Saunders, 1897. 2d ed. (with slight change of title), revised and enlarged. 1901, pp. 432, with 137 illustrations. Compact and useful. (.97). MICULA, W. System der Bakterien. Hand- •buch der Morphologic, Entwickelungsge- schichte und Systematik der Bakterien. i Bd. Allgemeiner Teil. 8vo. pp. vin, 368, with six plates, five of them from photo- micrographs. Jena, Gustav Fischer, 1897. ('97). FISCHER, ALFRED. Vorlesungen iiber Bakterien. 8vo. pp. vin, 186, with 28 figures. Jena, Gustav Fischer, 1897. For criticisms on that portion of this book which re- lates to plant diseases, see Centralb. f Bakt., 2 Abt. 1899 p. 271, 809, and 1901,88,128,190. English translation in 1900 by A. Coppen-Jones, under title, The structure and functions of bacteria. 2 German ed. in 1903. C97-'o3). LAFAR, FRANZ. Technische Mykologie. Ein Hand'buch der Garungsphysiologie fur technische Chemiker, Nahrungsmittel- Chemiker, Gahrungstechniker, Agrikultur- chemiker, Pharmaceuten und Landwirte. Jena, Gustav Fischer, 1897-1903. I lith. and 158 text figs. pp M The first volume, devoted to the bacteria, contains p. sit, 362. This was translated by Salter as Technical ycology, etc. London : Chas. Griffin & Co., and Phila- delphia : Lippincott, 1898. A new ed. is being published. ('98). DUCLAUX, E. Traite de Microbiologie. T. i, Microbiologie general. Paris, Masson et Ci-e. 1898. pp. in, 632, 61 text figures. ('98). Procedures recommended for the study of bacteria, with especial reference to greater uniformity in the description and differen- tiation of species. Being a report of a com- mittee of American bacteriologists to the Committee on the Pollution of Water Sup- plies of the American Public Health Asso- ciation. (Submitted at the meeting of the association in Philadelphia, Sept., 1897.") Concord, N. H., The Rumford Press, 1898. pp. 47, with charts. This pamphlet should be iu every laboratory, and in the hands of every student. ('98). HEIM, L. Lehrbuch der Bakteriologie mit bespnderer Berucksichtigung der bakterio- logischen Unitersuchung und Diagnostik. 2 Aufl., pp. xvn, 604. Stuttgart, Ferdinand Enke, 1898. ('98). PEARMAIN, T. H., AND MOOR, C. G.. Applied bacteriology, pp. xv, 464, with text figures and 8 lithographic plates (the latter copied from Lehmann and Neumann), also folded plate, after Stoddard, showing 3 "halo cul- tures." 2d ed., 1898. London, Bailliere, Tindall & Cox. Coo). MOORE, VERANUS A. Laboratory directions for beginners in bacteriology. Cornell Uni- versity (Ithaca, N. Y.). ad ed., revised, 1900. Ginn & Co., The Athenaeum Press, Boston, Mass. pp. xvi, 143. I'oo). MIGULA, W. Specielle Systematik der Bak- terien. 2 Bd. 8vo. pp. ix, 1,068, with 18 tables from photomicrographs and 35 text figures. Jena, Gustav Fischer, 1900. (Is- sued two months ahead of time.) This book should be in every laboratory. No other general work deals as carefully with the morphology of the bacteria. It is designedly less complete in its treat- ment of cultural characters. 206 BACTERIA IN RELATION TO PLANT DISEASES. (!oo). LEVY, ERNST, AND KLEMPERER, FEUX. Ele- ments of clinical bacteriology for physicians and students. Second enlarged and revised edition. Authorized translation by Augus- tus A. Eshner, Philadelphia. W. B. Saun- ders, 1900. pp. 441, 92 figs. ('01). ABBOTT, A. C. The 'hygiene of transmissible diseases, their causation, mode of dissemina- tion, and methods of prevention. W. B. Saunders & Co., Philadelphia and London, 1901. 2d ed. pp. 350, with 46 ill. and charts. Popular. ('01). ALMQUIST, E., OCH TROILI-PETERSSON, G. Mikroorganismerna i praktiska Lifvet Bak- teriologiens utreckling och untidastand- punkt. 184 pp. Svo. Stockholm, 1901. ('oi). CHESTER, FREDERICK D. A manual of deter- minative 'bacteriology. New York, The Macmillan Co.; London, Macmillan & Co., Ltd., 1901. pp. vi, 401. ('oi). CONN, HERBERT W. Agricultural bacteriol- ogy. A study of the relation of bacteria to agriculture, with special reference to the bacteria in the soil, in water, in the dairy, in miscellaneous farm products, and in plants and domestic animals, pp. VI, 412. Philadelphia, P. Blakiston's Son & Co., 1901. ('oi). FROST, WILLIAM D. A laboratory guide in elementary bacteriology. pp. vm, 205. Tracy, Gibbs & Co., Madison, Wis., 1901. ('02). International catalogue of scientific literature. First annual issue. R. Bacteriology. Pub- lished for the International Council by the Roy. Soc. of London, vol. vm, 1902 (De- cember), London, Harrison & Sons, 45 St. Martin's Lane. At least oue additional volume has been published. ('02). BOWHILL, THOMAS. Manual of bacteriologi- cal technique and special bacteriology. 2d ed., revised and enlarged. New York, Will- iam Wood & Co., 1902. pp. xvi, 324, 136 figs. ('02). EYRE, J. W. H. The elements of bacteriolog- ical technique, a laboratory guide for the medical, dental, and technical student. 170 illustrations. Philadelphia and London, W. B. Saunders & Co., 1902. pp. 371. ('02?). Collection de planches murales destinecs a 1'enseignement de la bacteriologie. Pub- liees par 1'Institut Pasteur de Paris. Cette collection touche comme principally sujets ; char bun, rouge!, cholera des ponies, pneumonie, iepre.suppu ration, peste, gonocoque, cholera, fievre typhoide, raorve tuberculose, lepre, actinomycose, diptherie, tetanos, etc. ut les maladies a protozoaires : coccidies, paludusme nialadie de la mouche tse-tse, trypanosomes, etc. Condi- tions de la publication. La collection compreiid actuel- lement 65 planches du format 80 x 62 centimetres, tirees en couleurs sur papier toile tres tort, munies d'oeillets per- mettant de les suspendre sur deux pitous et reuuies, dans un carton dispose specialement a cet effet. Elle est ac- compagnee d'uu texte explicatif r£dige en trois langues (francais, allemand, anglais). Prix: 250 francs (port en sus i. I.es planches ue sout pas vendues separement. (' — ). PARK, WILLIAM H. Bacteriology in Medicine and Surgery. Lea Brothers & Co., New York. I2mo., 688 pages, with 87 illustrations in black and colors, and 2 colored plates. ('02). ROGER, G. H. Les maladies infectieuses. Paris. Masson et Cie, 1902, T. I, pp. xiv, 664; T. IT, pp. 665-1,520; av. 117 fig. ('02). MATZUSCHITA, TEisi. Bacteriologische Diag- nastik. Zum Gebrauche in den bacteriolo- gischen Laboratorien und zum Selbstun- terrichte. Fur Aerzte, Tierarzte v.id Bo- taniker. Jena (G. Fischer), 1902, pp. xvn, 692, mit i Taf. ('02). HEWLETT, RICHARD T. A Manual of Bac- teriology, Clinical and Applied, 2d Edition. London (Churchill), 1902, p. 533, with 20 plates. ('02-'04). KOLLE, W., UND WASSERMAN, A., unter Mitwirkung von 54 Fachmanner. Handbuch der pathpgenen Mikroorganismen. Jena, Gustav Fischer. Begun in 1902. 20 Liefer- ungen to Sept., 1904. Fourlarge volumes, with an atlas containing 288 photo- graphs. Monographic iu character. ('03). STERNBERG, GEO. M. Infection and immunity, with special reference to the prevention of infectious diseases. G. P. Putnam's Sons, New York and London, 1903. pp. ix, 293. Popular. ('03). FRANKLAN-D, MRS. PERCY. Bacteria in daily life. Longmans, Green & Co., London, New York, and Bombay, 1903. pp. 216. No illustrations. Popular. ('03). WILLIAMS, HERBERT U. A manual of bac- teriology. Ninety-nine illustrations. 3a ed., revised and enlarged. Philadelphia, P. Blakiston's Son & Co., 1903. pp. xv, 351. ('03). McFARLAND, JOSEPH. A text-book upon the pathogenic 'bacteria for students of medi- cine and physicians. With 153 illustrations, a number of them in colors. 4th ed., re- written and enlarged. W. B. Saunders & Co., Philadelphia, New York, and London, 1903. pp. 629. ('03). MUIR, ROBERT, AND RITCHIE, JAMES. Manual of bacteriology. Am. ed. (with additions'), revised and edited from the third English ed. by Norman MacLeod Harris. 170 illus- trations. Macmillan Co., New York. Mac- millan & Co., Ltd., London, 1903. pp. xx. 565. An excellent book. IV. Physical, Chemical, Zoological, and Botanical Works of Special Use to the Plant Pathologist. ('61). GRAHAM, THOMAS. Liquid diffusion applied to analysis. Philos. Trans, of the Roy. Soc., London, vol. 151, pt. I, 1861. pp. 183-224. 3 figs. Interesting long paper, in which the making of silicate jelly is described. ('83). MOLISCH, HANS. Ueber d. Mikrochem. Nachw. v. Nitraiten u. Nitriten in d. Pflanze mittelst Diphenylamin oder Brucin. Be- richte der Deutschen Botanischen Gesell- schaft, 1883. pp. 150-155. See also Bot. Centralb., Bd. xiv, 1883, pp. 355-356. ('84). GAUTIER, ARMAND. Traite de chimie appliquee a la physiologic et a Fhygiene. Paris, 1884. Not seen. PHYSICAL, CHEMICAL, ZOOLOGICAL, AND BOTANICAL WORKS. 207 ('84). DE VRIES, HUGO. Over eene methode om in planten-sappen gebonden zurcn te befolen. Mnandblad voor Natuunvetenschappen, No. '), 1884. Rev. in Botan. Centralb., Bd. xxiv, 1>. 249. This author gives a method of estimating the quantity of acid in plants when not free, i. e. , wheu united with bases. ('84). MOLL, J. W. Ecne nicuwe niikrocbemische looizuurreactie. Maandblad voor Natuur- wetenschappen, 1884. Rev. in Bot. Centralb. Bd. xxrv, p. 250. liest method I micro-chemical) of detecting tannin in cells. He soaks small pieces of tissue in saturated solu- tion (- per cent i acetate of copper S-io days, and longer will not injure. Then they are sectioned and treated for a few minutes with a drop of o.spercent sol. iron ace- tate. Too long an exposure browns the cell walls. The sections are then washed in water, transferred to alcohol aud finally mounted in glycerine or glycerine jelly. If the test cannot be made at once, the specimens will not be injured by preserving in alcohol, after the soaking in acetate of copper. The tannin-bearing cells become green or bint-. ('85). HANAUSEK, T. P., AND PAMMER, L. Ueber die Loslichkeitsverhaltnisse des Kautschuks. Zeitschrift des allg.- osterr. Apotheker- Vereins, 1885, No. 31, pp. 486-488, with a table. Rev. in Bot. Centralb., Bd. xxv, 1886, p. 308. Note on best solvents for rubber. i '*5 t . STRASBURGER, ED. Zur mikroskopischen Teohnik. Bot. Centralb., Bd. xxiv, 1885, pp. 156-157. According to Strasburger, Faber's yellow pencils are the best for writing on glass or porcelain. To find par- ticular places in a preparation, he makes rings on the stage of the microscope to each side of the opening, and, when the desired field is in view, corresponding ones on the slide. The best method oi making eau de Javelle is said to be the following : (a) Take 20 parts of 25 percent chloride of lime (Chlor- kalkes), stir up in loo parts of water, aud let stand for a time. (6) Dissolve 15 parts of pure potash in loo parts of water. Add b to a, and, after one to several days, filter and use the filtrate. If lime still remains in the fluid, add a few drops of potash solution and filter out precipitate. ('85). LEHMANN, O. Physikalische Technik. Spe- cielle Anlekung zur Selbstanfertigung phy- sikalischer Apparate. pp. xn, 416, with 882 wood cuts and 17 tables. Leipsic, Wm. Engelmann, 1885. ('85). NOLL, F. Eau de Javelle, ein Aufhellungs- und Losungsmittel fur Plasma. Botan. Centralb., Bd. xxi, 1885, pp. 377-3§o. Authortell-hov,- to make and how to use eau de Javelle. Alcoholic material is best suited for treatment. Fresh protoplasm does not dissolve completely, but leaves detritus and granules. Strasburger, Ibid., Band xxiv, p. 157, says this is not eau de Javelle. ('85). KRAUS, C. Ueber amphotere Reaction der Pflanzen-Safte. Botan. Centralb., Bd. xxiv, 1885, p. 287. A M-jyer summarizes some of Kraus' results obtained from the pith parenchyma of twenty plants. Kraus claims that neutral litmus would not answer. He used red and blue litmus. Little pieces were thrown into the sap and left a long time. He got both reactions. ('86). PFEFFER, W. Ueber Aufnahme von Anilin- farben in lebende Zellen. Untersuchungen a. d. Bot. Inst. zu Tuebingen, Leipzig, 1886, Bd. n, Heft 2, pp. 179-329. ('86). BACHMANN, E. Spektroscopische Unter- suchugen von Pilzfarbstoffen. Plauen, 1886. Not seen. ('86). SHENSTONE, W. A. The methods of glass blowing for the use of physical and chemical students, pp. x, 86, with figures. I2mo. Rivingston's, Waterloo Place, London, 1886. ('89). BEHRENS, W., KOSSEL, A., UND SCHIEFFER- DECKER, P. Das Mikroskop und die Metho- den der mikroskopischen Untersuchungen. pp. VIH, 315, with 193 wood cuts. Braun- schweig, Harald Bruhn, 1889. ('90). CURTMAN, CHARLES O. Chemical reagents and the spectroscope. I2mo. pp. 256, with 12 plates. John L. Boland Book and Stationery Co., St. Louis, 1890. A useful little book. ('90). NICKEL, EMIL. Die Farbenreactionen der Kohlenstoffverbindungen. 2d ed. pp. VIH, 134. Berlin, Verlag von Hermann Peters, 1890. ('9l-'92). OSTWALD, WILHELM. Lehrbuch der allge- meinen Chemie. Zweite ganzlich umgear- beitete Auflage. Leipzig, 1891-1892. 2 Bd. The fourth book of the second edition was translated into English by M. M. Pattison Muir, under the title " Solutions." London, 1891, pp. xiii, 316, 8 vo. ('92). TRIMBLE, HENRY. The tannins, pp. 168. J. B. Lippincott Co., Philadelphia, 1892. ('92). ZIMMERMANN, A. Die botanische Mikrptech- nik. Ein Handbuch der Mikroskopischen Praparations- Reaktions- und Tinktions- methoden. Mit 63 Abbild. im Text. Tubin- gen, 1892. pp. x, 278. English translation by James Ellis Humphrey. Henry Holt & Co., New York, 1893. ('93). LEA, A. SHERIDAN. The chemical basis of the animal body. New York, Macmillan & Co., 1893. pp. 288. Forms Part v of 6th ed. of Foster's larger Physiology. ('93). BENDER, ADOLF, UND ERDMANN, HUGO. Chem- ische Praparatenkunde. 2 vols. Bd. I. Anleitung zur Darstellung anorganischer Praparate, von Bender, pp. vm, 530, with 102 figures, 1893. Bd. 11. Anleitung zur Darstellung organischer Praparate, von Erd- man. pp. xn, 610, with 41 figures. Stutt- gart, Ferdinand Enke, 1894. C93-'95)- SCHMIDT, ERNST. Ausfiihrliches Lehrbuch der Pharmaceutischen Chemie. Braunsch- weig, Fr. Vieweg & Sohn. 3d ed. Inorg., 1893. Organic, 1895. pp. 648, vi. 4th ed., 1901. pp. xxxm, 1,944. ('94). BEHRENS, H. A manual of microchemical analysis. With an introductory chapter by John W. Judd. With 87 illustrations, pp. xxv, 246. London and New York, Mac- millan & Co., 1894. ('94). LANDOLT UND BOERNSTEIN. Physikalisch-chem- ische Tabellen. 2d ed. pp. XI, 563. Berlin, Julius Springer, 1894. ('94) The National Dispensatory. 5th ed. 1894. Lea Brothers & Co., Philadelphia and New York. pp. VIH, 1903- ('94). SCHULZ, G., AND JULIUS, P. Systematic sur- vey of the organic colouring matters. Trans- lated from the German and edited with ex- tensive additions by Arthur G. Green, pp. VHI, 205. London and New York, Mac- millan & Co., 1894. Very useful. 208 BACTERIA IN RELATION TO PLANT DISEASES. ('94). LIDFORSS, B. Ueber die Wirkungssphare der Glycose u. Gerbstoff-Reagenten. Sep. Abdr. aus Lunds Univ. Arsskr. T. xxvm, 14 PP- Rev. Bot. Centralb., 1894, Bd. LIX, p. 281. Author says tannins give most of the protein reactions, and may thus lead investigators to many wrong conclu- sions. The following test is given for "grape sugar in plant tissues : The parts to be tested are soaked for some time in au alcoholic copper solution, and then brought to a boil over a water bath. This causes a pre- cipitate of copper oxide (oxydul ) in all of the cells which contained grape sugar. The alcoholic copper solution is made as follows ; Add some acetic acid and glycerin to an alcoholic solution of copper acetate. This is then mixed with an equal volume of alcoholic solution of caustic soda (Natronlosungl. This mixture is as sensi- tive as Fehliug's solution, and is not reduced by various substances which reduce Fehling. A great number of soluble reducing substances are removed by the alcohol from the plant cells, while the sugar remains behind. Bar feed's reagent consists of acetate of copper dis- solved in water and made acid by acetic acid. It is useful for detection of sugar in some cases where Fehliug's solution is worthless, i. e., in the presence of Phloroglu- cin, Aesculin, Quercit, which reduce the latter. The for- mer, on the contrary, is reduced by Hydrochinou aud Resorcin. ('95). CROSS AND BEVAN. Cellulose. An outline of the chemistry of the structural elements of plants, with reference to their natural his- tory and industrial uses. pp. vi, 320, with 14 plates. London, Longmans, Green & Co., 1895. ('95). MEYER, ARTHUR. Untersuchungen fiber die Starkekorner. Svo. pp. xvi, 318, with 9 lithographic plates and 99 text figures. Jena, Gustav Fischer, 1895. ('95). VON LIPPMAN, E. O. Die Chemie der Zuck- erarten. Svo. Second edition, pp. xxvi, 1,174. Braunschweig, Fr. Vieweg u. Sohn, 1895. The third edition appeared in 1904 in two large vol- umes, pp. xxxviii, 2003. Indispensable. ('(.=,). BEHRENS, H. Anleitung zur mikrochemischen Analyse der wichtigsten organischen Ver- bindungen. I Hft. Anthracenegruppe, Phenole, Chinone, Ketone, Aldehyde, pp. vni, 64, with 49 figures, 1895. 2 Hft. Die wichtigsten Faserstoffe. pp. vni, 108, with 18 figures in text and three colored plates. 1896. 3 Hft. Aromatische Amine. pp. vn, 135, with 77 figures, 1896. 4 Hft. Karba- mide, aliphatische Karbonsauren, Aroma- tische Karbonsauren. Hamburg and Leipsic, Leopold Voss, 1897. Very useful. ('96). BEYERINCK, M. W. Kulturversuche mit Amoben auf festem Substrate. Centralb. f. Bakt., i Abt., Bd. xix, pp. 257-267, 1896. ('96). COMEY, ARTHUR M. A Dictionary of Chem- ical Solubilities. Inorganic, pp. xx, 515. Macmillan & Co., London and New York, 1896. ('96). Merck's Index. An encyclopaedia for the physician and the pharmacist; stating the names amd synonyms ; source or origin ; chemical nature and formulas ; physical form, appearance, and properties ; melting and boiling points; solubilities; gravities and percentage strengths; physiological ef- fects; therapeutic uses; modes of adminis- tration and application; regular and maxi- mum dosage; incompatibles; antidotes; special cautions ; hints on keeping and (handling; methods of testing; market val- ues, etc., of the chemicals and drugs used in medicine, in chemistry, and in the arts. 2d ed. Merck & Co., New York, 1896. Very useful. ('96). LEFEVRE, LEON. Traite des matieres color- antes organiques artificielles, de leur prepa- ration industrielle et de leur applications. 2 vols. 8vo. pp. xx, 1,648, with 31 text figures and 261 dyed or printed samples of silk, wool, cotton, paper, and leather, pre- pared under the direction of the author especially for this edition. Paris, G. Mas- son, 1896. ('97). DAVENPORT, C. B. Experimental morphology. Part i. Effect of chemical and physical agents upon protoplasm, 1897. The Mac- millan Co., New York. pp. xiv, 280, 74 figs. ('97). MILLER, WALTER. Scientific Names of Latin and Greek Derivation. Proc. Calif. Acad. Sci. Zoology. Third Ser. Vol. I, No. 3, pp. II5-I43, 1897. ('97). SCHNEIDER, A., AND ALTSCHUL, JULIUS. Rea- gents and reactions known by the names of their authors. Milwaukee, Wis., 1897. Pharm. Review Pub. Co. This boob was first written by A. Schneider. The Ger- man edition was revised and enlarged by Julius Altschul. It was translated into English by Richard Fischer. ('97). BAUSCH, EDWARD. Manipulation of the micro- scope. Ban sell and Lomb Optical Co., Rochester, N. Y. 3d ed. 200 pp. 1897. ('97-'o4>. PFEFFER, WILHELM. Pflanzenphysiologie. Ein Handbuch der Lehre vom Stoffwechsel und Kraftwechsel in der Pflanze. Bd. i. Stoffwechsel. pp. x, 620. mit 70 Holzschnit- ten. Leipzig, 1897. Bd. n, Kraftwechsel. i Halfte, 1901, pp. 353, mit 31 Holzschnitten. II Halfte, 1904, pp. xi, 353-986, mit 60 Holz- schnitten. Verlag von Wil'helm Engelmann. English translation by Alfred J. Ewart, Oxford, Clarendon Press. Vol. i, 1900. Vol. II (Part i), 1903. Vol. ni in prepara- tion. ('98). BEHRENS, WILHELM. Tabellen zum Gebrauch bei mikroscopischen Arbeiten. Braun- schweig, Harald Bruhn. pp. vn, 237. 3d ed., revised, 1898. Extremely useful. ('98). VON BUNGE, GUSTAV. Lehrbuch der physi- ologischen und pathologischen Chemie. In neunundziwanzig Vorlesungen fiir Aerzte und Studierende. 4 vermehrte und verbes- serte Aufl. Leipzig, Verlag von F. C. W. Vogel, 1898. pp. rv, 510. English translation, from 4th German ed. by Starling, London, 1902. ('98). SCHROEDER VON DER KOLK, J. L. C. Kurze Anleitung zur mikroscopischen Krystal- bestimmung. pp. 60, with text figures. Wiesbaden, C. W. Kreidel's Verlag, 1898. PHYSICAL, CHEMICAL, ZOOLOGICAL, AND BOTANICAL WORKS. 209 CgS-'oj). ALLEN, ALFRED H. Commercial organic analysis. A treatise on the modes of assaying tlio 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. r. Alcohols, neutral alcoholic derivatives, etc., ethers, vegetable ncids, starch, sugars, etc. 3d ed.,i898, pp. xii, 557. Vol II, Part r. Fixed oilsaud 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. ii, Part in. Terpenes, essential oils, resins, cnm- phors, etc. ,id ed. preparing. Vol. in, Part I. Tannins, dyes, and coloring matters. 3d ed., enlarged and rewritten. Illustrated. 1900, pp. xvi, Vol. HI, Part n. The amines, hydrazines and deriva- tives, pyridiue bases, the antipyretics, etc. Vegetable Alkaloids, tea, coffee, cocoa, etc. 8 vo. ad ed., 1892, ]>p. viii, 584. Vol. in, Part in. Vegetable alkaloids, non-basic vege- table bitter principles. Animal bases, animal acids, cyanogen compounds, etc. id 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. ('oo). SCHOENICHEN, WALTER, UNO KALBERLAH, ALFRED. B. Eyferth's Einfachste Lebens- formen des Tier- und Pflanzenreiches. Naturgeschichte der mikroskopischen Sitss- 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. Sth ed., enlarged and improved, 1900. pp. XI, 640, with figures. London, J. and A. Churchill. This book should be in every laboratory. ('oo). VAN RiJN, J. J. L. Die Glykoside chemische Monographic der Pflanzenglykoside, nebst •systematischer Darstellung der kiinstlichen Glykoside. Gebriider Borntraeger, Berlin, 1900. pp. xvi, 511. ('oo). BEHRENS, H. Mikrochemischc Technik. Ham- burg u. Leipzig, Leopold Voss, 1900. pp. viii, 68. ('oo). BUECHLER, MAX. Die Diastasen, 1900. Not seen. Coi). GAGE, S. H. The Microscope. An introduc- tion to microscopic methods and to his- tology. Sth ed., revised, 1901. pp. IV, 299, with over two hundred figures. Comstock Publishing Co., Ithaca, N. Y. Deserves a place in every laboratory. Coi). 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 Microtomies 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 Lee and Henneguy. Paris, Octave Doin, 1902. Coi). CROSS, C. F., AND BEVAN, E. J. Researches on cellulose, 1895-1900. Longmans, Green & Co., London, New York, and Bombay, 1901. pp. vn, 180. Coi). 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 im dyeing and calico printing. London, Chas. Griffin & Co., Ltd.; Philadelphia, J. B. Lippincott Co., 1901. pp. 372. ('02). OMELIANSKI, W. Ueberdie 'Garungder cellu- lose. Centralb. f. Bakt., 2 Abt., Bd. vm, Nos. 7-13, pp. 193-201, 225-231, 257-263, 289- 294, 321-326, 353-36i, 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 anaero- 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 oi 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. xn, 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 II. 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 Wdley & 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). NUTTALL, GEORGE H. F. Blood immunity and blood relationship; a demonstration of cer- tion blood-relationships amongst animals by means of the precipitin test for bloo_d. pp. xn, 444. Cambridge (England) University Press, 1904. V. ('28). ('30). ('32). ('38). ('40). 'I i. Books and Papers of More or Less General Interest. EHRENBERG, C. G. Symbolae physical, seu icones et descriptiones animalium everte- bratorum sepositis insectis quae ex itinere per Africam Borealem et Asiam Occiden- talem Friderici Guilclmi Hemprich et Chris- tian! Godofredi Ehrenberg medicinae et chirurgiae doctorum studio novas aut illus- tratae redierunt percensuit et regis iussu et impensis edidit Dr. C. G. Ehrenberg. Decas prima. Berolini ex OfHcina Academica Venditur a mittlero MDCCCXXVIII. 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. 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. EHRENBERG, C. G. Ueber die Entwickelung und Lebensdauer der Infusionsthiere, etc. Abhandl. d. K. Akad. d. Wissensch. zu Berlin, 1831. Printed January, 1832. EHRENBERG, C. G. Die Infusionsthierchen als vollkonunene Organismen. Ein Blick in das tiefere organische Leben der Natur, von Christian Gottfried Ehrenberg zu Ber- lin. Nebst einem Atlas von vierundsechszig oolorirten Kupfertafeln gezeichnet vom Verfas-ser. Leipzig, Verlag von Leopold Voss, 1838. pp. 548, folio. HENLE, J. Pathologische Untersuchungen. Berlin, August Hirschwald. pp. vi, 274. 1840. PCHROEDER, 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 he preserved indefi- nitely if protected from the floating matter of the air by means of cotton plugs, Jioiled 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 mean^ nf many ingenious experi- ments. ('54). COHN, FERDINAND. Untersuchungen iiber die Entwicklungsgeschichte der mikroscopischen Algen und Pilze. Der Akademie tibergeben den i Mai, 1853. Nov. Act. Aoad. 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- macie, Bd. cix (Neue Reihe Bd. xxxni), Hft. i, pp. 35-52, 1859. Leipzig und Heidel- berg. C6o). 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 Chimie et Physique, 1862. 3 serie, T. LXIV, pp. 5-110. ('65). DAVAINE, C. Sur 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 iiber 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. xni. pp. 380-408. ('73). PASTEUR, Louis. Etudes sur !e 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 theorie nouvelle de la fermentation. 12 plates, 85 text figures. Paris, 1876. Gau- thier-Villars. pp. yin, 387. ('77). VON NAEGELI, C. Die niederen Pilze in ihren Beziehungen zu den Infectionskrankheiten und der Gesundheitspflege. Miinchen. R. Oldenbotirg, 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- tcrien 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. HI, 1880-1882. pp. 188-194. BOOKS AND PAPERS OF MORE OR LESS GENERAL INTEREST. 211 ('82). ENGELMANN, TH. W. Zur Biologie der Schi- zomyceten. Bot. Zeitung, 1882, col. 321-325 and 337-341- ('84). METSCHXIKOFF, E. Ucber die Beziehung der Phagocyten zu Milzbramlbacillen. Archiv f. patholog. Anat. u. Physiologic u. f. Klin. Med., Bd. LXXIX, 1884. pp. 502-526. 2 plates. CS6). BOLTON, MEADE. Ueber das Verhalten ver- schiedener Bacterienarten im Trinkwasser. Ztschr. f. Hyg. Bd. i, 1886. pp. 76-114. ('86). 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., UNO BANDLER, A. Die En-twicke- lung von (pathogenen) Spaltpilzen unter dem wechselseitigen Einfluss ihrer Zerset- zungsprodukte. Fortschr. d. Med. Bd vi 1888. pp. 769-773. Treats of growth of bacteria in media exhausted for other organisms. ('88). BANTI, Guipo. Sulla distruzione dei batterii nell'organismo. Arch, per lo sc. med., 1888. Vol. xii, 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. ('90). 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 u, 1893-1896. pp. 1-4. Review in Rev. Soi., 1894, pp. 437-438. ('94). KOCHS, W. Giebt es ein Zellleben ohne Mikroorganismen? Biol. Centralb., 1894. No. 14, pp. 481-491. Answer, yes. Plants are grown from sterilized seeds and kept fourteen mouths without contact with bacteria. Plums refused to decay when the surface had been ster- ilized. ('94). FRANKLAND. Die Bakteriologie in einigen ihrer Beziehungen zur chemischen Wissen- schaft. Centralb. f. Bakt., Bd. xv, 1894, pp. 101-112. ('9S)- 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. xxviii, 1895, pp. 205-268. ('95). ACHARD, CH., ET PHULPIN, E. Contribution a 1'etude de Fenvahissement 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 sur la penetration des mi- crobes intestinaux dans la circulation generate pendant la vie. Ann. de 1'Inst. Pasteur, T. ix, 1895, pp. 199-209. ('95-'96). NUTTALL, GEORGE H. F., 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. n, 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 Studium 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. triol. 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. (!oo). HoF, A. C. Untersuchungen fiber 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. ('oi). FISCHER, ALFRED. Ueber Protoplasmastruktur. Antwort an O. Buetschli. Archiv f. Ent- wickelungsm. d. Org., 1901, Bd. xm, pp. 1-33. ('02). JoEST, ERNST. Unbekanrate 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 ir), 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- phyce'ae (Plates xxni-xxv, pp. 237-335. Bibliog. Trans, and Proc. Bot. Soc. Penn- sylvania, Vol. i, No. 3, 1904. Fiuds short cillia on side wall* of Cyanophyceze. 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 "TOeuvre 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 sole, moyen pratique assure de la combattre et d'en prevenir le retour. Tome I, La pebrine et la flacherie, pp. xn, 322, illustrated, and Tome n, Notes et docu- ments, pp. 327, Paris, 1870. Gautliier- Villars. ('76). KOCH, ROBERT. Aetiologie der Milzbrand- krankheit begriindet auf die Entwicklungs- geschichte des Bacillus anthracis. Cohn's Beitrage z. Biol. d. Pflanzen. Bd. n, p. 277, Breslau, 1876. ('77). PASTEUR, L. Etiologie des maladies charbon- neuses. Archives Veter. T. n, Paris, 1877, pp. 668-671. ('So). PASTEUR, CHAMBEKLAND 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 ?ur 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 iiber die Arbeiten des K. Gesundheit- samtes, welche zur Entdeckung des Bacillus der Rotzkrankheit gefuhrt haben. Deutsch. med. Wochenschr. Bd. vni, 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 Typhusbaoillus 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. xcvin, Paris, 1884, pp. 1229-1231. Earlier papers will be found in the same journal, T. xcvin. p. 4^7 . T. xc v. p. 1 1.^7, and T. xcn, p. 1259. ('84). LOEFFLER, FRIEDRICH. Untersuchungen iiber die Bedeutung der Mikroorganismen fiir die Entste_hung 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 be read hy every student who desires to know how a good piece of investigation is conducted. ('84). NICOLAIEK, ARTHUR. Ueber infectiosen Teta- nus. Deutsche med. Wochenschr., 1884. Bd. x, pp. 842-844. ('84). KOCH, ROBERT. Vortrag iiber die Cholera. Berliner Win. 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. xin, 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. L, 1887, pp. 19-29. ('88-'go). 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 in, 1889. pp. 273- 288. 36 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. vn, 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 Sinii h. ('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. Experimentelle Untersuchungen iiber 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. Z;ir Kenntniss des hog- cholerabacillus. Centralb. f. Bakt, Bd. ix, PP. 253-257, 307-311, and 339-343- ('92). PFEIFFER, R. Vorliiufige Mittheilungen iiber die Erreger der Influenza. Deut. med. Wochenschr., Bd. xvin, 1892, p. 28. ('93). BEHRING. Die Geschichte der Diphtherie. Leipzig, 1893. Not seen. SPECIAL HUMAN AND ANIMAL DISEASES. 213 ('93). BRIEGER, LUDWIG, AND COHN, GEORC. Unter- suchungen iiber das Tetanusgift. Zeitschr. f. Hyg., Bd. xv, pp. i-io, 1893. As small a quantity of the tetanus poisou as 0.000,23 gram would be a lethal dose for a man weighing 70 kilos. This is au 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 Aetiologie der Influenza. Zeitschr. f. Hyg., Bd. xm, 1893, pp. 357-386, 8 plates. ('94). Now, F. G. Ein neuer anaerober Bacillus des malignen Oedems. Zeitschr. f. Hyg., Bd. xvu, 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. O. 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 waters of the reservoir containing the sick fish, its inflow and outflow pipes, an anaerobic, gas forming (COz), motile, short organism (Bacillus piscicictUH (ttjilis), pathogenic to cold-blooded animals, especially fish aud frogs. Or- ganism coagulates milk and grows readily at 37.5° C. It 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 Dysenterie in Japan. Centralb. f. Bakt, I Abt., Bd. xxm, 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. in, 1898, pp. 451-511. ('98). LOEFFLER. Bericht der Commission zur Er- forschung der Maul- und Klauenseuche bei dem Institut fiir Infektionskrankheiten in Berlin. Erstattet an den Cultusminiister von dem Vorsitzenden 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., 1903. 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, L/ondon, vol. XLVII, pp. 147-152. Authors describe as the cause of pleuro-pneumonia in cattle au 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 aud 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, aud 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 aud Salimbeui 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- hienues 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 Chamberlaud 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- pneumorae. Ann. de 1'Inst. Pasteur, 1898, T. xn, pp. 240-262. ('99). GELPKE, THEODOR. Bacterium septatum und dessen Beziehungen zur Gruppe der Diph- therienbacterien (B. diphtheriae [Klebs-L6f- fler], B. pseudodiphtheriticum [Loffler] 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. Pluila. 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. Jn 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. ('oo). 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. ('og-'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. ('oi). 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 P'ate. Organism not sporiferous. Author made various tests from old cultures kept in sealed tubes of bouillon, and of very small round grains. ('oi). 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. viii, 1901, pp. 441-460, 6 figs. Confirms the work of Nocard and Roux. ('02). MARSH, M. C. Bacterium truttae, 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. 3S5-368. ('02). MOORE, V. A. The pathology and differential diagnosis of infectious diseases of animals. Ithaca, N. Y., 1902. pp. xiv, 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. 411-415, 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 + 15. Growth is also inhibited by an alkaliuity 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). MONFAI.LET, D. Bibliographic abregee des infections. Paris et Santiago (Chili), Ch. Goffi, editeur, 1903, pp. 1-65. About iqoo classified references to papers on human and animal diseases of a communicable nature. ('04). RF.MLINGER, P. Les travaux re-cent sur la rage. Bull, de 1'Inst. Pasteur, 1904, T. II, pp. 753- 764. " We have shown that if one filters au emulsion of rabies virus through a Berkefeld V bougie and then cen- trifuges the filtrate, the upper layers are deprived of vir- ulence, but the virulence is kept in the lower layers. Barratt has obtained the same results with au unfiltered emulsion of a rabid brain." ('04). FLEXNER, SIMON, HOLT, L. EMMETT, and as- sistants. Bacteriological and Clinical Studies of the Diarrhea! Diseases of Infancy, with Reference to the Bacillus Dysenteric (Shiga). Studies from the Rockefeller In- stitute for Medical Research, New York. Vol. n, 1904, PP- 7-202. Predisposition, Conditions Favoring Infection or Immunity. CHAUVEAU. See XXH. LoEFFLER, FRIEDRICH. Zu.r Immunitatsfrage. Mitth. a. d. K. Gesundheitsarrvte. Bd. I, 1881, pp. 134-187. Also a separate, 54 pp. METCHNIKOFF. See xxi. NUTTALL. See xxxvm. NUTTALL. See xxi. FLUEGGE, C. Studien iiber die Abschwachuns virulenter Bakterien und die erworbene Immunitat. Zeitsch. f. Hygiene. 1888. Bd. IV, pp. 208-230. BUCHNER. See xxi. NISSEN. See xxi. LEHMANN. See xxi OGATA. See xxi. BUCHNER, HANS. Ueber Immunitat, deren natiirliches Vorkotmnen und kiinstlichc Erzetigung. Munch. Med. Wochenschr. 1891, 38 Jahrg., pp. 551-554, 574-579- EHRLICH AND WASSERMANN. See xxi. HuEPPE, FERDINAND. Perlsucht und Tuber- culose. Berliner klin. Wochenschrift, 1901, No. 34, pp. 876-878. Also a separate, pp. 10. . ZABOLOTNY, D. Recherches sur la peste. 2 mem. Experiences d'inoculation, d'im- munisation et de traitement des animaux. Arch des Sci. Biol. publiees par 1'Inst. Im- perial de Med. Exper. a St. Petersbourg. T. vin, No. 4, 1901, pp. 390-427. r 2 plates. 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. Co2-'o4). KOLLE AND WASSERMAN. See in. VII. C8o) C8i) ('87) C88) C88) CSS) ('89) C89) ('90) ('9i) ('90 ('94) Coi) ('oi ) Coi) VIII. Symbiosis, Antagonism. C8i). KEKN, 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 u. sp. Plates good. i '84) KRANNHAI.S, II. Ueber das Kumys-ahnliche getrank "Kephir" und ueber den "Kephir - pilz Deutsch. Archiv. f. klin. med. Bd xxxv, pp. 18-37, i P^te. Bibliography of IB titles. SYMBIOSIS; CARRIERS OF INFECTION; MORPHOLOGY. 2I5 (84). STRI-VI:. Hi IN-KICH. Ueber Kephir. Ber. d deu1 m. Gesellsch. Bd. xvn 1884 , >o , PP- 314-3 16 and 1,364-1 ,368. (87). METCHNIKOFF. See xxi. ('87. C, \i;i;i . C. Ueber Antagonisten unter den •-Tien. Correspondenzbl. f. Schweizer rzte, Jajhrg. xvn, 1887, pp. 385-392. C88). XfiTAi.L. See xxi. ('88). HE FREUUENREICH. E. De I'aratagonisme des bacteries et de I'immunite qu'il confere aux milieux de culture. Ann. de 1'Inst. Pasteur T. u, iSSS, 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. 262-200 ('89). BEYERINCK, M. \V. 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 maybe- come pathogenic when inoculated together. ('90) ('90 ('93). (94). '96 ('97). Coo). BLACOVESTCHENSKY, 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. xvin, 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. GALTJER, V. Nouvelle recherches stir 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 iiber den Kefir. Landw Jahrbuch d. Schweiz, 1896. Bd. x, pp. 1-20. 2 text figs, and I heliotype plate. VON- FHEUDENREICH, ED. Bakteriolosjische Un- tersuchungen iiber den Kefir. Centralb f Bakt., 2 Abt. Bd. in, 1897. PP. -17-54. 2 figs. ; 87-95, I35-MI. 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. xii, Torino. 1888, pp. 279-292. ('91). WAITE, M. B. Results from recent investiga- tions in pear blight. Proc. .Am. Asso. Adv. Sci.. 40th 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-Kon" Ann. de 1'Inst. Pasteur. T. vm, 1894, pp 662-667. The pest is carried by rats. ('95)- SMITH, ERWIN F. Bacillus tracheiphilus etc Central!), f. Bakt.,etc. 2 Abt. i Bd, 1805 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 (Pammel). The cause of a brown rot in cruciferous plants. Centralb. f. Bakt. 2 Abt., Bd. in, pp. 409-410. Disease communicated by slugs and bv 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 vin, No. 1-2, pp. 1-154. 3 plates. Bibliography of 366 titles. Noticed in Nature, Dec. 14, 1899. Coo). GALLI- VALERIC, BRUNO. Les puces des rats et des souris jouent-elles tin role important dans la transmission de la peste bubonique a 1'homme? Centralb. f. Bakt, I A.bt Bd xxm, 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. ('04). BRENNER, W. Die Schwarzfaule des Kohls Centralb. f. Bakt. 2 Abt. Bd. xii, p. 729. Disease said to be communicated by aphides. X. General Morphology of the Bacteria. Cytology. ('74). BILLROTH, THEODOR. Untersuchungen fiber die Vegatationsformen von Cocco-bacteria sep- tica und den Antheil welchen sie an der Entstehung und Verbreitung der accidentel- len Wundkrankheiten haben. Berlin, 1874. Verlag von Georg Reimer. Quarto, pp xrv 244. 5 plates. ('77). BILLROTH, TH., AND EHRLICH, F. Unter- suchungen iiber 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. Pp.iltpilze und Spaltalgen, I.cipsic, i88j. vi, 74. Verlag von Veit and Conip. 7 plates. 2l6 BACTERIA IN RELATION TO PLANT DISEASES. ('86). HUEPPE, FERDINAND. Die Fortnen der Bak- tericn und ihre Beziehungen zu den Gat- fnngen und Anten. 1886. pp. vm, 152. 24 figs. Wiesbaden. C. W. Kreidel's Verlag. CSX). POMMER, GUSTAV. Ein Beitrag zur Kenntniss der fadenbildenden Bakterien. Mitth. bot. Inst. Graz, Jena, iSSS, pp. 93-112. I plate. Bacillus brassicas, a sporiferous form, isolated from an imperfectly sterilized decoctioii of cabbage leaf. ('89). METCHNIKOFF, EL. Contributions a 1'etude du pleomorphisme des bacteries. Ann. de 1'Inst. Pasteur, T. in, 1889, pp. 61-68. I plate. ('89). ERNST, PAUL. Ueber Kern-und Sporenbildung in Bakterien. Zeitschr. f. Hyg. Bd. v, 1889, pp. 428-486. 2 plates. C8o). BABES, VICTOR. Ueber isolirt farbbare Antheile von Bakterien. Zeitschr. 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. i 'So). METCHNIKOFF, EL. Note sur le pleomorphisme des bacteries. Ann. de 1'Inst. Pasteur, T. in, 1889, pp. 265-267. ("90). ALMQUIST, E. Untersuchungen tiber einige Bakteriengattungen mit Mycelien. Zeitsch. f. Hyg. Bd. vm, 1890, Heft 2, pp. 189-197. Coo). 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 raraosa. Bulletin de 1'Acad. de St. Petersbourg. Nouvelle serie (n), 1891, T. .xxxiv, p. 481. Not seen . i ''lit. DANGEARD. See xxni. ('91). PROTOPOPOFF. Sur la question de la structure des bacteries. Ann. de 1'Inst, Pasteur, 1891, Tome v, p. 332-336". ('91). 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. 51-55. ('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 dis- tinguished." Coj>. THAXTER, ROLAND. On the Myxobacteriacea?. a new order of Schizomycetes. Botanical Gazette, vol. xvn, No. 12, pp. 389-406. Pis. xxn-xxv, 1892. ' 'i-1' SAUVAGEAU, C., ET RADAIS. Sur les genres Cladotbrix, 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 cine merkwiirdige Erschei nun n-omatium Oketiii Ehrbg. sp ' f. Bakt.. xi Bd., 1892, pp. 257 164, mit i t:if. i rulnred). Atilh"i how curious bauds MI lnid^fs cou- terial cells sidewise as well as t ml ti> t-ml. ('92). TRAMBUSTI, A., AND GAI.EOTTI, G. Neuer Beitrag zum Sttidium der inneren Struktur der Bakterien. Centralb. f. Bakt., xi Bd., 1892, pp. 717-722, mit i Taf. (colored). ('92). KLEIN, E. Zur Geschichte des Pleomorphis- ITHIS des Tuberculoseerregers. Centralb. f. Bakt., xii Bd., 1892, pp. 905-906. ('92). BUETSCHLI, O. Untersuchungen tiber 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. C9S). WAGER, HAROLD. Preliminary note upon the structure of bacterial cells. Annals of Botany, vol. ix, 1895, pp. 659-661. ('9S)- COPPEN-JONES, A. Lleber die Morphologic und systematische Stellung des Tmberkelpilzes und iiher die Kolbenbildung bei Aktinomy- kose und Tuberkulose. Centralb. f. Bakt.. xvii 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., 1805, 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. Beobadhtungen iiber die mctachro- 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. ('96). COPPEN-JONES, A. UehtT die Nomeuclatur des sog. " Tuhcrkelbacillus." Centralb. f. Bakt., i Abt, Bd. xx, 1896, No. 10-11, pp. 393-395- ('96). KANTHACK, A. A. Ueber verzweigte Diph- theriebacillen. Centralb. f, Bkt., xx Bd., 1896, pp. 296-297. ('96). ZETTNOW. Bilder von Spirillum undtila majus bei freiwilligem Ahstrrhen. Centralb. f. Bakt., xix Bd., 1896, pp. 177-180, with i plate, 16 figs. ('97). THAXTER, ROLAND. Contributions from the Cryptogamic Laboratory of Harvard Uni- versity, xxxix. Further Observations on the Myxobacteriaceae. Botanical Gazette, vol. xxiit. No. 6, 1897, pp. 393-411, 2 plates. Co/). MARPMANN, G. Zur Morphologic und Biologic des Tuherkelbacilliis. Centralb. f. Bakt.. xxn Bd., 1897, pp. 582-586, with I plate. GENERAL MORPHOLOGY OF THE BACTERIA; CYTOLOGY. 217 (.'97). JOHAN-OLSEN, OLAV. Zur Pleomorphismus- frage. Centralb. f. Bakt., 2 Abt., Bd. in, 1807, pp. 273-284, 2 plates. Kinds branching forms. |V,;). Km. Die Streptothrixform des Rotlaufbacil- lus. Centralb. f. Bakt., xxn Bd., 1897, pp. 726-732, with 4 figs. ('98). SCHULTZ. See xxxvni. ('98). STOLZ, ALBERT. Ueber besondere Wach&tums- 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. Bafct, xxiv Bd., 1898, pp. 945- 955, i plate. The author's figures show the body of the spirillum blue enclosing 1-5 carmiii colored granules. ('98). RUZICKA, VLAD. Zur Frage von der inneren Struktur der Mikroorganismen. Centralb. f. Bakt., xxni Bd., 1898, pp. 305-307, with i plate. Finds granules, which take stains, in bacterial body reserves conclusions as to their nature, but thinks they are not pleomorphic or degeneration phenomena. ("98). GRASSEERGER, 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 zur 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- tans, B. perittomaticuni, B. flexile. B. turgesceus, B. bracliysporum, B. implectans, B petroselini.B. augulans. Bacillus goniosporus, B. pectocutis, B. paucicutis, B. cyliudrosporus. 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., xxni Bd., 1898, pp. 433-438, and pp. 489-492. with 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. 'i'.)). SCHULZE, OTTO. UiTitersuchungen iiber die Strahlenpilzformen des Tuberculoseerregers. Zeitsohr. 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. M). BERESTXEW, X. Zur Frage der Klassifikation und systematischen Stelhmg der Strahlen- e. Centralb. f. Bakt., xxvi, 1899, p. 390. ('99). MOEI.I.ER, ALFRED. Ein neuer saure- und alko- holfcster Bacillus aus der Tuberkelbacillen- gruppc, welcher echte Verzweigungsformen bildet. Centralb. f. Bakt., Bd. xxv, 1899, pp. 369-373. with i plate. ('99). SPIRIG, W. Die Streptothrix (Actinomyces) 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- phologic und Entwickelungsgeschichte der Bakterien nach Studien an drei Korner- bacillen. 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. ('oo). 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- ingmethod i 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. ('oo). ZETTNOW. Romanowski's Farbung bei Bak- terien. Centralb. f. Bakt., xxvn Bd., 1900, pp. 803-805. Says Dr. Feinberg's papers contribute " nichts Neues " <"oo). NAKANISHI, K. Vorlaufige Mitteilungen iiber eine neue Farbungsmethode zur Darstellung des feineren Baues der Bakterien. Munch, med. Wochenschr., 1900, No. 6. Rev. in Centralb. f. Bakt., xxvii 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- bakteriums. 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. ("ot). MEYER. ARTHUR. Ueber die Verzweigung der Bakterien. Centralb. f. Bakt.. I Abt., xxx Bd., 1901, No. 2, pp. 49-60, 2 plates. COT). ROSENFELD. See xv. COT). RETCHENKACH. II. Ueber Verzweigung bei Spirillen. Centralb. f. Bakt., i Abt., Bd. xxix. IQOI. pp. 553-557- i heliotype plate. Many of the spirilla are shown with distinct branches. ('02). BUETSCHI.I, OTTO. Bemerkungen iiber Cyano- phyceen und Bacteriaceen. Archiv. f. Pro- thtenkunde, Bd. i, Heft. I. Jena, Gustav Fischer, 1902. pp. 41-58, I plate. On the nature of the " Centralkoerper." ('02). ERRERA, LEO. Sur une bacterie de grandes dimensions: Spirillum colossus. Recueil de 1'Inst. botanique (Univ.de Bruxellesl, T. v, 1002, pp. 347-357. Also a separate. 2l8 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., vin Bd., 1902, pp. 689-699. Also a separalc. I 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 Luftbacillus. (Vorl. Mit- teilung). Centralb. f. Bakt., Abt. 2, Bd. ix, 1902, pp. 257-260, mit I Taf. ('03). HILL, II. W., AND RICKARDS, 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. 3Oth Ann. meet- ing iat New Orleans, Dec. 9-12, 1902, Co- lumbus, Ohio, 1903. Also a separate, 4 pp., 2 figs. ('03). WOI.BACH, 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- 313-333. 13 plates. Also a separate. ('04). THAXTER. ROLAND. Contributions from the cryptogamic laboratory of Harvard Univer- sity, LVI, Notes on the Myxobacteriaceae. Bat. Gaz., June, 1904, vol. xxxvn, pp. 405- 416, with 2 plates. Cystobacter becomes Polyaugium. ('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. xn, pp. 641-648, 'and Bd. xm, pp. 13-22, 20 figs. Describes a branched form. XI. Spores. [See also some papers under X, XV, and XXXIV.] i ';i 1 1 . CHUN, FERDINAND. Untersuchnngen iiber Bac- terk-n. 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- liche Intelligenzbl., Jaihrg. xxxi, 1884, pp. 370. i'N7». LEHMANN, K. B. Ueber die Sporenbildung bei Milzbrand. Munch, med. Wochenschr., 1887, pp. 485-488, No. 26. I'RAZMOWSKI, A. Ueber Sporenbildung bei den Bakterien. Vet-hand, d. k. k. Akad. d. \Vissenschaften in Krakau. Math.-naturw. Si-ktion, Bd. xvm, 1888, p. 35, I Tafel. Author thinks existence of " arthrospores " is not proved. ('88). GLOBIG. See xxxm and xxxiv. < iRUBEE. Si'C XXXIII. ('88). BUCHNER, II. Ueber die vermeintlichen Sporen kl . isss. Bd. iv, pp. 385-390. ('89). KLEIN, LUDWIG. Ueber einen neuen Typus der Sporenlbildung 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 one 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 minutes It is subsequently plunged into 5 per cent chromic acid and again thor- oughly washed in water, after which carbol-fuchsiu is dropped upon it and warmed over the flame to boiling for 60 seconds. The carbol-fuchsiu 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 off. 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. xm, pp. 71-112. Resistance is due to extreme dryness of the spores, and to the f.ict 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 acid method universally applicable. Author some-times substitutes chloriodid zinc. He has also found hydrogen peroxide very useful. He also uses auiliu water fuchsiu. A pure, colorless, toluidin free anilin may be had from Joh. Wolff, in Bres- lau. In case ot anthrax spores, the proper time of expos- ure is i}:2 minutes with chromic acid; 2tO2,1.j minutes with concentrated chlorzinc iodide solution, and 3 min- utes with hydrogen peroxide. Here also the author says he obtained the best results with H2O2. ('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 .s minutes) into steaming hot water contain- ing 10 per cent ammonia water, to which has also been added 10 to 20 dropsof some alcoholic aiiilin 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 safrauin ; the contrast stains are vesuviu, chry- soidin, metliylene blue, malachit green or safrauin. The preparations are said to be clear and satisfactory. ('93). PHYSALIX. See xxxm. ('94). ERNST, PAUL. Farbungsvcrsnche 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 bacteries 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., 1806, pp. 360-362. ('95). BUNGE, R. Ueber Sporenbildung bei Bakter- ien. Fortschr. d. Med., Bd. xm, 1895, No. 20 and 21. Rev. in Centralb. f. Bakt., xvm Bd., 1895, p. 718. SPORES ; FLAGELLA. 219 ("96). SCHREIBER, OSWALD. Ucber die physiologis- chen Bedingungen der endqgenen Sporen- bildung bei Bacillus anthracis, subtilis, und tumescens. Centralb. f. Bakt., XX Bd., 1896, 353-374 and 429-437. Forty-five papers cited at close of this article. ('96). BUCHNER, H. Ueber die physiologischen Be- dingungen der Sporenbildung beim Milz- brandbacillus. Centralb. f. Bakt., xx Bd., 1896, pp. 806-807. ('98). CATTERINA, G. Ricerche sull'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. Hochschule zu Karlsruhe, n Bd., I Heft, 1898, pp. 65-72. ('98). ACJESZKY, ALADAR. Eine einfache Sporen- fdrbungsmethode. Centralb. f. Bakt., xxni Bd., 1898, pp. 329-33L The unfixed covers are placed in a boiling hot 3-3 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 cent sulphuric acid, and couuterstaiued I to 2 minutes in malachit green or methylene blue. ('99). STEPHANIDIS, PHILOPIMIN. Ueber den Einfluss des Nahrstoffgehaltes von Nahrboden auf die Raschheit der Sporenbildung und die Zalil und Resistenz der gebildeten Sporen. Arch. f. Hyg., Bd. xxxv, 1899, pp. l-io. Review in Centralb. f. Bakt., xxvi Bd., 1899, p. 568. lu 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). KLEIN, ALEX. Eine einfache Methode zur Sporen farlbung. Centralb. f. Bakt., xxv Bd., 1899, PP- 376-3/9- 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 percent H2SO4 for I to 2 seconds, washed in water and counterstained 3 to 4 miuutes in alcoholic methylene blue solution diluted with water. ('99). DANNAPPEL. See xxxm. Coo). SMITH, R. GREIG. The double staining of spores and bacilli. Proceedings of .the I.in- nean 3 « "f Xc\v South Walo-^, 1900, Part 3, June 27, pp. 394-397- Also a separate (issuiM Nov. 22, 1900). ('02). SCHAUDINN, FRITZ. Beitrage zur Kenntnis der Bakterien und verwandter Organismen. i. Bacillus butchlii. 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 given, illustrating iuner structure, stages in the formation of the spores lone in each pole of the rod I, polar germination of the spores, etc. The organism is 24 to So /j. long by 3 to 6 M. broad, mostly 50 to 60 n x 4 to 5 n. The bacillus is flagellate, after the manner of B. subtilis. ('76). ('77). ('78). 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. WARMING, EUG. Om nogle ved Danmarks Ky«ter levende Bakterier. Kjobenhavn, 1876. KOCH. See i.v. DALLINGER, W. H. On die 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 LEON. 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). LOEFFLER, 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). TRF.XKMANN. Die Farbung der Geisseln von Spirillen und Bacillen. Centralb. f. Bakt., vi Bd., Oct. 15. 1889. No. 16-17, pp. 433-436. i 90) MESSEA. See LVI. ('90). LOEFFLER, F. Weitere Untersuchungen iiber dir Bc-ixung und Farbung der Geisseln bei den Bakterien. Centralb. f. Bakt., Bd. vu, 1890, pp. 625-639. ('90). TRENKMANN. Die Faribung der Geisseln von Spirillen und Bacillen. n. Mitth. Centralb. f. Bakt.. 1890. Bd. vni, 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 lo 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. i 'MI i. lIi'MFHREY, J. E. Notes on Technique, n. I', it. Gazette, 1891, pp. 71-73. Cilia of zoospores of algce and fungi are stained very readilyand sharply ''in a drop of moderately strong solu- tion (in qo per cent alcohol t 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 yivant des oils ou flagella de certaines bacteries mobiles. C. R. de la Soc. de bioJogie, 1892, No. 23, pp. 542-543. Also Bull. Med., 1892, p. 1,003. (93)- SCLAVO. Di un rapido processo per le colora- zione delle ciglia di alcuni microorganism!. Ministera dell'intern. L-aboratori scientifica della direzione di Santa-Roma. Rev. in Ceutralb. f. Bakt. Bd. xv. p. 507, 1893. ('93). NICOLLE, M., ET MORAX, V. Technique de la coloration des cils, ' ' 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. Ci>S). BOWHILL, Tn. Eine neue Methode der Bak- iterien-Geisselfarbung bei Gebrauch einer Orceinbeize. Hyg. Rundschau, 1898, No. I. Rev. in Centralb. f. Bakt., xxm Bd., 1898, pp. 667-668. STEPHENS, J. \V. 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 largiii snlu- i. 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 Krmengem. ('99). ZETTNOW. Ueber Geisselfarbung bei Bakter- ien. Zeitscihr. f. Hyg., Bd. xxx. I Heft., March, 1899, pp. 95-106. Discusses van Erinengem'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" iu a watch glass. A drop or two of this is then placed ou 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 staiu consists of tatmic 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. Counterstaiu for the bod}- 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, 1900, pp. 597-605, i plate and I fig. ('oi). WILLIAMS, 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. Unterstichung iiber die Gallerl- bildungen des Zuckerriibensaftes. Resume allemand du memoire russe, Charkow, 1878. Not seen. ("85). RIBBERT. Zur Farbung der Pneumoniekokken. Deutsche med. Wochenschr., 11 Jahrg., 1885, p. 136. Gives a method for staining capsules. ('85). FRIEULAENDER, C. Ueber Pneumonie-Micrococ- cen. Fortsch. d. Med., Bd. in. 1885, pp. 91-93. Replies to criticism of Germain See. and gives his method of staining the capsule. ('85). FRIEDLAENDER, C. Notiz, die Farbung der Kap- selmicrococcen betreffend. Fortsch. d. Medicin, Bd. m, 1885, No. 23, pp. 757-760. The author's plan for staining capsules is as follows : Pass the dried covers three times through the flame, and then expose from one to several minutes in i per cent acetic acid water. Remove from the cover by blowing through a glass tube drawn to a point, aud dry in ail- quickly. Then expose iu a saturated solution of'anilin- water gentian violet for a very few seconds, wash iu water aud examine. The exposure to the gentian violet should be barely long enough to staiu the capsule without stain- ing the interior protoplasm. CAPSULES; STAINS AND STAINING METHODS. 221 ('92). LIESENBERG, C., u. ZOPF, W. Ueber den sogen- annten Froschlaichpilz (Leuconostoc) der europaischen Riibenzucker- und der javan- ischeu Rohrzuckerfabriken. Zopf's Beitraege, Hit. i, 1892, pp. 1-29, with 2 plates, and Hft. ii, 1892, pp. 1-2. Rev. in Am. Nat., March, 1897, p. 228. ('92). WKLCH, WM. H. A gas-producing bacillus (B. aerogenes capsulatus, nov. spec.) capable of rapid development >in the blood-vessels after death. Bull. Johns Hopkins Hospital, vol. 3, No. 24, July-August, 1892, pp. 81-91. Also a .separate. ('94). KOCH, ALFRED, AND HOSAEUS, HANS. Ueber einen neuen Froschlaich der Zuckerfabriken. lYntralb. f. Bakt, Bd. xvi, 1894, pp. 225- 228, mit i fig. Describes a branching gelatinous organism as Bact. pediculatum. The branching is due to a terminal split- ting or one-sided development of the capsule. (*9S). STIFT, A. Ueber die in den Produkten der Zuckerfabrikation auftretenden Bakterien. Centralb. f. Bakt, 2 Abt., Bd. I, 1895, pp. 277-283. ('96). WILDE, MAX. Ueber den Bacillus pneumonias Friedliinder's und verwandte Bakterien. Inaugural Dissertation, Bonn, 1896, pp. 74. Bibliography of 89 titles. Printed by Carl Georgi. ('96). FRICKE, CARL. Ueber den sogenannten Bacil- lus mucosus capsulatus. Zeitschr. f. Hyg., Bd. xxni, 1896, pp. 380-451, with a bibliog- raphy of 25 titles. ('98). PANE, N. Ueber die Genesis der Kapseln des Pneumococcus. Centralb. f. Bakt., xxiv Bd., 1898, pp. 289-294, with £ figs. The capsule is the swollen outer part of the bacterium. ('98). KAI.I'.MANN. Eine neue Methode zur Farbung von Bakterienkapseln. Hyg. Rundschau, 1898, No. 18. Abstr. Centralb. f. Bakt., I Abt., Bd. xxv, p. 32. ('99). WARD. See XLVII. ('99). MOORE, A. Capsule staining. Trans. Jenner Inst., 2(1 sen, pp. 244, 1899. Recommends a contrast stain for the capsule. The preparation is first fixed with dilute acetic acid, then stained in carbol fuchsin for about i minute, washed in distilled water and dried. It is then stained, with or without gentle heat, in night blue for i or 2 minutes, washed and dried. The night blue solution is Morton's modification of McCrorie's stain. (See Morton, Flagella staining. ) By this method the capsule is stained blue and the body dark red. ('99). STRONG, LAWRENCE WATSON. Ueber die Kap- selbacillen. Centralb. f. Bakt., xxv Bd., 1899, pp. 49-52. ('oo). BONI, ICILIO. Methode zur Darstellung einer " Kapsel " bei alien Baktcrienarten. Centralb. f. Bakt., xxvni Bd., 1900, pp. 705-707. ('01). WARD. See XLVII. foi). BONI, I. Ricerche sulla capsula dei batteri. Giorn. Soc. ital. igiene, Milano, vol xxni, 1901, pp. 417-430. ('02). SMITH, R. GREIG. An Ascobacterium from the sugar-cane, with notes upon the nature of the slime (Bacterium sacohari, n. sp.). I': Breedings of Linnean Soc. of New South \\ riles, vol. xxvii, 1902, part i, pp. 137-14^. i plate. Also a separate (issued Aug. 22, 1902). See also Central!), f. Bakt, 2 A'bt Bd. ix, p. 806. XIV. Stains and Staining Methods. (See also XI, XII, and XIII.) EHRI.ICH. Beitrage zur Kenntniss der Anilin- farbungen und ihrer Verwendung in der mikroskopischen Technik. Arch. f. mikr. Anat. Bd. xm, p. 263. ('75). WEIGERT. Farbung von Bakterien. Ber. iiher d. Sitzungen d. sehlesisch.cn Gesellsch. f. vatenl. Cultur, 10 Dec., 1875 ('77). KOCH. See LV. ('81). WEIGERT. Zur teohnik der mikroskop. Bak- terienuratersuchungen. Virdi. Archiv. Bd. LXXXIV, iSSi, p. 275. ('82). EHRLICH. Farlbung der Tuberkelbacillan Verhdlgn. d. Ver. f. i. Med., i Mai, 1882. D. mecl. Woch., 1882, p. 269. ('82). ZIEHL. Zur Farbung des Tuberkelbacillus. Deutsche med. Wochensc.hr., 1882, No. 33 P- 451- ('82). SZYSZYLOWICZ, J. Das Corallin als mikro- chenusches Reagens in d. Pflanzenhistologie, R. i. S. Ak. Krakau, T. x, 1883, pp. 97-114. Not seen. Rev. Bot. Centralb., 1886, Bd xxvin, p. 51. See also Bot. Centralb., li.l xn, 1882, p. 139. This substance should be dissolved in sodium carbon- ate. It is iised as a test for slime derived from starch which stains diffusely purple or rose. After staining the preparation should be subjected to hot alcohol, which removes the stain from all but the starch grains and slime derived therefrom. Gum slitne and cellulose slime are bleached, the former even in cold alcohol. Pure gum does not stain. Mount the preparation in Canada balsam. ('83). ZIEHL. Zur Lehre von den Tuberkelbacillen, insbesondere iiber deren Bedeutung fur Diagnose und Prognose. Deutsche med. Woohenschr., 1883, p. 62. ('84). KOCH. See vi. I 'N \ ) . LOEFFLER. See VI. ('84). GRAM, C. Ueber die isolirte Farbung der Schizomyceten in Schnitt-und Trocken- praparaten. Fortschr. d. Med, Bd n No 6, 1884, pp. 185-189. ('86). PFEFFER, W. Ueber Aufnahme von Aniliu- farben in lebende Zellen. Untersuchungen a. d. hot. Inst. Tubingen, n, p. 179, 1886. ('87). UNNA. Die RosaniJine und Pararosaniline, eine bacteriologische Farbenstudie. Derma- tologischen Studien. Heft 4, pp. 9-73. _ Hamburg, Voss, 1887. ('87). KUEHNE. Ueber ein combinirtes Universal- verfahren, Spaltpilze im thierischen Gewebe naohzuweisen. Dermatol. Studien, heraus- geg. von Unna. Heft 6. Hamburg, L. Voss, 1887. ('87). HEIM, L. Die Neuerungen auf dem Gebiete der bakteriologischen Untersuchungsmeth- oden seit dem Jahre, 1887, I. Farbunsjs- methoden. Centralb. f. Bakt., x Bd., 1891, pp. 260-265, 288-296, and 323-328. ('88). KUEHNE, H. Praktische Anleitungzum mikro- skopischen Naohweis der Bakterien im thierisohen Gewebe. Leipzig (Giinthers), 1888, pp. 15-23, Methylenblaumethode ('88). UNNA, P. G. Die Entwicklung der Bak- terien farbung. Eine historisch-kritische Uebersicht. Centralb. f. Bakt., 1888, in Bd., pp. 22-26 ; also pp. 61-63, 93-99, 120-125, IS3- 158, t89-I9S, 2I8-22T. 254-2^9, 285-291, 312- 320, 345-31^. At the end 71 paper* on this subject are mentioned by title. 222 BACTERIA IN RELATION TO PLANT DISEASES. ('91 ). PREGL, FRITZ. Ueber eine neue Karbolmethy- lenblau-Method. Ceatralb. f. Bakt., x Bd., 1891, pp. 826-829. Pregl finds the following method of staining sections on slides satisfactory : " Exposure for one-half to one minute to carbol methylen blue, sometimes with the aid of heat; second, brief washing in water; third, bleaching in 50 per cent alcohol until the section has become pale blue passing into greenish ; fourth, removal of the water by means of absolute alcohol ; fifth, clearing in xylol ; sixth, enclosure in balsam." Kuehne's carbol methylen blue is made as follows : 1.5 grins, methylen blue, 10 grins, absolute alcohol, loogramss percent carbolic-acid water. The alcohol is poured over the mtrthylcu blue ; the carbolated water is then added and rubbed up thoroughly with it. If only a small quantity is needed from time to time, it is better to make it up in less amounts, as, with time, the staining power of the solution decreases. ('92). KuEHNE, H. Das Malachitgriin als Auszieh- ungsfarbe. Centralb. f. Bakt., xi Bd., 1892, PP. 7S6-7S8. Author finds malachit green dissolved in anilin oil very useful for treatment of sections stained in carbol-fuch- siu, etc. The bacteria stand out distinctly deep red on a bluish background. See paper for details. ('93)- NICOLLE, ET CANTACUZENE, J. Proprieties colorantes de I'oxychlorure de ruthenium ammoiuacal. Ann. de 1'Inst. Pasteur, T. vii, pp. 331-334- ('9S). UNNA, P. G. Ueber Verwendung von Anilin- mischungen zur tinktoriellen Isolierung von Gewebselementen. Monatshefte f. prakt. Dermatologie, Bd. xxi, 1895. Rev. in Cen- tralib. f. Bakt., I Abt., xx Bd., 1896, p. 406. ('95)- NICOLLE, M. Pratique des colorations micro- biennes, Methode de Gram modifiee et methode directe. Ann. de 1'Inst. Pasteur, T. ix, 1895, No. 9. Rev. in Centralb. f. Bakt., XVHI Bd., 1895, p. 552. Carbol violet is substituted for nniliu violet. Excess of color is removed withalcohol-aceton. ('ho]<-igischen Veranderungen der Milz- brandbacillen bei ihrer Auflosung durch Pyocyanase. Centralb. f. Bakt., xxvn Bd., 1900, pp. 776-787, with I colored plate, ('oo). KKAUSF., PAUL. Beitrage zur Kenntnis des Bacillus pyocyaneus. Centralb. f. Bakt., xxvii Bd., 1900, pp. 769-775. Considers effect of electricity (Tesla stream); pigment formation when in symbiosis with streptococci ; beha- vior in hydrogen, carbon dioxide, illuminating gas, hy- drogen sulphid ; in vacuo ; nature of the pigments. Coo). SMITH, THEOBALD. Die Bedeutung von Varie- taten IKM pathogenen Bakterien. Centralb. f. Bakt., xxvii Bd., 1900, pp. 676-677. Coo). MATZUSCHITA, TEISI. Ueber die Verander- lichkeit der Eigenschaft des Bacillus an- thracis, Gelatine zu verflussigen. Cenitralb. f. Bakt., xxvin Bd., 1900, pp. 303-304. ('oo). MATZUSCHITA, TEISI. Die Einwirkung des Kochsalzgehaltes des Nahrbodens auf die Wuchsform der Mikroorganismen. Zeits. f. Hyg., 1900, Bd. xxxv, p. 495. CQI). ROSENFELD, A. Ueber die Involutionsformen einiger pestaihnlicher Bakterien auf Koch- salzagar. Centralb. f. Bakt., I Abt, Bd. xxx, 1901, pp. 641-653. ('02). SMITH, THEOBALD. The relation between •bovine and human tuberculosis. The Medi- cal News, New York, vol. LXXX, Feb. 22, 1902, pp. 342-346. Also a separate, pp. 14. ('02). LEPOUTRE, L. Reoherches sur la transforma- tion experimentale de bacteries banales_ en races parasites des plantes. Annales de 1'inst. Pasteur, T. xvi, 1902, pp. 304-312. ('04). MAASSEN, ALBERT Die teratologischen Wuchsformen (Involutionsformen) der Bakterien und ihre Bedeutung als diagnos- tisches Hilfsmittel. Arbeiten a. d. Kaiser- lichen Gesundheitsamte, Berlin, 1904, Bd. xxi, Heft 3, pp. 385-400. pi. x to xv. XVI. Culture-Media. ('59). PA YEN. Sur la gelose et les nids de salangane. C. R. des se. de 1'Acad. des sci., Paris, T. XLIX, 17 Oct., 1859, pp. 521-530. According to Payen. agar-agar has the following aver- age composition : Carbon, 42.770; hydrogen, 5. 775; oxygen, 51.445; total, 100.000. ('82). 'N.AEGELI, C. v. Untersuchungen ueber Niedere Pilze. a. d. Pflanzenphys. Inst. i. Muenchen. 1882, pp. 1-285. ('86). GUILLEBEAU, A., ET DE FREUDENREICH, ED. Preparation des gelees a base d'agar-agar. Archiv. des sci. phys. et nat, 3e Periode, T. xv, Geneve 1886, pp. 466-468. This author describes a method of making agar with- out filtering. Cook for a quarter of an hour a water solution of agar (2 per cent), salt 0.5 per cent, and pep- tone i per cent. This is alkaline ; neutralize. Add an equal quantity of bouillon prepared the day before. This bouillon contains salt and peptone in the same pro- portion (I kilo, of meat for 2 litres of water). Cook in the autoclave for two hours at a temperatuoe of 120° to 125° C. Do not pass 130°, as agar changes color. Remove and let stand. After 4 to 5 hours one has in the upper part of the flask a limpid liquid with all impurities at the bottom. During this time the temperature must be kept above 42°. Decant and sterilize at 110° C. ('86). MIQUEL, P. "De la culture des bacteries" an Septieme Memoire sur les organismes micro- so ipique de 1'air et dex eaux. Annuaire de 1'iibserv. a Montsouris pour 1885. See espe- cially pp. 569-570. This deals with use of Fucus crispus as basis ior a solid culture-medium. ('87). SCHOTTELIUS, M. Einige Neuerungen an bac- teriologischen Apparaten. 2 Vollstandig klarer Agar-Nahrboden. Centralb. f. Bakt., 1887, it Bd., pp. 100-101. Agar is soaked about five minutes in 2 per cent hydro- chloric acid and then washed in frequent changes of water. Five to ten per cent of this agar is then macerated over night in bouillon at room temperature ; cooked ; peptone and salt added ; neutralized with carbonate of soda or potash ; again cooked, and finally passed through filter paper. 224 BACTERIA IN RELATION TO PLANT DISEASES. ('87). DAL Pozzo, D. Das Eiweiss der Kiebitzeier als Nahrboden ftir Mikroorganismen. Med. Jahrb. Jahrg., 1887, pp. 523-529; ('87). ABBOTT, A. C. An improvement in .the method of preparing blood serum for use in bac- teriology. Medical News, 1887, vol. i, p. 207. Rev. in Centralb. f. Bakt., II Bd., 1887, PP. 424-425- ('87). RASKIN, M. Zur Ziichtung der pathogenen Mdkroorganismen auf aus Milch bereiteten fcsten und durchsicbtigen Nahrboden, St. Petersburger med. Woclienschr, xn Jahrg., 1887, pp. 357-360. ('88). VON FREUDENREICH, E. Zur Bereitung des Agar-Agar. Centralb. f. Bakt., 1888, III Bd., pp. 797-798. Recommends filtering nutrient agar in the autoclave at about i io°C. Time required 30 to 60 minutes. ('88). VAN PUTEREN. Ueber die Herstellung von festen Nahrboden aus Milch zu Mikro- organismen Kulturen. (Russian.) 1888. ('88). HUEPPE, FERDINAND. Ueber die Verwendung von Eiern zu Kulturzwecken. Centralb. f. Bakt., 1888, iv Bd., pp. 80-81. Cultures are made in the uncooked eggs after shaking and proper surface sterilization. ('88). Roux, E. De la culture sur pcwnme de terre. Ann. de 1'Inst. Pasteur, T. II, 1888, pp. 28-30, 2 figs. ('89). PETRI, R. J. Ueber den Gehalt der Nahrgela- itine an Salpetersaure. Centralb. f. Bakt., v Bd., 1889, pp. 457-460. Commercial gelatin generally contains nitrates in con- siderable quantities. (>'ij). VOIGTLAENDER, FELIX. Ueber die Diffusion in Agargallerte. Zeitschr. f. Physik. Chemie., Bd. in, 1889, pp. 316-335- A study of some of the physical properties of agar. The rate of diffusion of many acids and other substances was determined. The conclusions are : 1. The diffusion in agar jelly from watery solutions is not disturbed by the process of imbibition. 2. The validity of pick's law for dilute solutions was amply demonstrated. 3. The rapidity cf diffusion of a substance in different concentrations of the agar jelly is the same. The diffusion constants observed in the jelly are like those of water or greater or less. 4. With rise of the temperature the constant does not increase in linear relation, but the amount of the salt which enters in increases. ('89). REIN, J. J. The industries of Japan; together with an account of its agriculture, forestry, arts, and commerce ; from travels and re- searches undertaken at the 'cost of the Prus- sian Government; with 44 illustrations and 3 maps. London, Rudder & Stoughton, 27 Paternoster Row, 1889, pp. xn, 570. 2d re- vised German edition, 1905. The cartilaginous Florideie, particularly species and varieties of Gigartineie, Caulacantheie, Gelide, Sphiero- coccea;, and Tylocarpece, are distinguished for their high proportion of pararabin, and furnish, with boiling water algse-jelly. They are gathered in great quantity on all the coasts of the Malay Archipelago and the waters of China and Japan, and are utilized in part direct as food, partly in the preparation of algii'-glue, Jap. Fu-nori, or .illiM.-jelly, Jap. Kanteu. In trade these articles, both raw and dried, and when further prepared, are designated by the Malay word Agar-Agar, i. e., vegetable. This name 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 «'t them Hai-Thao, a transparent glue, with which they stiffen silk 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 Nahrgelatine an Salpetersaure." Cen- tralb. f. Bakt., v Bd., 1889, pp. 679-680. ('90). KUEHNE, W. Kieselsaure als Nahrboden ftir Organismen. Zeitsohr. f. Biol. Neue 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 fur Agar ; 2, Ersatz fur Gelatine. Centralb. f. Bakt, x Bd., 1891, pp. 122-124. ('92). PETRI, R., UND MAASZEN, ALBERT. Ueber die Bereitung von Nahrbouillon fur bakteriolo- gische Zwecke. Arbeiten aus d. kaiserl. Gesundheitsamte, Bd. vm. No. 2, 1892, pp. 3H-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 chromogeuic 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. overwith 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 stir 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-August, 1892, p. 92. A useful and easy method. ('93). USCHINSKY. Ueber eine eiweissfreie Nahr- losu-ng fiir pathogcne Baikterien, nebst einigen Bemerkungen iiher Tetanusgift. Centralb. f. Bakt., Bd. xiv, 1893, No. 10, pp. 316-319- ('93). NASTIUKOFF. Ueber Nahrboden aus Eigelb fiir 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 ilrs Nahrbodens auf das Wachsthum der l',.ikterien. Zeitschr. f. Hygiene, Bd. xv. 1893, pp. 183-191. 3 pla.u-. ('94). FRAENKEL, C. Beitrage zur Kenratniss des I lakterienwachsthums auf eiweissfreien Niihrlosungen. Hyg. Rundschau, Jahrg. iv. 1804, pp. 769-776. CULTURE- MEDIA. 225 ('94). WESCNER, F. Die Bereitung ernes festen un- durcksdphtigen Nahrbodens fiir Bakterien aus Hiihnereiern. 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 Streptokokkenziicbtung auf sauren Nahrboden. Centralb. f. Bakt., Bd. xvn, i Abt., 1895, pp. 865-874. ('95). SEDGWICK AND PKESCOTT. See XLVI. ('95). ELSNER. Untersuchungen iiber eleatives 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 fiir Bakterien. Centralb. f. Bakt., xvni 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- 245. ('95). 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., xvni 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 agar powder is then added and cooked i hour in streaming steam. ('95). MAASSEN, ALBERT. Die organischen Sauren als Nahrstoffe und ill re Zersetzbarkeit durch die Bakterien. Arb. a. d. kaiserl. Gesundheitsamte, Bd. xn, Zweites Heft, 1895, pp. 340-411. ('96). CAPALDI, ACHILLE. Zur Verwendung des Eidotters als Nahrbodenzusatz. Centralb. f. P.akt., 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 autbor obtains sterile nutrient gelatin melting at 2g°-3O° C. ('97). STODDART, F. WALLIS. New meithod 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- dunnung 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, mouopotassium 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. Schnelle 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. n, 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. 379-380. 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. xxxn, 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. Uelber das verschiedene Verhalten 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. ( oo). GLAESSNER, PAUL. Ueber die Verwertbarkeit einiger neuer Eiweisspraparate zu Kultur- zwecken. Centralb. f. Bakt., xxvn Bd., 1900, PP- 724-732. Comparative tests of somatose, nutrose, etc. ('02). YENDO, K. Uses of marine Algre in Japan. Postelsia, St. 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 Herstelhmg von Nahrboden imd zur Bakterienziichitung. Ztschr. f. Hyg., 1904, Bd. XLVI, pp. 1-22. Discusses effect of adding various acids aud 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 Keuntniss der Micro- coccen. Arch. f. exp. Path. u. Pharmakol., 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. ('Si). KOCH, ROBERT. Zur Untersuchung von patli- 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 media, viz : by use of gelatine for streaks and poured plates. See pp. 24 and following, especially page 27. In 1881, orearlier, Buchner diluted his anthrax fluid until it 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. i'X4>. HOFFMANN, F. W. Einfachcr Einbettungs- apparat. Zoologisoher Anzeiger, vn 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 Chine en Microscopic. Bull. Soc. Beige d. Microscop., 1884. Not seen. The ink is used to form a contrasting back- ground, ('86). VON ESMARCH, EfiwiN. Ueber eine Modifika- ition des Koch'schen Plattenverfahrens zur Isolierung und zum quantitative!! Nachweis von Mikroorganismen. — Zeiitsch. f. Hygiene, 1886, Bd. I, pp. 293-301, 3 figs. The method of roll cultures is here described. ('87). PETRT, R. J. Eine kleine Modification des Koch'schen Plattenverfahrens. Centralb. f. Bakt., i Bd., 1887, No. 9, pp. 279-280. Description of the now everywhere common I'etri- dishes. I'S;). ESMARCH. See xxxni. ('89), I'.EYEKINCK, M. W. L'auxanographie on la mstjhode de I'hydrodiffusion dans la gelatine appliquee aux recherches microbiologiques. Arch, neerlandaises de sci. ex. et nat., 1889, T. xxin, pp. 367-372. ''•/MI KUEISLER. I'liirr-iu-hungen iiber die Brauch- liarki-it iK-r "Filtres san-- prcsMun, Systeme Chamberland-Pasteur." Zeitschr. f. Hyg., r.'i. VIM, iX'io, pp. 48-54. ('91). NUTTAIX, GEORGE H. F. A method for the estimation of the actual number of Tubercle bacilli in tuberculous sputum. With a note on the general application of the method to bacteriology. Bull, of the Johns Hopkins Hospital, vol. II, 1891, pp. 67-76. By use of this method flasks or tubes of media can be inoculated, it is said, with predetermined exact num- bers of bacteria. ('91). BUJWID, O. Eine einfaohe Filtervorrichtung zum Filtriren sterilisirter Fliissigkeit. Cen- tralb. f. Bakt., ix Bd., 1891, pp. 4-5, mit i Abbildung. ('91). GABRITSCHEWSKY, G. Zur Technik der foak- teriologischen Untersuchungen, I. Graduirte Kapillarpipetten zum Abmessen sehr kleiner Fliissigkeitsmengen. Centralb. f. Bakt., x Bd., 1891, pp. 248-250, mit 2 figs. ('91). NORDTMEYER, H. Ueber Wasserfiltration durch Filter aus gebranniter Infusorienerde. — Zeitsoh. f. Hygiene, 1891, Bd. x, pp. 145- 154, I fig. ('91). SMITH, THEOBALD. Kleine bakteriologische Mittheilungen. Centralb. f. Bakt., x Bd., 1891, pp. 177-186, mit 2 figs. First two notes relate to apparatus; the second, to his inside-out method of filtering through Chamberland bougies (figured). ('91). KNAUER, FRIEDRICH. Eine bewahrte Methode zur Reinigung gebrauchter Objekttrager und Deckglasdien. Centralb. f. Bakt., Bd. x, 1891, pp. 8-9. Even old stuck-together slides are said to be cleaned perfectly by boiling 30 minutes in 10 per cent lysol, with occasional stirring. They should then be flooded with tap water, and finally wiped with a soft, clean, oil-free cloth. " In recent preparations, 14 days old, I obtained even by 15 minutes boiling in 5 per cent lysol solution a perfect cleaning of the glass." ('91). ALTMANN, P. Thermoregulator neuer Kon- struktion. Centralb. f. Bakt., ix Bd., 1891, pp. 791-792. ('91). BEYERINCK, M. W. Die Kapillarhebermikro- skopirtropfenfiasche. Centralb. f. Bakt., ix Bd., 1891, pp. 589-590, mit i Abbildung. An easily constructed useful water flask, furnishing drops of any size (see fig. 16) or a fine stream. ('92). HASWELL, WM. A. On a simple method of substituting strong .alcohol for a watery solution in the preparation of specimens. Proc. Linn. Soc. N. S. W., vol. VI, for 1891, pp. 433-436, i fig. Sydney, 1892. This method was worked out 10 years later, indepen- dently, in my laboratory, by Deane B. Swingle, who had his paper and drawings ready for publication when this earlier paperwas discovered. Swingle's device, which is a considerable improvement on Haswell's, is showu in Fig. 146. By means of the expanded base of the inner tube a quite equable diffusion of the alcohol is secured. ('92). GILTAY, E., u. ABERSON, J. H. Methode zur Prnfung von Filtereinrichtungen wie die Chamberland-Bougies. Cenitralb. f. Bakt., iScu, Bd. XII, pp. 92-95, I fig- ('92). v. FREUDENREICH, ED. Ueber die Durchlas- sigkeit der Chamherland'schen Filter fiir Bakterien. Centralb. f. Bakt., xn Bd., 1892, pp. 240-247. The author's experiments lead to the conclusion that the Chamberland filter cannot be used safely more than a week for the continuous filtration of water. It must then be sterilized. METHODS OF WORK, APPARATUS, ETC. 227 ('92). SMITH. THEOBALD, AND MOORE. V. A. Zur Priifung 'der PasteU'r-Ohamberland Filter. Centralb. f. Bakt., xn Bd., 1892, pp. 628-629, mit i Abbildung. ('92). ALTMANN, P. Ein neuer Thermoregulator fiir Petroleumheizung bci Thermostaten. Centralb. f. Baht., xn Bd., 1892, pp. 654-655. mit 2 figs. ('92). ARLOING, G. De I'uifluence des filtres mineraux sur les iliquides contenant des substances d'originc microbienne. C. R. des se. de 1'Acad. des sci., T. cxiv, 1892, pp. 1,455-1.457. ('92). DE FREUDENREICH. De la permeabilite des filtres Chamberland a 1'egard des bacteries. Annales de Micrographie, 1892, Tome iv, PP. 559-568. ('92). HOLM, JUST CHR. Sur les methodes de culture pure et specialment sur la culture sur plaques de M. Koch et la 1 unite des erreurs de cette im'thode. Meddelelser fra Carlsberg Lab. Tredie Bind. Andet Hefte. Kjobenhavn, 1892, pp. 1-23. Danish edition, Tredie Bind. Forste Hefte. 1891, pp. 1-32. ('92). NENCKI AND ZAWADZKI. See XLVII. ('92). ALTMANN, P. Neue Mikrogaslampen nls Sicberheitsbrenner. Centralb. f. Bakt., xn Bd., 1892, pp. 786-787, 3 figs. ('93). ZETTNOW. Reinigung von neuen Deckglasern. Centralb. f. Bakt., Bd. xiv, 1893. pp. 63-64. The last traces of fat are best removed from covers by burning. The clean covers are placed on anSto lo cm. square piece of sheet iron, which is then heated for a few minutes in the open flame of a Bunsen burner. ('93). SCHEPILEWSKY, E. A. Ein Regulator zum Thermostaten mit Wasserheizung. Cen- tralb. f. Bakt., Bd. xrv, 1893, pp. 131-138, miit I fig. ('93). GRUBER, MAX. Gesichtspunkte fiir die Priifung und Beurtciking von Wasserfiltern. Cen- tralb. f. Bakt., Bd. xiv, 1893. pp. 488-493. ('93). KIRCHNER, MARTIN. Gesichtspunkte fiir die Priifung und Beurteilung von Wasserfiltern. (Entgegnung auf die gleichnamige Arbeit von Prof, Max Gruber in Wien). Centralb. f. Bakt., Bd. xiv, 1893, pp. 516-527- ('93). BRUNNER, G., AND ZAWADZKI, A. Zahlplatte zu den Petri'schen Schalen. Centralb. f. Bakt., Bd. xiv, 1893, pp. 616-618, mit I fig. A method is given for dividing a circle mem. in diame- ter into 64 equal parts. The circles and sections are to be in white ink on black paper. (-93)- SCHOEFER, HANS. Ueber das Verhalten von pathogenen Keimcn in Kleinfiltern. Cen- tralb. f. Bakt., Bd. xiv, 1893, pp. 685-693. ('93). LAFAR, FRANZ. Fine neue Zalilvorrichtung fiir Plattenktilturen in Petrisohalen. Zeitsch. f. Nahrungsmitteluntersuchung u. s. w., W'ien, 1893. No. 24, p. 429. Rev. in Centralb. f. Bakt., Bd. xv. 1X04. pp. 331-333, mit I fig. ('94). LoEFFLER, F. Eine sterilisirbare Injections- spritze. Centralb. f. Bakt., Bd. xvi, 1894, PP. 729-731. To be had from Wittig, instrument maker in Greifs- wald, Germany. ('94). HOUSTON, A. C. Note on a simple apparatus for collecting samples of water for bac- teriological purposes, ait different doplh-. from the surface. Jour, of Path, and Bart., vol. II, 1894, pp. 496-497, I fig- ('94). VALLIN, E. Le regeneration par agents chimiques des filtres Chamberland. Revue d'hygiene eit de police sanitaire, 1894, No. 11, p. 946. Rev. in Centralb. f. Bakt., xvn Bd., 1895, pp. 496-497- Author recommends a sodium bisulphite solution (1:20) for cleaning filters. ('94). KUPRIANOW, J. Zur Methodik der keimfreien Gewinnung des Blutserums. Centralb. f. Bakt., xv Bd., 1894, pp. 458-462, I fig. Describes and figures a device for filling test tubes with exact amounts of fluid culture media. ('94). FUNCK, ERNST. Zur Frage der Reinigung der Deckgluser. Centralb. f. Bakt., Bd. xvi, 1894, pp. 113-114- Author cleans slides and covers which have been used with oil .Canada balsam, stains, etc., by first placing them for a time in turpentine, separating the covers from the slides as far as possible. They are then put into a broad beaker, covered with hydrochloric acid, to which is added a few grams (2 to 3 knife-points) of chlorate of potash . This is then covered with a glass plate and heated on a water bath for a few minutes. The glasses are then washed in hot water. Equal parts of soda, talcum, and sieved sawdust are now added with water enough to make a thick fluid mixture, which is now heated for one- half hour on the water bath, with frequent shaking of the contents of the beaker. The glasses are then washed again in hot water, to which a little hydrochloric acid has been added, especially, in case some calcium carbon- ate has been deposited on the glass. Finally, wash in hot water or ethyl alcohol and dry with a soft cloth. ('94). VAN HEST, J. J. Bakterienluftfilter und Bak- terieniluftfilterversohluss. Centralb. f. Bakt., Bd. xvi, 1894, pp. 435-447, also pp. 495-49?). mit 11 figs. ('95). KNAUSS, K. Eine einfache Vorrichtung zum Abfiillen von je 10 ccm. Nahrsubstanz. Cenitralb. f. Bakt., xvn Bd., 1895, pp. 878- 8/9, with I fig. ('95). BANTI, G. Eine einfaohe Methode die Bak- iterien auf dem Agar und dem Blutserum zu isolieren. Centralb. f. Bakt., xvn Bd., 1895, PP. 556-557. Agar is slanted, in broad tubes (diam. 2-3 cm). The material containing the bacteria is diluted to the proper amount with sterile bouillon or water. The condensation water is then inoculated by needle or loop, and subse- quently tilted over the whole surface of the agar and allowed to drain back again. ('95). BLEISCH, MAX. Ein Apparat zur Gewinnung klaren Agars ohne Filtration. Centralb. f. Bakt., xvn Bd., 1895, pp. 360-362. ('95). LODE, ALOIS. Eine automatische Abfiillburette fiir Niihrlosungen und Heilserum. Centralb. f. Bakt., xvin Bd., 1895, pp. 53-54, with 3 figs. ('95). BRUNNER, CONRAD. Notiz zur Methode der Isolierung von Bakterien auf Agarplatten ini Reagensglase. Centralb. f. Bakt., xvin Bd., 1895, p. 59. ('95). BUJWID, O. Bemerkungen iiber die Filtration bakterienhaltiger Fliissigkeiten. Centralb. f. Bakt., xvm Bd., 1805, pp. 332-333. Considers the Chamberland a less fragile and safer filter than the Berkefeld. ('95). ABEL, RUDOLF. Ein Halter fiir Objekttrager und Deckglaschen. Centralb. f. Bakt., xvm Bd., pp. 782-783, with I fig. ('96). MELNIKOW-RASWEDENKOW, M. Ueber die Einstellting des d'Arsonvalschen Thermos- taten. Centralb. f. Bakt., xix Bd., 1896, pp. 709-712, with I fig. ('96). CZAPLEWSKI. Bakteriologische Notizen. Cen- tralb. f. Bakt., P.d. xx, 1896. pp. 307-313. Sixteen notes on methods of work. 228 BACTERIA IN RELATION TO PLANT DISEASES. ("96). KRETZ, RICHARD. Eine handliche und leicht sterildsierbare Abfiillvorrichtung fiir Kultur- tlussigkeiten. Centralb. f. Bakt., xix Bd., 1896, pp. 73-74, with I fig. ('97). VAN'T HOFF, H. J. Eine schnellere und quan- titativ bessere Methode der bakteriologis- chen Platitenzahlung. Centralb. f. Bakt., xxi Bd., 1897, pp. 731-733, with I fig. Pours a suitable dilution of the bacteria on surface of gelatin or agar, and spreads fluid as widely as possible. ('97). BOLLEY, H. L. An apparatus for the bac- teriological sampling of well 'Waters. Cen- tralb. f. Bakit., xxn Ed., 1897, pp. 288-290, with i fig. ('97). KASPAREK, THEODOR. Ein Vacuum- Apparat zum Abdampfen von Kulturen mit Ehmann'- scher Wasserheizung. Centralb. f. Bakt., xxn Bd., 1897, PP- 6-7, with i fig. ('97). KISCHENSKY, D. Ein Verfahren zur schnellen mikroskopisclien Untersuchung auf Bak- terien in Deckglas- und Objekttrager- Praparatem. Centralb. f. Bakt., xxi Bd., 1897, pp. 876-877. ('97). SMITH, THEOBALD. Uelber Fehlerquellen bei Priifung der Gas- und Saurebildung bei Bakterien und deren Vermeidung. Centralh. f. Bakt., xxn Bd., 1897, pp. 45-49. ('97). Now, F. G. Neue Apparaite zum Filtrieren und zum Sterilisieren durch Dampf. Cen- tralb. f. Bakt.. xxn Bd., 1897, pp. 337-340, with 3 figs. ('97)- SCHUERMAYER, B. Eine Abanderung des automabischen Gasabschlusses beim Ver- loschen der Flammen an Briitschranken. Centralb. f. Bakt., xxi Bd., 1897, pp. 400- 401, with i fig. ('97). ROBERTSON, SIGISMUND. Ueber Objekttrager- und Deckglashalter. Centralb. f. Bakt., xxi Bd., 1897, pp. 589-591, with 2 figs. ('98). PIORKOWSKI. Ein neuer heizbarer Parbetisoh. Deutsch. med. Wochenscbr., 1898, No. 20. Rev. in Centralb. f. Bakt., xxrv Bd., 1898, pp. 902-903, i fig. ('98). SMITH, THEOBALD. One of the condition^ under which discontinuous .sterilization may be ineffective. Journal of Experimental Medi- cine, vol in, 1898. Rev. in Centralb. f. Bakt.. xxvi Bd.. 1899, p. 585. ('98). NOVY, F. G. Ein neuer Thermoregulator, Centralb. f. Bakt., xxm Bd., 1898, pp. 1,054- 1,056, with 2 figs. Made by Greiuer and Friedrichs, Stuetzerbach, Thurin- gia, Germany. ('98). MURRILL, PAUL. Ein wirksamer Gasdruckreg- ulator. Centralb. f. Bakt., xxm Bd., 1898. pp. 1,056-1,059, with 2 figs. ('99). KERN, FERDINAND. Eine nutomaitische Mess- pipette fiir keimfrcie Fliissigkeiten. Cen- tralb. f. Rakt., xxv Bd., 1800. pp. 7.5-77, with i fig. ('99). NOVY. F. G. Collodium Sacs. See his book entitled Laboratory work in bacteriology, pp. 496-501. ('oo). BULLOCH. WILLIAM. A simple apparatus for obtaining plate cultures nr surface growths of obligate anaerobes. Centralb. f. Bakt., xxvil Bd., 1900, ,pp. 140-142, with I fig. Author uses the unguentum resince of the British Phar- macopoeia as a luteing material. This consists of resin in powder 200 grams, or 8 ounces ; yellow beeswax 200 grams, or R ounces : olive oil 200 grams, or S ounces, and lard l.so grams, or 6 ounces. Melt together with gentle heat the resin and wax, add the other ingredients, strain through muslin, and cool with stirring. Coo). WRIGHT, JAMES H. A simple method for anaerobic cultivation in fluid media. Cen- tralb. f. Bakt., xxvii Bd., 1900, pp. 74-75, •with i fig. Coo). STEWART, C. BALFOUR. Apparatus for heating cultures to separate spore-bearing micro- organisms. Centralb. f. Bakt., xxvn Bd., 1900, pp. 366- 367, with i fig. This is a modification of Meyer's hot-air bath. Coo). PETRI, R. J. Eine einfache Vorrichtung zum Abfullen der Nahrgelatine. Centralb. f. Bakt., xxvn, Bd., 1900, pp. 525-526, i fig. Coo). NUTTALL, GEORGE H. F. Ein Apparat zur Herstellung von Rollkulturen. Centralb. f. Bakt., xxvn Bd., 1900, pp. 605-609, with 2 figs. Coo). PIORKOWSKI. Ein Apparat zur Ermittelung von Desinfektionswirkungen. Centralb. f. Bakt., xxvn Bd., 1900, pp. 609-610, with i fig. Coo). EPSTEIN, STANISLAUS. Ein neuer TJiermoreg- ulator. Centralb. f. Bakt., xxvin Bd., 1900, PP- 503-504, with i fig. Coo). PETRI, R. J. Ein neuer Reagenzglasstander fiir Kulturen. Centralb. f. Bakt., xxvin Bd.. 1900, pp. 747-748, with I fig. ('oo). SMITH, R. GREIG. The measurement of bac- teria. Proceedings Linnean Soc., New South Wales for 1900, Sydney, 1901, vol. xxv, pp. 533-536. Three figures dn text. Also n separate (issued Nov. 22, 1900). Coo). PETRI, R. J. Neue verbesserte Gelatine- Sohhlchen f verbesserte Petri-ScMlchen). Centralb. f. Bakt., xxvin Bd., 1900, pp. 79- 82,with 3 text figs. This Petri-dish has a cover of yellow brown glass, of such a form that when they are piled one above the other all are protected from the action of the violet rays of the spectrum. These are made in two forms by Paul Alt- maun, Berlin. ('oo). PETRI, R. J. Neue anaerobe Gelatine-Schal- chen-Kultur (verbesserte Patri-Schalchen). Centralb. f. Bakt., xxvin Bd., 1900, pp. 196- 199, with 2 text figs. Coo). WRIGHT, J. H. A simple method of cultivat- ing anaerobic bacteria. Jour. Boston Soc. Med. Sci., vol. v, 1900, pp. 114-115. The plug is pushed part way down the test-tube con- taining the culture. The cotton is then partly saturated with strong pyrogallic acid water I equal bulks of water and acid). Sodium hydrate solution (i NaOH, 2 water) is then pipetted on, and the tube immediately plugged air- tight with a soft rubber stopper. The media should con- tain i per cent glucose. It should be fresh, and its reac- tion not more acid than -f- 15. It may be used for fluids, or roll cultures, and other forms. ('02). HILL, HIBBERT WINSLOW. "Hanging block" preparations for the microscopic observa- tion of developing bacteria. Journal of M«d. Research, Boston, vol. vn, 1902 (new ser., vol. n), pp. 202-212, 3 figs. Also a separate. ('02). WHERRY, WM. B. Experiments on the per- meability of the Berkefeld filter and the Pas- teur-Chamberland bougie to bacteria of small size. Jour, of Med. Research, vol. vni (n. s.. vol. in). 1902, pp. 322-328, I fig. ('02). KELLERMAN, KARL. A method for fixing and sectioning bacterial colonies, fungous my- celium, etc. Tour. App. Micro., vol. v, 1902, p. 1,080. Also a separate. ('02). KELI.ERMAN, KARL F. An improved method for making collodion tubes for dialyzing. Jour. App. Micro., vol. v, 1902, p, 2,038. METHODS OF \V<)U|<; MKANS OF DIFFERENTIATING. 229 ('02). CARNOT, PAUL, ET GARNIER, MARCUL. Sur la technique des cultures en tubes de sable. Paris, C. R. soc. biol., T. LIV, 1902, pp. 748- 750. ('02). CARNOT, PAUL, ET GARNIER, MARCEL. De IVmploi des tubes de sable comme inethode gOnerale d'etude, d'isolement et de selection des microorganismes mobiles. Paris, C. R. soc. biol., T. LIV, 1902, pp. 860-863. ('02). RECAUD, CL. Nouveau bain de paraffine a cbauffage et regulation electriques. J. anat. physiol., Paris, T. xxxvm, 1902, pp. 193- -M4, av. fig. ('02). GRIJNS, G. Eine einfache Vorrichtung, inn zu verliindern, dass beim Gebrauch des Briitapparates fiir konstante niedrige Tem- pcratur, System Lautenschlager (Katalog No. 60, No. 117), wenn das Eis im Behalter ausgeht, das ungekiiblte Wasser in den kalten Schrank fliesst. Centralb. I. Bakt., Abt. I, Bd. xxxi, Originate, 1902, pp. 430-432. ('02). HARRIS, NORMAN MACLEOD. Concerning an improved method of making collodium sacs Centralb. f. Bakt., Abt. I, Bd. xxxn, Orig- inale, 1902. pp. 74-80. Dr. Harris makes his sacs around gelalin capsules. ('03). GORSLINE, CHARLES S. On the preparation and use of collodium sacs. Vaughan Quarter Century Book, 1903, pp. 390-394. Ann Arbor, Mich., George Wahr. ('03). WIN SLOW, C. E. A., AND NIBECKER, C. P. The significance of bacteriological .methods in sanitary water analysis. Technology Quar- terly, vol. xvi, 1903, pp. 227-239. Also a separate. ('04). REMLINGER, P. Le passage du virus rabique a travers les filtres. 2e mem. Ann. de 1'Inst. Pasteur, T. xvin, 1904, pp. 150-164. ('04). Report of the [English] committee appointed to consider the standardisation of methods for the bacterioscopic examination of water. Journal of State Medicine, August, 1904. See also Chemical News, vol. xc, Oct. 7, 1904, pp. I/7-I79- Distilled-water agar and distilled-water gelatin are recommended for use along with nutrient agar and gelatin. In the search for B. colt the committee recom- mends either the glucose-formate broth of Pakes or the bile-salt broth of MacCoukev. XVIII. Special Means of Differentiating Bacteria. ('84). GRAM. See xiv. ('87). VON ROZSAHEGYI, A. Ueber das Zuchten von Bakterien in gefarbter Nahrgelatinc. Cen- tralb. f. Bakt., n Bd., 1887, No. 14, pp. 418-424. Author added various substances to his culture media e. g. fuchsin, methylen blue, gentian violet, vesuvin, etc. Different bacteria behave very differently as respects growth, absorption of pigment, and change of color in the pigment. Organisms may be differentiated in this way. ('88). BU.TWID, O. Neue Methode zum Diagnos- ticiren und Isoliren der Cholerabakterien Centralb. f. Bakt., 1888, iv Bd., pp. 494-496. _ In 24 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 iudol and a trace of nitrite. ('90). PETRUSCHKY, JOHANNES. Die Farbenreaktion bakterieller Stoffwechselprodukte auf Lack- mus als Beitrag zur Charakteris.tik und als Mittel zur Unterscheidung von Bakterien- arten. Centralb. f. Bakt., vn Bd., 1890, pp. 1-8 and 49-53- ('92). BUJWID, ODO. Eine neue biologische Reaktion fiir die Cholerabakterien. Centralb. f. Bakt., xn Bd., 1892, pp. 595-596. ('92). BEYERINCK, M. W. Notiz fiber die Cholera- roMireaktion. Centralb. f. Bakt., xn Bd 1892, pp. 715-718. ('93). SCHILD. Formalin zur Diagnose des Typhus- bacillus. Centralb. f. Bakt., Bd. xiv~ 1893, PP. 7I7-7I8. 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 corn-munis, 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. Author tests suspicious water by poured plates. After 14 hours at 37.5° C. all blue colonies resembling typhoid are tested further, i. e., in milk, gelatin, bouillon, nitrate solution, and sugar media. ('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- terienfarbstoffe 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 abdominaliis vom Bacillus coli com- mune. Centralb. f. Bakt., Bd. xvi, 1894, pp 817-820. Uses various " chromo-agars " for differentiating bac- teria, i.e., agar with fuchsin, malachit green, nigrosiu and indohn. Sodium bisulphit must be used with tin- the first two. ('94). LUNKEWICZ, M. Eine Karbenreaktion auf die salpetrigc Saure der Kultnren der Cholera- bacillen und einiger anderer Bakterien Centralb. f. Bakt., Bd. xvi, 1894, PP- 945-949. Describes the preparations of the Griess-IIosvav re- agent. ('94). ABEL, RUDOLF. Ueber die Brauchbarkeit der von Sohild angegebenen Formal inprobe zur Differential-Diagnose des Typhus bacillus Centralb. f. Bakt., Bd. xvi, 1894, pp. i 041- 1,046. Finds some bacteria more tolerant of formalin than was stated by Schild. It is not a satisfactory method for separating typhoid bacilli irom 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. 4I9-4.U. Notes on synthetic media. ( '( i- i . 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. Central*), f. Bakt, xxiv Bd., 1898, pp. 518- 525, colored diagram. CyS). FERMI. See xxxvi. ('98). ROTHBERC.ER, C. JULIUS. Differential diagnos- tische Unte.rsuchungen mil gefarbten Nahr- boden. Centralb. f. Bakt., xxiv Bd., 1898, PP- 5i3-5i8. 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 10 cc. of fluid agar, and then \. cc. of a 24-hoiir 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'novo colorito in verde-cupo dal caffe crudo, come mezzo diagnosttco di svUuppi batterici. Gazz. degli ospedal.i e della cliniche, 1898, No. 31. Rev. in Centralb. f. Bakt., xxv Bd., 1899, p. 257. ('99.) ROTHBERGER, C. Juuus. Differential diagnos- tische Unters'uchungen mit gefarbten Nalir- boden. II, Mitteilung. Centralb. f. Bakt., xxv Bd., 1899, pp. 15-17 and 69-75. Author tested the effect of various bacteria oil 35 anilin dyes, 13 of which proved useful in agar media. The loss of color in methylen blue, safranin, Toluidin blue, Or- seille extract and indigo carmiu is due to reduction pro- cesses. In case of the indigo carmiu, the blue was first changed to dark green. ('oo). ScHEFFLER, W. Das Neutralrot als Hilfsmittel zur Diagnose des Bacterium coli. Centralb. f. Bakt., xxviii 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 II Moel- ler, Butterbacillus Petri-Rabinowitsch, Thimotheebacillus Moller). Miinchener med. Wochenschr., Bd. XLVIII, 1901, pp. 1,483-1,484- i'oj). 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. VIH, 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 lo, Lie big'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 yed Indvielsen af Statens Serum- institut. Kjobenhavn, 1902, pp. 1-39. ('02). BUXTON, B. H. A comparative study of the bacilli intermediate between B. coli com- munis 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., 1902. Pub. Columbus Ohio, 1903, vol xxvni, pp. 402-412. Also a separate, pp. 11. ("03). SCHUEDER. Zum Nachweis der Typhusbak- terien im Wasser. Zeitschr. f. Hyg., Bd. XLII, 1903, 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 infusoires vivant sans gaz oxygene libre et determinant des fermentations. — 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 Now, F. G. Die Kultur anaerober Bakterien. Centralb. f. Bakt., Bd. xiv, 1893, pp. 581- 6oO, 2 figS. The " Novy jars " are here figured and described, and 49 references to literature are given at the end of the article. <'i>3). BKYERINCK, M. W. Ueber Atmungsfiguren benveglicher Bakterien. Centralb. f. Bakt., Bd. xiv, 1893. pp. 827-845, mit i Tafel. ('93). SMITH. See xx. ('94). NICOLAIER. Bemerktmg zu der Arbeit von Prof. F. G. Novy: "Die Kultur anaerober Bakterien" (Centralb. f. Bakt., Bd. xiv, i*' i.O- 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 Sauerstoffaussoheidung chromophyllhal- tiger Zellen im Licht bei Anwendung der Bacterienmethode. 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. xyi, 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. Bine 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 M_ethode zur Ziichtung anaerober Kulturen in flussigen Nahrboden. Centralb. f. Bakt., xvii Bd., 1895, pp. 460-461, i fig. ('95). BRAATZ, EGBERT. Einiges tiber die Anaero- biose. Centralb. f. Bakt., xvii Bd., 1895, pp. 737-742, with I fig. ('95). KEDROWSKI, W. Ueber die Bedingungen, miter welohen anaerobe Bakterien auch ln-i Geqi/nwart 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- anaerober Bakterienarten. Arbeiten aus dem Bakteriologisohen Institut der Tech- niscben Hochschule zu Carlsruhe, Bd. I, 1895. H. 2, pp. 148-183, with 2 Taf. ('96). KASPAKEK, THEODOR. Ein •einfacher Luftab- schluss fliissiger Nahrboden beim Kulti- vieren anaerober Bakterien. Centralb. f. 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. Ill, 1896, pp. 231-236. I'M;). BECK, M. Zur Ziichtung anaerober Kulturen. Centralb. f. Bakt., xxn Bd., 1897, pp. 343- 345, with 2 figs. ('98). SCHOLTZ, W. Ueber das Wachvtum 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 Tetamisbacillus. Centralb. f. Bakt., xxiv 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. ('98). KLEIN, ALEX. Bin Apparat zur bequemen Herstellung von anaeroben Plattenkulturen. Centralb. t. Bakt.. xxiv Bd., 1898, pp. 967- 971, 2 figs. The reviewer has uot had that trouble with Novy's ap- paratus complained of by Mr. Klein. The author's siphou arrangement for mixing the potash water with the pyrogallol after exhaustion of the air appears to be very good. (98), C98) CoS) ('98) ('98) ('98) ('98) ('99) s ('99) (•oo) Coo). Cool Coo) Coo) ('02) FERRAN, J. Ueber die Verwendung des Ace- tylens bei der Kultur anaerober Bakterien. Centralb .f. Bakt., xxrv Bd., 1898, p. 29. ZUPNIK, LEO. Ueber eine neue Methode anaerober Zuchtung. Centralb. f. Bakt.. xxiv Bd., 1898, pp. 267-270, with I fig. OPRESCU. Zur Teohnik der Anaerobenkultur. Hyg Rundschau, 1898. No. 2. Rev. in Cen- tralb. f. Bakt., xxni Bd., 1898, p. 668. UCKE, ALEXANDER. Bin Beitrag zur Kenntnis der Anaeroben. Centralb. f. Bakt., xxin Bd., 1898, pp. 996-1,001. TRENKMANN. Das Wachstum der anaeroben Bakterien. Centralb. f. Bakt., xxin Bd., 1898, pp. 1,038-1,043 and 1,087-1,090. MARPMANN. Eine neue Methode zur Her- stellung von anaeraben Rollglaskii'lturen mit Gelatine oder Agar. Centralb. f. Bakt., xxin Bd., 1898, pp. 1,090-1,091. EPSTEIN, STANISLAUS. Apparat zur Kultur anaerober Bakterien. Centralb. f. Bakt., xxiv Bd.. 1898, pp. 266-267, I fig. KABRHEL, GUSTAV. Zur Frage der Ziiohtung anaerober Bakterien. Centralb. f. Bakt., xxv Bd., 1899, pp. 555-56i, •with i fig. As an oxygen indicator author uses tnethylene blue in sugar gelatin. 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. 34°-343- A's° a separate. 4 pp. EPSTEIN, STANISLAUS. Ein vereinfachtes Ver- fahren zur Zuchtung anaerober Bakterien in Doppelschalen. Centralb. f. Bakt., xxvm Bd., iQoo, p. 443, with i fig. PETRI. See xvn. KRAUSE. See xv. BULLOCK. See xvn. \YKTI-.IIT. See xvn. OMEUAXSKT. W. Ein einfarhcr Apparat zur Kultur von Anaeroben im Reagenzglase. Centralb. f. Bakt.. Abt. 2, Bd. vm, 1902. pp. 711-714. XX. Fermentations, Gas-Formation, Enzymes, Etc. (See also XIX and XLVII1.) ('57). PASTEUR, L. Memoire stir le fermentation appelce lactique. C. R. des se. de 1'Acad. des sci., T. XLV, 1857, pp. 913-916. C77). VINES, S. H. On the digestive ferment of Nepenthes. Journ. Linn. Soc. (Bot.), vol. xv, 1877, pp. 427-4.V- (*7g). v. NAEC.ELI, CARL WILHELM. Theorie der Garung. Ein Beitrag zur Molecularphysiol- Miinchen, 1879, pp. IV, 156. ('79). PRAZMOWSKI. A. Zur Entwicklvmgsgeschichte und Fermentwirkung oini^cr Bacterien- Arten. Vorlhufipre 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 ohemis- ohen Fermenten oder Enzyme-logic, pp. vi, 124. Heidelberg, 1882. ('82). BECHAMP, A. Sur les microzymas comimr 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. xcrv, pp. 1,653-1,656. ('82). BERT, B., ET REGNARD, P. Action de 1'eau oxygenee stir les matieres organiques ct 1< 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, S serie, 1882, pp. 14-17. ('82). WORTMANN, J. Untersuchungen iiber das 'diastatisehe Ferment der Bacterien. Zeitschr. f. pbysiol. chem., Bd. vi, pp. 287-329, 1882. Also Jour. Chem. Soc., XLIV, pp. 930-938, 1883. ('82). MARCANO, V. Fermentation directe de la fi'cule. Mecanisme 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 T'lieterogenie, I'histogenie, la physiologic et la pathologie, pp. xxxvm, 992, with 3 pi. Paris, 1883. Prior to 1883 Becharnp isolated from yeast au L'iix\ mu capable of inverting cane sugar. See pp. 69 et seq. ("83). VIGNA, A. Ueber Bafcteriengahrung des Glycerins. Ber. d. deutsch. chem. Gesells., xvi, pp. 1,438-1,439; Gazz. Chim. Ital, vol. xin, pp. 293-296, 1883. ('83). TAPPEINER, IT. Ueber Cellulosegahrungen. Ber. d. detrtsch. chem. Gesells., Bd. xvi, 1883, pp. 1,734-1,740. ('83). TAPPEINER, H. Ueber die Sumpfgasgahrung im Schlamme der Teiche, Sumpfe und Kloaken. Ber. d. deutsch. chem. Ge^rll . Bd. xvi, 1883, pp. 1,740-1,744. ('86). HopPE-SEvi.ER, FELIX. Ueber Gahrung der Cellulose mit Bildung von Methan und Kohlensaure. Zeitschr. f. physiolog. Chemic., 1886, Bd. X, pp. 201-217 and 401-440. ('S')i. KRAMER, ERNST. Stiidieii iiber selileimige Gaihrung. Sitzungsb. d. k. Ak. d. Wissen- schaften. Math.-naturw. Classc, Bd. xcvin, 1889, IV Hft., Abt. ii b. Chemie. pp. 358-396. Wien. An interesting paper. C.SQ). FRANKLAND, PERCY F.. AND Fox, J. J. ( )u ,i pure fermentation of inannite and glycoriiu1. Proc. Royal Soc., Lond., 1889, vol. XLVI, pp. 345-357- ('89). CURTMAN, CHAS. O. Nachweis der Glvci^e durch Safranin. Pharmaceutische Runds- ohau, Bd. vn, 1889, p. 132. ("89). TISCIIUTKIN, N. Die Rolle der Bakterien bei der Veranderung der Eiweissstoflfe auf den P.lattern von Pinguicula. Ber. d. deutsch. hot. Gessellsch., Bd. vn. 1889. pp. 346-355- ('90). SCLAVO, ACHII.I.E, UND Gosio, B. Ueber cine neue Garung der Stiirke. Staz. speritn. agrar. ital., 1890, vol. xix, p. 540. FERMENTATIONS, GAS-FORMATION, ENZYMES, ETC. 233 Cgo). REINITZER, FR. Ueber die wahre Natur des Gummifermentes. Zeitschr. f. physiol. Ohemie, Bd. xiv, 1890, pp. 453-470. This author states that Wiesuer's ferment, which changes cellulose into gum and slime, does not exist Nickel, of Rerliu, has come to the same conclusion inde- pendently. (See Hot. Centralb., 1890, Bd. XLIII, p. 118). The "orciu salzsuure" reaction of Reichl is a furfurol reaction. I'oo). SMITH, THEOBALD. Das Gahrungskolbchen in der Bakteriologie. Centralb. f. Bakt, vn Bd., 1890, pp. 502-506, rnit I Abbildung. ('90). SMITH, THEOBALD. Einige Bemerkungen iiber Saure- 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., vn 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 1'Acad. des Soi. 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 tine fermentation visqueuse de 1'encre. — Annales de Micrographie, 1891, T. iv, pp. 13-21. ('91). FERMI, CLAUDIO. Weitere Untersuchungen iiber die tryptischen Enzyme der Mikroor- ganismen. Centralb. f. Bakt., x Bd., 1891, pp. 401-408. ('91). RITSERT. Bakteriologische Untersuchungen iiber das Schleimigwerden der Infusa. Ber. d. pharm. Gesellsch., I Bd., 1891, pp. 389- 399. Rev. in Centralb. f. Bakt., 1892, xi, PP- 730-733. ('91). SUCHSLAND. E. Ueber Tabaksfermentationen. Ber. d. d. bot. Gesellsch., Bd. ix, 1891, pp 79-81. f'oi). RKYERIXCK, M. W. Verfahren zum Nachweise der Saureabson.derung bei Mikrobien. Cen- tralb. f. Rakt., 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 opaque plate. Colonies which secrete acids are soon surrounded by a transparent dif- fusion field. Other carbonates may be used, e. g , zinc carbonate. This methodmay also be used forthe demon- stration of alkali production. ('91). TISCHUTKIN, N. P. Ueber die Rolle der Mikroorganismen bei der Ernahrung der insektenfressenden Pflanzen. Arbeit, d. St. Petersburg naturf. Gesell., vol. for 1890, Scot. d. Bot. pp. 33-37, St. Petersburg, 1891. in Bot. Centralb., Bd. L. 1892, p. 304. l"«uV CUNX, H. W. Isolirung eines "Lab"' fer- mentes aus Bakterienkulturen. Centralb. f. Bakt., Bd. xii, 1892, pp. 223-227. ('92?). FKAXKLAND AND FREW. The fermentation of calcium glycerate by the Bacillus ethaceticus. Trans. Roy. Chem. Soc., 1891. Rev. in Centralb. f. Bakt., xii Bd., 1892, p. 724. ('92). FRANKLAND, P.. AND MACGRECOR. J. Fermen- • in of arabinose with the Bacillus cili.i (vticus. Trans. Chem. Soc., 1892. Rev. in Centralb. f. Bakt., xn Bd., 1892. p. 725. ('02). FRANKLAND. P. F., AND FREW, W. A pure fermentation of mannitol and dulcitol. Trans. Chem. Soc., 1892, pp. 254-277. ('92). FRANKLAND, P. F., AND LUMSDEN, J. S. De- composition of mannitol and dextrose by the Bacillus ethaceticus. Trans. Ohem. Soc., 1892, pp. 432-444. ('92). FERMI, CLAUDIO. Beitrag zum Stadium der von den Mikroorganismen abgesonderten diastatischen und Inversionsfermente. Cen- tralb. f. 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 Ernahrung insekten- fressender Pflanzen. Acta Horti Petro- politanie, Bd. xn, St. Petersburg, 1892, pp. 1-19. Abstract in Bot. Centralb., Bd. LIII, 1893, P. 322. ('92). FRANKLAND, P. F. Cantor lectures on recent contributions to the chemistry of bacteriol- ogy of the fermentation industries, gr. Svo., 31 pp. London, W. S. Trounce, 1892" Not seen. ('93). HILDEBRANDT, H. Weiteres iiber hydrolytische Fermente. etc. Arch. f. path. Anat. ti. Physiol., Bd. cxxxi, 1893, pp. 5-39. ('93). GOEBEL. Pflanzenbiologisohe Schilderungen n, 1893, p. 186. Criticises views of Dubois and Tischutkin. ('93). HAPP. Bakteriologiisohe und ehemische Unter- suchungen iiber die sohleimige Garung, p. 31. these inaugurale. Berlin, 1893. Not seen. Describes Micrococcus gummosus. ('93). CAVAZZANI, EMIL. Zur Kenntnis der diasta- tischen Wirkung der Bakterien. Centralb. f. Bakt., Bd. xm, 1893, pp. 587-589. ('93). PERE, A. Sur la formation des acides lactiques isomeriques par I'action des microbes sur les substances hydrocarbonees. Ann. de 1'Inst. Pasteur, T. vn, 1893, pp. 737-750. ('93). HESSE, W. Ueber .die gasformigen Stoff- \vech selproducte 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. Wrlder Quarter Century Book, Ithaca, N. Y., 1893, pp. 187-232. ('93V FRANKLAND, P., AND MACGRECOR, J. Sarco- lactic acid obtained by the fermentation of inactive Lactic acid. Trans. Chem. Soc., London, 1893, pp. 1.028-1,035. i '93 I. GRIMBERT. L. Fermentation anaerobic produitc par le Bacillus prthobutylicus. ses variations sous certaines influences biologiques. Ann. de I'lnst. Pasteur, T. vn, 1893, pp. 353-402. ('93). GREEN, J. R. On Vegetable Ferments. An- nals of Botany, 1893. Vol. vn, 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. LTeber die Enzyme. Zeitschr. f. Hyg., Bd. xvni, 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. xm, pp. 218-222. 234 BACTERIA IN RELATION TO PLANT DISEASES. Cy4). GOTTSTEIN, ADOLF. Ueber die Zerlegung des Wasserstoffsuperoxyds durch die Zellen, init Bemerkungen iiber eine makroskopische Reaktion fiir Bakterien. Virohow's Archiv., Bd. cxxxm, p. 296. Rev. in Centralb. f. Bakt, Bd. xvi, 1894, pp. 518-519. ('94). TIMTE, 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 rnakroskopische Wasser- untersuchung durch Anwendung von Was- serstoffsuperoxyd. Centralb. f. Bakt., Bd. xvi, 1894, pp. 180-182. This substance, which causes liberation of oxygen from H2d>2, is said to be nuclein. Author failed to confirm Gottstein's results. (Virchow's Archiv., Bd. cxxxin, Heft 2.) ('94). GREEN, J. R. Tihe dnfluence of light on dias- tase. Abstr. of a paper read before the British Assoc. at Oxford, Aug., 1894. Ann. of Bat, 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 Buttersauregahrung. Centralb. f. Bakt., 2 Abt., Bd. i, 1895, pp. 84-87 and 118-120. Cos). 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 scientinques), 1895. Micro- organisms and Fermentation. English trans- lation bv Alex. K. Miller and A. E. Lenn- holm. Third edition completely revised, with 8^ 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. I/95). Gosio, B. Zersetzungen zuckerhaltigen Nahrmateriales durch den Vibrio cholera asiaticae 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. (''15). 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. ym, 220. 8vo. Paris, Societe d'editions scientifiques, 1896. CiXi). PFEFFER, W. Ueber regulatorische Bildung von Diastase. B>er. u. d. Verhandl. d. K. sach. Gesellsch. d. Wiissenschaften zu Leip- zig. Mathematisch-Physik. Classe, 1896, pp. 513-518. Also a separate. (''17). BUCHNER, H. Die Bedeutung der activen loslichen Zellprodukte fiir den Ohemismus der Zelle. Miinchen. med. Wochenschr., 1897, No. 12, pp. 299-302. ('97). BUCHNER, ED. Alkoholische Garung ohne 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 Treniiurjg der Gahrwirkuug von den lebeuden Hefezellen ist bishemicht 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. n 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 I'inst. imp. de med. exper. a St. Petersbourg, T. vn, pp. 411-434. i heliotype plate. ('99). FERMI, CLAUDIO, AND BUSCAGI.IONI. Die pro- teolytisohen Enzyme am Pflanzenreiche. Centralb. f. Bakt., ate 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 u. Diastases, toxines et venins. Paris. Masson et Cie, 1899, pp. in, 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). BuLLER, A. H. R. Die Wirkung von Bak- terien 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 chemische Dynamik des Wiasserstoffsuperoxyds. 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. Alcoholischc Gahrung ohne Hefezellen. Ber. d. deutsch. chem. Gesellsch., Jahrg. xxx (1897), 2,668; Tahrg. xxxi (1898), 209. 1,084, l,°9°, i,53i; Jahrg. xxxn (1899), 127-137. ('99). GREEN, J. REYNOLDS. The soluble ferments and fermentation, pp. xui, 480. Cambridge (England), at the University Press, 1899. zd ed., 1901, pp. xv, 512, with a bibliography of 896 titles. An excellent book, and the only complete, authorita- tive one in English. ('oo). DUCLAUX, E. Traite de microbiologie. Tome in. Fermentation alcoolique. Paris. 1900, pp. in, 760, 84 text figures, 8vo. Masson et Cie. ('oo). 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. Coi). 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). BOUFFARD, A. Les maladies microbiennes des vins. Fermentation alcoolique ; maladies miicrobiennes ; ca.rre des vins; hygiene des vins ; traitement des vins malades. Nancy, 1901, I2mo., avec 6 planches et fig. Not seen. for). DUCLAUX, E. Traite de microbiologie. Tome iv. Fermentations variees des diverses sub- stance- ternaires. Paris, Masson et Cie., 1901, pp. in, 768, 45 text figures. Valuable. ('oi). GESSARD, C. fitudes sur la tyrosinase. Ann. de 1'Inst. Pasteur, T. xv, 1901, pp. 593-614. (,'ni). BEI.TERI.XCK, M. W. Anhaufungsversuche mit Ureumbakterien. Ureumspaltung duroh Urease und durch Katabolismus. Centralb. f. Bakt., Bd. VH, 1901, pp. 33-61, with I plate and 4 figures. ('02). ROLLY. Weiterer Beitrag zur Alkali- und Saureproduktion der Bafcterien. Arch. f. Hyg., Bd. XLI, 1902, pp. 406-412. ('02). SCHREIBER, KARL. Fettzersetzung durch Mikroorganismen. Arch. f. Hyg., Bd. xu, 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 1001, Pt. 4, pp. 674-683, 684, Sydney, 1902. Also a separate (issued May 20, 1902). ('02). OMELIANSKI, W. Sur la fermentation for- 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). EFFRUNT, JEAN. Enzymes and their applica- tions. English translation by Samuel C. Pres- cott, vol i. The enzymes of the carbohy- drates. The oxidases. New York. John Wiley & 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 Bakterienflora der Sauern Gahrung einiger Nahrungs-und Genussmittl. Arb. a. d. Bact. Institut der tech. Ilochschule zu Karlsruhe, n 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. mucilagiueus. M. vulgaris. M. piliformis, M. expressus, M. granulatus, M. sublutens. M. teuer. M. ill.-inilitlosus. M. vesicosus. M. mucilaginosus. M. fulvus, Bacilhis fortissimus, B. opacus, B. spirans, B. fuliginosus, B. eminans, B. ventricosus, B. citricus, B. odoratus. B. amygdaloides, B. robustus, B. tuherostis, B. globul'isu--. B. fuiigosus. B. fiavescens. B. gracilescens. Bacterium variosum. B. spiuosuui. B. crenntum, B. plicativum, B. brevissimum, B. ramificaus. B. gibbosum, B. gracillimum. B. squamatum. B. iimforme. B. iusiilstim, B. subcitriniiin. B. grauulosum, Pseudomonas lactica, Ps. Listen. ('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. xvil, 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, !9O2-'o3. Co.?). Lii-pMANN, E. O. VON. Zur Nomenklattir der Enzyme. Berichte d. deutsch. Chem. Gesell- schaft, 1903, Bd. xxxvi, p. 331. XXI. Ptomaines, Toxins, Antitoxins, Serums, Phagocytosis, etc. ('86). BKIEGER. Untersuchungen iiber Ptomaine. Berlin, 1886. Traduction par Roussy et Winter as Microbes, ptomaines et maladies. Paris, 1887, pp. xu, 235. ('87). METCHNIKOFF, £LIE. Sur la lutte des cellules de 1'organisme centre 1'invasion des microbes. Ann. de 1'Inst. Pasteur, T. i, 1887, pp. 321-336. ('88). NUTTALL, GEO. Experimente iiber die bac- terienfeindlichen Einfliiss des thierischen 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 Eigenscliaft des Blutes. Zeitschr. f. Hyg., Bd. vi, 1889, pp. 487-520. ('89). BUCHNER, H. Ueber die baoterientodtende \\ irkung 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 frischen Harns des gesunden Menschen. Centralb. f. Bakt., Bd. vn, 1890, pp. 457-460. ('90). JACQUEMART, F. Les ptomaines, histoire et caracteres chimiques, memoire couronne par la Societe royale des sciences medicales et naturelles de Bruxelles. Jour, de med. de chir. et de pharm., Bruxelles, 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., 1890, Bd. vm, pp. 768-773. ('90). LE\VANDO\VSKI, A. Ueber Indol- und Phenol- bildung durch Bakterien. Deutsche mediz. Wociiensohrft, Bd. xvi, 1890, p. 1,186. ('90). GABRITCHEVSKV, G. Sur les proprietes chimio- tactiques des leucocyies. Ann. de 1'Inst. 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. BACTERIA IN RELATION TO PLANT DISEASES. ('91). 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 immunisirter Thiere. Zeitsoh. f. Hyg., Bd. xvm, 1894, pp. 239-250. ('95). BRIEGER, L. Weitere Erfahrungen iiber Bak- teriengifte. Zeitsch. f. Hyg., Bd. xix, 1895, pp. 101-112. ('96) BKIEGER UND BOER. Ueber Antitoxine und Toxine. Zeitsch. f. Hyg., 1896, Bd. xxi, pp. 259-268. ('96). GAUTIER, ARMAND. Les toxmes microbiennes et animales, 1896, pp. vn, 617. Soc. d'edi- tions scientifiques, Paris. ('96). EHRLICH, P. Die staatliche controle des Diph- therie serums. Berl. klin. Wochenschr., Bd. xxxin, 1896, 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. Contaius 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. vill. 495. ('96). METCHNIKOFF, EL., Roux, E., ET TAURELLI- SAUMBENI. 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. 11, 1897, pp. 543-547- Coo). BORDET, J. Les serums Jiemolytiques, leurs antitoxines et les theories des serums cytoly- tiques. Ann. de 1'Inst. Pasteur, 1900, T. xiv, pp. 257-206. ('oo). METCHNIKOFF, E. Sur les cytotoxines. Ann. de 1'Inst. Pasteur, T. xiv, pp. 369-377, 19°°- Coo). FISCHER, ALFRED. Die Empfindliohkeit der Bakterienzelle und das baktericide Serum. Zeitschr. f. Hyg., Bd. xxxv, 1900, pp. 1-58, i plate. Coi). ZABOLOTNY. See vn. Coi). 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. Coi). BORDET, J., ET GENCOU, O. Sur 1 existence de substances sensibilisatrices dans la plupart des serums antimicrobiens. Ann. de 1'Inst. Pasteur, 1901, T. xv, pp. 289-302. Cm). I'.UKDET, J. Sur le mode d'action des serums cytolytiques et sur 1'unite de 1'alexine dans tin meme serum. Ann. de 1'Inst. Pasteur, T. xv, 1901, pp. 303-318. Coi). GRUBER, MAX. Zur Theorie der Antikorper. T. Ueber die Antitoxin-Immunitat. it. Ueber Bakteriolyse und Haemolyse. Miin- ner mod. Wochenschr., Bd. XLVIII, 1901, pp 1.8:7-1.830, pp. 1880-1884, pp. 1,924-1,986. Nut -i en. Co2). MAKX, K. Die experimentelle Diagnoitik, Serumtherapie und Prophylaxe der Infec- •i -kranklu-iten. [Bibliothek v. Coler, Bd. xi. 1 Berlin (A. Uirschwald), 1902, pp. vn, i 1 1 lit 2 Taf. ('04). WASSERMANN, A. 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De 1'attenuation des effets des inoculations yirulentes par 1'emploi de _tres petites quantites de virus. C. R. des se. de i'Acad. des sci., T. xcn, 1881, pp. 844-848. CHAMBERLAND ET Roux. Sur 1'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, ELIE. Sur 1'attenuation des bacteridies charbonneuses dans le sang des moutons refractaires. Ann. de 1'Inst. Pas- teur, T. i, 1887, pp. 42-44- MACE. See xxin. D'ARSONVAL AND CHARRIN. See xxxn. ROGER. See xxxn. KLEPZOFF. See xxxni. FUHRMANN, FRANZ. Ueber Virulenzsteigerung eines Stammes des Vibrio Cholerae asia- ticae. Sitzungsber. d. kaiserl. Akad. d. Wissensch. Mathematisch-Naturwissensch. Klasse., Bd. cxn. Heft. vni. 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 Ccolored). ('So). 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 xxvu. 1880. pp. I/4-I79- ('82). ENGELMANN, TH. W. Zur Biologic der Schizomyceten. Bot. Zeitung, 40 Jahrg., 1882, col 321-325 and 337-341. Describes a gffeen organism as Bacterium chloriuum. It is believed to contain chlorophyll C8/). SCHOTTEUUS. See xv. CS-1). PROVE, OSKAR. Micrococcus ocliroleucus eine neue chromogene Spaltpilzform. Beitr. ?.ur Biol. der Pflanzen., Bd. IV, Hft. 3, 1887, pp. 409-439, I plate. I'MiMKNTS, GKKKN 15ACTERIA. 237 '89). \1 MI':. Sur la recuperation de la vitalite des cultures de bacteries par passages sur cer- tains milieux. Soc. des sc. de Nancy pour 1888, Series n, T. ix, Fasc. xxn, pp. xxix- xxx and 79-83, Paris, 1889. The medium used was potato. Bacteria which have lost their power to form pigmeut ou agar or to infect frogs will often regain these functions if cultivated for a time on potato. ('89). XDIT. \V. Ueber Pilzfarbstoffe. iv. Vor- kommen einer Lipochroms bei Spaltpilzen. Bot. Zeitung, Bd. XLVII, 1889, col. 89-92. i'Sj). PETROW, N. Uber einen neuen roten Farb- stoff-bildenden Bacillus. Arb. a. d. Bact. Institut der tecbn. Hochschule zu Karlsruhe, it Bd., 3 Heft, 1902, pp. 271-291, with i plate. Describes Bacillus subkilieusis. ('02). LoEW, O., AND KOZAI, Y. Ueber Ernahrungs- verhaltnisse beim Bacillus prodigiosus. Bull, of the College of Agric., Tokyo Imperial Univ., vol. v, 1902, No. 2, pp. 137-141. Also a separate. A favorable medium for production of pigment and bacteriolytic enzyme is composed of peptone i percent, sodium acetate 0.2 per cent, and asparagin 0.2 per cent in water. ('03). MARSH. See vi. ('03). PAPENHAUSEN, HUBERT. Uber die Bedingun- gen der Farbstoffbildung bei den Bakterien. Arb. a. d. Bact. Inst. der tech. Hochschule zu Karlsruhe, in Bd., I Heft, 1903, pp. 43-79. Bibliog. of 20 titles. T \veuty-two species experimented upon. Oxygen is very necessary for the production of the pigments. The other conditions for optimum production of pigment vary greatly in different species. ('04). HEFFERAN, MARY. A comparative and ex- perimental study of bacilli producing red pigment. A dissertation submitted to the faculties of the Graduate Schools of Arts, Literature and Science, in candidacy for the degree of Doctor of Philosophy. The Uni- versity of Chicago. Printed in Jena by Gustav Fischer, 1904, pp. 55. Bibliog. of 77 tides. ('04). LEONARD, ETHEL L. Bacterium cyaneum : A new chromogenic organism. The Johns Hopkins Hospital Bulletin, vol. xv, 1904, pp. 398-400. XXIV. Reduction and Oxidation. ('^7). SPINA, A. Bacteriologische Versuche mit gefarbten Nahrsubstanzen. Centralb. f. Bakt., ii Bd.. Jena, 1887, Nos. 2-3, pp. 71-75. Reduction processes of bacteria in presence of methy- leu blue, etc. t'S/). CAHEN, FRITZ. Ueber das Reduktionsver- mogen der Bakterien. Zeitschr. f Hyg , Bd. n, 1887, PP. 386-396. Cgi). WlNOGRADSKY. Se« XXV. ('94). FERMI, CLAUDIO, AND MONTESANO, GIUSEPPE. Ueber die Dekomposition des Amygdalins 'durch Mikroorganismen. Centralb. f. Bakt., Bd. xv. 1894, pp. 722-727. ('96). SMITH. THEOBALD. Reduktionsersoheinungeu bei Bakterien und ihre Beziehungen zur Baktcrienzelle, nebst Bemerkungen iiber Re- duktionserscheinungen in steriler Bouillon. Centralb. f. Bakt., xix Bd., 1896, pp. 181-187. ('99). MUELLER, FRIEDR. Ueber reduzierende Eigen- schaften von Bakterien. Centralb. f. Bakt., xxvi Bd., 1899, pp. 51-63. TliL- pigment used must be soluble in water, and must iint poison the bacteria. Author uses methyleue blue and litmus. Under literature, n papers are cited. ('99). ROTHBERGEK. See xviii. ('99). MUELLER, FRIEDRICH. Ueber das Reduktions- vermogen der Bakterien. Centralb. f. Bakt., xxvi Bd., 1899, pp. 801-819. Author used methyleue blue, litmus, indigo-carmiu and rosanilin acetate in various media with many organisms. C'oi). GRAN. See xxv. ('02). CATHCART, EUUARD, UND HAHN, MARTIN. Ueber die reduzierenden Wirkungen der Bakterien. Arch. Hyg., Miinchen, Bd. XLIV, 1902, pp. 295-321. ('02). EMMERLING, OSCAR. Die Zersetzung stick- stofffreier organischer Substanzen durch Bakterien. Braunschweig (F. Vie\veg & S.), 1902, pp. ix, 141, mit 7 Taf. Not seen. ('03). VAN DELDEN, A. Beitrag zur Kenntniss der Sulfatreduction durch Bacterien. Centralb. f. Bakt., 2 Abt., xi Bd., 1903, pp. 81-94 and 113-119, i heliotype pi. XXV. Nitrifying and Denitrifying Organisms, Use of Free Nitrogen. ('66). WORONIN. Ueber die bed der Sohwarzerle und der igewohnlichen Lupine auftretenden Wurzelanschwellungen. Memoires de 1'Acad. imp. St. Petersb., 7 Serie, T. x, 1866. See also Ann. des sci. nat. Bot., 5 se., T. VH, pp. 73-86, i plate. ('77). SCHLOESING, TH., ET MUENTZ, A. 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Proces-verbaux se. du 23 nov., 1882, pp. ni-iv. ('83). SPRINGER, A. Reduction of nitrates by fer- ments. Am. Chem. Jour., vol. rv, pp. 452- 453, 1883. ('86). GAYON ET DUPETIT. Recherches sur la reduc- tion des nitrates par les infiniments petits., 1886. Paris? Publisher? Not seen. 240 BACTERIA IN RELATION TO PLANT DISEASES. ('86). MUNRO, J. H. M. The formation and destruc- tion of nitrates and nitrites in artificial solu- tions, ami in river and well waters. Jour. Ohem. Soc. Trans., London, 1886, vol. xux, pp. 632-681. ('88). BEYERINCK. Die Bakterien der Papiliona- ceenknollohen. Bat. Zeitung, Jahrg. XLYI, 1888, col. 725-735, 741-750, 757-771, 781-790, 797-804, 2 plates. Review in Centralb. f. Bakt., Bd. v, 1889, pp. 804-805. ('88). HELLRIEGEL, H., UND WILFARTH, H. Unter- suchungen uber die Stickstoffnahrung der Gramineen und Leguminosen, 1888. Beila- geheft zu der Zeitsohr. d. Vereins f. d. Rubenzucker-Industrie d. Deutschen Reichs. Berlin, pp. 234, with 6 plates. ('90). FRANKLAND, PERCY F. The nitrifying process and its specific ferment, Pt. I. Phil. Trans. Roy. Soc., Lond., vol. 181 B., 1890, pp. 107- 128. ('90). WINOGRADSKY, S. Recherchcs sur les organ- ismes de la nitrification. Ann. de 1'Inst. Pasteur, T. iv, 1890, pp. 213-231, 257-275, and 760-771. Nitromonas can live on purely inorganic matters, although destitute of chlorophyll. His " nitratferment " consists of pear-shaped rods, which do not exceed 54 n in length, and are lio to 2 times less in breadth. The " nitritorganismus " is 4 to 5 times larger than the " nitratbilduer," and has a roundish form. ('90). LAURENT, EM. Experiences sur la reduction des nitrates par les vegetaux. Ann. de 1'Inst. Pasteur, T. iv, pp. 722-744, 1890. ('91). GILTAY, E., UND ABERSON, J. H. Recherches sur un mode de denitrification et sur le schizomycete qui la produit. Archives neer- landaises des sci. ex. et nat., 1891, T. xxv. PP- 341-361, i plate. ('i)l). SCHLOESING, TH., FILS, ET LAURENT, EMILE. Sur la fixation de 1'azote litre par les plantes. C. R. des se. de 1'Acad. des soi. Paris, 1891, T. cxm, pp. 776-778 and 1,059- 1,060, 1892, T. cxv, pp. 659-661 and 732-735. Coi). WINOGRADSKY, S. Recherches sur les organ- ismes de la nitrification. Ann. de 1'Inst. Pasteur, T. v, 1891, pp. 92-100 and 577-616. Winogradsky's fourth and fifth memoirs on nitrifying bacteria. The fourth deals with the question of solid media suited to their culture, and describes the making of silicate jelly. The fifth deals with the formation and the oxidation of nitrites in the nitrification. BEIJERINCK, M. W. Over pphooping van at- mospherlsohe stikstof in culturen van Bacillus radicicola. Versl. en Mededeel. der Koninkl. Akad. van Wetensch. zu Amster- dam, Afd. Natuurkunde, III, 8, 1891. pp. i l'»i)-(475), I text fig. Abstract in Koch's .Tali res.b., Ill Bd., p. 205. WINOGRADSKY, S. Contributions a la mor- phologic des organismes de la nitrification Arch. d. sci. biol. publiees par 1'Inst it nt Im- perial de med. exper. a St. Peterslirmri;. Tome I, 1892, p. 108. Reviewed in Ann, dr 1'Inst. P.'iMi'-ur. T. VI, 1892, pp. 458-462. Srm.nKsiNr,. Tn., 1'ii.s, ET LAURENT. F.MII.E. Recherrhr- snr hi fixation de 1'azote libre par les plantes. Ann. de 1'Inst. Pasteur. iR<)->, T. vi. pp. 65-115. I'.i KTIIEI.OT. Nouvelle recherches sur la fixa- linn dc 1'azote par les microbes. C- 1\ di •• se il.- 1'Arail. di-s sci., ifVu, T. rxv, pp. 5(10 :,7 | ('g i ('93). BERTHELOT. Recherches nouvelles sur les imicroorganismes fixateurs de 1'azote. C. R. des se. de 1'Acad. des sci., Paris, 1893, T. cxvi, pp. 842-849. ('94). DUCLAUX, E. Sur la fixation de 1'azote at- mospherique. Revue critique. Ann. de 1'Inst. Pasteur, 1894, T. vm, pp. 728-736. ('94). WINOGRADSKY, S. Sur ['assimilation de 1'azote gazeux de I'atmosphere par les microbes. C. R. des se. de 1'Acad. des sci., Paris. T. cxvi, 1893, p. 1,385, and T. cxvni, 1894, Feb. 12, p. 353. Rev. in Centralb. f. Bakt., Bd. xvi, 1894, pp. 129-130. ('95). WINOGRADSKY, S. Recherches sur I'assimila- tion de 1'azote libre de 1'atrnosphere par les microbes. Arch, des sci. biol. pub. par 1'In- stitut. imperial de medeoine experimentale a St. Petersbourg, Tomie ill. No. 4, 1895, pp. 297-352. A very interesting and valuable paper. ('96). WINOGRADSKY, S. Zur Mikrobiologie des nitrifikaitionsprozesses. Centralb. f. Bakt., 2 Abt, Bd. 11, 1896, pp. 415-428. ('97). MAZE. Fixation de 1'azote libre par le bacillc des nodosites des legumineuses. Ann. de 1'Inst. Rasteur, T. xi, 1897, pp. 44-54. The second and third memoirs are continued in the Annales for 1898, under the title, " I*es microbes des nodosites des legumineuses." ('98). KoNVAi.EwSKi, S. Beitrage zur Frage iiber die Assimilierung von freiem Stickstoff seitens der Bakterien. Russ. Arch. f. Pathol., Bd. vi, 1898, p. 251. Rev. in Centralb. f. Bakt., xxv Bd., 1899, pp. 771-772. ("98). MAZE. Les microbes des nodosites des legum- ineuses. Ann. de 1'Inst. Pasteur, T. xn, 1898. Second memoir, pp. 1-25, I fig. Third memoir, pp. 128-155, 2 plates. The second memoir deals with the physiology of the organism. The third memoir deals \vitli the morphology of the root-tubercle organism. Maze- says it is patho"- genic for certain animals. In the animal body it is said to become nearly round, to be much reduced in diame- ter, and to have a tendency to grow in chains. ('99). OMELIANSKY, V. Sur la nitrification de 1'azote organiqtie. Arch, des sci. biol. publiees par 1'inst. imp. de med. «xper. a St. Petersbourg, T. vn, 1899, pp. 272-290. ('99). OMELIANSKY, V. Sur la culture de_s microbes nitrificateurs du sol. Arch, des sci. biol. pub- liees par 1'inst. imp. de med. exper. a St. Petersbourg, T. vn, 1899, pp. 291-302. (99). WINOGRADSKY u. OMELIANSKY. Ueber den Einflus der organischen Substanzen auf die Arbeit der nitrificierenden Mikrobien. Centralb. f. Bakt., 2 Abt., Bd. vi, 1899, p. 329. ('99). WINOGRADSKY, S., ET OMELIANSKY, V. L'in- fkience des substances organiques sur le travail des microbes nitrificateurs. Arch, des sci. biol. puhliees par 1'inst. imp. de med. exper. a St. Petersbourg, T. vn, 1899, pp. 233-271. ('oo). KRUEGER, W., AND SCHNEIDEWIND, W. Ursache und Bedeutung der Salpeter-Zersetzung im Boden. Landwirtschaftliche Ja-hrbiichcr. Ztschr. f. wiss. Laiidwirtschaft. Berlin, 1900, Bd xxix, pp. 747-7/0, 2 plates. Coi). BKIIERINCK, M. W. Ueber oligonitrophile Mikroben. Centralb. f. Bakt., etc.. Bd. vn, Mini, pp. 561-582, with i heliotype plate. I >i -, iil.es A/otobacter. NITRIFICATION, ETC.; USE OF FREE CO2 ) LUMINOSITY. 241 ('oi). GRAN, H. H. Studien iiber Meeresbakterien. i. Reduction von Nitraten und Nitriten. IliT^rns Museums Anrbog., 1901, No. 10, PP. 23. Three new species are described : Bacillus repens, R. trivialis, B. Heusenii. ('oi). MAASSEN, ALBERT. Die Zerzetzimg der Nitrate und der Nitrite durch die Bakterien. Ein Beit rag zum Kreislauf des Stickstoffs in der Natur. Arbeit, a. d. K. Gesundheitsamt, Bd. xvm, 1901, pp. 21-77. ('02). MOORE, GEO. T. Bacteria and the Nitrogen Problem. Yearbook Dept. Agric., 1902, pp. 333-342, 6 plates. Also a separate. Pure cultures of the root-tubercle organism are ab- sorbed on cottou, which is then dried, wrapped in tin foil, and thus distributed to the agriculturist. He is directed to throw the cotton into a pail of water, to which is added small packages of nutrient salts. After 48 hours the fluid is filled with the bacterial growth. The legum- inous seeds are then drenched with it, and sown after dry- ing in the shade. ('02).' SMITH, R. GKEIG. Notes on Vibrio denitrifi- cans, Sewerin. Proc. Linneau Soc., New South Wales, for the year 1901. Sydney, 1902, vol. xxvr, Pt. i, pp. 118-121. i plaite. ('02). OMELIANSKI, W. Kleinere Mitteilungen uber Nitrifikationsinikroben. I. Die Kultur des Nitritbildners auf Papierscheiben. n. Wird schweflige und phosphorige Saure durch Nitrobacter oxydiert? in. Scheiden die Nitritmikroben eine Oxydase aus? Cen- •tralb. Bakt., Abt. 2, Bd. vin, 1902, pp. 785- 787; Bd. ix, 1902, pp. 63-65, pp. 113-117, mit i Taf. ('02). BEIJERINCK, M. W., UND VAN DELDEN, A. Ueber die Assimilation des freien Stick- stoffs durch Bacterien. Centralb. Bakt., Abt. 2. Bd ix, 1902, pp. 3-43. ('02). P.UHLERT, H. Untersuchungen iiber die Art- einheit der Knollchenbakterien der Legumi- nosen und iiber die landwirtschaftliche Be- deutung dieser Frage. Habilitationsschr. Halle (Druck v. Wischan u. Wettengel), 1902, p. 55. Centralb. Bakt, Abt. 2, Bd. ix, 1902, pp. 148-153, 226-240, and 273-285. ("02). HII/TNER, L. Ueber die Impfung der Legumin- osen mit Reinkulturen. D. landw. Presse, Berlin, Bd. xxix, 1902, pp. 119-120. ('02). NOBBE, FRIEDRICH, UND RICHTER, L. Ueber den Einfluss des Nitratstickstoffs und der Humussubstanzen auf den Impfungserfolg bei Leguminosen. Landw. Versuchstat, Berlin, Bd. LVI, 1902, pp. 441-448. ('03). BERSTEYN, P. Uber einige in den Kulturen zur Reinziichtung der Nitratbildner regel- massig auftretende Bakterienarten. Arb. a. d. Bact. Inst. der tech. Hochschule zu Karls- ruhe, in Bd., i Heft, 1903, pp. Sr-ioo. The following new species are described : Bacterium comes, B. modestum. B. debile and Ps. humicola. None oi these species will grow in media entirely free from organic matter. ('03). BENECKE, W., UND KEUTNER, J. Ueber stick- stoffbindende Bakterien aus der Ostsee. Ber. d. deutsch. 'bot. Gesellsch., Bd. xxi, Heft 6, pp. 333-346, Berlin, 1903, 4 text figs. ('05). MnoKE, GEORGE T. Soil Inoculation for Le- gumes. Bureau of Plant Industry, !'. S Department of Agriculture, Bull. 71, Jan. 23, 1905, pp. 72, pi. 9. XXVI. Use of Free Carbon Dioxide. ('87). HuEPPE, FERDINAND. Ueber Chlorophyllwirk- ung chlorophyllfreier Pflanzen. Tageblatt der 60 versammlung deutscher Naturforscher u. Aerzte in Wiesbaden, 1887, pp. 244-245. ('99). WlNOGRADSKY U. OMEUANSKY. See XXV. ('02). NATHANSOHN. See LI. ('03). BEIJERINCK, M. W., UND VAN DELDEN, A. Ueber 'eine farblose Bakterie, deren Kohlen- stoffnalirung aus der atmospharischen Luft herrii'hrt. Centralb. f. Bakt., 2 Abt., Bd. x, No. 2, 1903, pp. 33-47. See also Versl. Wis. Nat. Afd. K. Acad. Wet., Bd. xi, 1903, pp. 450-465 (Dutch); and Proc. Sci. K. Acad. Wet., Bd. v, 1903, pp. 398-413 (English), Amsterdam. " We will use the name Bacillus oligocarbophilus to designate a colorless bacterium whose carbon needs, in the dark as well as in the light, are satisfied by a not yet exactly known carbon compound, or compounds, of the air, out of which compound this organism must also cre- ate the necessary energy for its life processes." It is stated that this substance is not carbon dioxide. ('04). BEIJERINCK, M. W. Ueber die Bakterien, welche sich im Dunkeln mit Kohlensaure als Kohlenstoffquelle ernahren konnen. Centralb. f. Bakt., 2 Abt., xi Bd., 1904, No. 20-22, pp. 593-599- ('75). ('So). ('85). C87). C87). ('87). ('87). C88). ('88). ('89). XXVII. Luminous Bacteria. PFLUEGER, E. Beitrage zur Lehre von der Respiration. Sec. 5. Die Phosphorescenz der lebendigen Organismen und ihre Bedeu- tung fur die Pnincipien der Respiration. Pfliiger's Archiv, 1875, Bd. x, pp. 275-300. LASSAH, O. Die Micrococcen der Phosphores- cenz. Pfliiger's Arch. f. die gesam. Physio!., 1880, Bd. xxi, pp. 104-108. NuEECH. Ueber leuohtende Bacterien. Bale, 1885. Not seen. FISCHER. Bakteriologische Untersuchungen auf einer Reise nach Westindien. n. Ueber einen lichtentwickelnden, im Meerwasser gefundenen Spaltpilz. Zeitschr. f .Hyg. Bd. u, 1887, pp. 54-92, and Nachtrag, pp. 92-95. FORSTER, J. Ueber einige Eigensohaften leuoh- tender Bakterien. Centralb. f. Bakt., Bd. u, 1887, pp. 337-340. KATZ, OSCAR. Preliminary remarks on phos- phorescent bacteria from sea-water. Proc. Linn. Soc., New South Wales, vol. n, Pt. 11, 1887, pp. 331-336. DUCLAUX. Sur les microbes phosphorescents. Revue critique. Ann. de 1'Inst. Pasteur, T. I, 1887, pp. 489-492. DUBOIS, R. Sur le role -de la symbiose -chez certains aiiimaux marins lutruineux. C. R. des se. de 1'Acad. des sci., T. CVH, Paris, 1888, pp. 502-504. FISCHER, B. Ueber einen neuen lichtentwick- elnden Bacillus. Centralb. f. Bakt., 1888, in Bd., pp. 105-108, and pp. 137-141. BEYERINCK, M. W. Le photobacterium luini- nosum, bactc?rie lumineuse de la mer du nord. Arcli. neer. des sci. ex. et nat., T. xxin, 1889, pp. 401-415. 242 BACTERIA IN RELATION TO PLANT DISEASES. ('89). BEYERINCK, M. W. Lcs bacteries luraineuses dans leurs raports avec 1'oxygene. Arch, neer. des sci. ex. et nat., T. xxm, 1889, pp. 416-427. ('89). GIARD, A., UND BILLET, A. Observations sur la maladie phosphorescente des Talitres et autres Crustaces. C. R. de la Soc. de biol., 1889, Tome i, pp. 593-597. ('89). I.EHMANN, K. B. Studien iiher Bacterium phosphorescens Fischer. Centralb. f. Bakt., v Bd., 1889, pp. 785-791. I'm) I. GIARD, A. Nouvelles reoherchcs sur les bac- teries lumineuses pathogenes. C. R. de la Soc. de biol., 1890, Tome II, pp. 188-191. ('90). BKIJERINCK, M. W. Over lichtyoedsel en plastisch voedsel van lichtbacterien. Versl. en Meded. d. Kon. Akad. v. Wetenschappen. Aft. Natuurk. Derde Reeks, Deel vn, 1890, pp. 239-302. i text figure. ('90). BILLET, A. Contribution a 1'etude de la mor- phologic et du developpement des bac- U'-riacees. Bull, scientifique de la France et de la Belgique, public par Giard, T. xxi, 1890, pp. 1-289, plates 9. Bibliography of 662 titles. Also a separate. The bacterium of luminous sand fleas is described iii a uote on p. 144 as Bacterium Giardi Billet. ('91). BEYERINCK, M. W. Sur 1'aliment photogene et 1'aliment plastique des bacteries lumi- neuses. Arch, neerl. des sci. ex. et nat., T. xxiv, 1891, pp. 369-442. ('91). KATZ, OSCAR. Zur Kenntniss der Leuchtbak- terien. Centralb. f. Bakt., 1891, IX Bd., pp. I57-I63, 199-204, 229-234, 258-264, 3"-3i6, 343-349- ('92). RusSELL, H. L. Impfungsversuohe mit Giard's pathogenem Leuchtbacillus. Centralb. f. Bakt., xi Bd., 1892, pp. 557-559- Author obtained no conclusive evidence as to the patho- genic nature of this organism. ('92). EIJKMANN, C. Lichtgevende Bacterien. GeneeskundigTijdschriftvoorNederlandsch- Indie. Deel xxxn, Aflevering 4. Batavia en Noordwijk, 1892. pp. 109-115. Also a sepa- rate. Rev. in Centralb. f. Bakt., xii Bd., 1892, pp. 656-657. ('93). DUBOIS, RAPHAEL. Extinction de la lumi- nosite du Pihotobacterium sarcophilum par la lu-miere. C. R. des se. et mem. de la soc. de Biologie, Paris, 1893, 9 sen, T. v, pp. 160- 161. This organism was isolated from the luminous flesh of a dead rabbit. It was cultivable in a synthetic medium made as follows : Ordinary water, 100 ; asparagine, i ; glycerin, i ; potassium phosphate, o.m ; sea salt, 3. After some months in this medium the luminosity diminished. Kxposed to the light for some days, at about io°C.,the culture takes on a fine orange yellow color, becomes opaque, and loses its luminosity except at the edges. Transfers may be made from this'yellow growth readily, but the resulting cultures are not luminous. In the dark, however, such cultures return after some days tn their orginal transparent color, and again become luminous. ('95). KuTSCHER. Zur Phosphorescens der Elbvib- rionen. Centralb. f. Bakt., xvm Bd., 1895, p. 424. t"bic plate. Centralb. f. Bakt., xxiv Bd.. 1808. pp. 609-612. Only the luminous sorts are able to act through glass. The action of tiou luminous species is probably dm- "to the evolution of volatile chemical substances." The lat- ter exposures were long — several days. ('99). HENNEBERG, W. Leuchtbakterien als Krank- heitserreger bei Schwammucken. Centralb. f. Bakt., xxv Bd., 1899, pp. 649-650. ('99). BARNARD, J. E. Photogenic Bacteria. Trans, of the Jenner Inst, London, 1899, second series, pp. 81-112. 2 plates. " Spectroscopically the light emitted by the photogenic organisms examined by me is confined to a very small portion of the visible spectrum ; never extending into the ultra-violet or infra-red. Visually it only includes the green and blue, and photographically it extends very slightly further into the violet. The economic value of such n light source is obvious, and it is n matter of great practical importance to determine its method of produc- tion." The writer experimented with 13 species. ('02). McKENNEY, R. E. B. Observations on the conditions of light production in luminous bacteria. Proc. of Biol. Soc. of Wash., Nov. 20, 1902, vol. xv, pp. 213-234. Also a separate. Bibliog. of 35 titles. ('02). BARNARD, J. E., AND MACFADYEN, ALLAN. On Luminous Bacteria. Annals of Botany, vol. xvi, Dec., 1902, pp. 587-588. ('03). MOLISCH, HANS. Vienna Acad. Sci., 1903. Luminous bacteria for Safety Lamp. Science, 1903, p. 719. Paper read, but not yet printed. Note in Science made from a Renter's telegram. ('04). GORHAM, F. P. See a preliminary uote in Central!*, f. Bakt., 2Abt., XIII Bd. XXVIII. Hydrogen Sulphide and Otherwise Unclassified By-Products. ('79). MIQUEL, P. De la fermentation sulfhydrique. Bull, de la Soc. ohim., T. xxxn. 1879, p. 127-131- ('89). HOLSCHEWNIKOFF. Ueber die Bildung yon SchwefehvasserstofF durch Bakterien. Fortschr. d. Med., 1889, Bd. vn, pp. 201-213. ('91). NENCKI, M. Die isomeren Milohsauren al< Erkennungsmittel einzelner Spaltpilzarten. Centralb. f. Bakt., Bd. ix, 1891, pp. 304-306. ('9i-'93). RAY-PAILHADE. Recherehes experiment- ales sur le pbilothion. Paris. 1891. Le philothion et le soufre. Assoc. franc., pour 1'av. des sci., Congres de Besangon, 1893. Part I, pp. 193, 250, and 302. ('92). SOMMARUGA, E. Ueber Stoffwechselproducte von Mikroorganismen. Zeitschr. f. Hyg., Bd. xii, 1892, pp. 273-297. ("92). PETRI, R. J., u. MAASSEN, A. Ueber die Bil- dung von Schwefehvas_•). BANNING, FRIEDRICH. Zur Kenntniss der Oxal- saurebildung durch Bakterien. Centralb. f. Bakt., Abt. 2, Bd. vm, 1902. pp. 520-525, pp. 556-567, mit I Taf. ('77) C/S). (,'Si). XXIX. Action of Light on Bacteria. DOWNES AND BLUNT. Researches on the ef- fect of 'light upon bacteria and other organ- isms. Proc. Roy. Soc., vol. xxvi, No. 184, 1877, 6 Dec., pp. 488-500. London. Downes and Blunt determined germicidal action to be associated chiefly with the actinic rays of the spectrum. i. DOWNES AND BLUNT. On the influence of light upon protoplasm. Proc. Roy. Soc., vol. xxvin. No. 191, 1878, 19 Dec., pp. 199-212. TYNDALL, J. Note on the influence exercised by light on organic infusions. Proc. Roy. Soc., London, vol. xxvm, pp. 212-217. Na- ture. vol. xix, 1879, p. 210. Negative results. Used flasks, and organisms grew after exposure to sunlight. TYNDALL, JOHN. On the arrestation of in- fusorial life by solar light. Br. asso. for the .advancement of sci. Rep., 1881, pp. 450-451. Nature, vol. xxiv, p. 466, 1881. Tyndall's results were due to the way in which he experimented, i. e., with flask cultures. ('82). ENGELMANN, TH. W. Bacterium photome- tricum. Ein Beitrag zur vergleichenden physiologie des Licht- und Farbensinnes. Onderzoekingen gedaan in het Physiologisch Laboratorium der Utreditsche Hoogeschool, Derde Reeks, vn, Aflev. n, 1882, pp. 252- 290, I plate. ('84). DOWNES, A., AND BLUNT, T. P. The influence of light on bacteria. Trans. Roy. Soc., Vic- toria, vol. xx, pp. 1-2, 1884. Reply to Jamieson. ('85). DUCLAUX, E. Influence de la lumiere du soleil sur la vitalite des germes de microbes. C. R. des se. de 1'Acad. des sci. T. C., 1885, pp. 119-121. ('85). DUCLAUX, E. Influence de la lumiere du soleil stir la vitalite des micrococcus. C. R. des se. de 1'Acad. des sci., Paris, T. ci, 1885, pp. 395-397- ('86). ARLOING, S. Influence de la lumiere blanche et de ses rayons constituants sur le devel- oppement et les proprietes du Bacillus an- thracis. Arch, de physiol. normale et pathol., 1886, 3 ser., T. vn, pp. 209-235. ('86). STRAUS, I. Note sur 1'action de la lurniere solaire sur les spores du Bacillus anthracis. C. R. des ise. et mom. de la soc. de Biologie. Paris, 8 se., T. in, 1886, pp. 473-474. ('86). DOWNES, ARTHUR. On the action of sunlight on Micro-organisms with a demonstration of the influence of diffused light. — Proceed- ings of the R. Society, London, 1886, vol. XL, pp. 14-22. ('87). DUCLAUX, E. Action de la 'lumiere sur les •microbes. Ann. de 1'Inst. Pasteur, 1887, T. i, pp. 88-92. ('87). Roux, E. De 1'actioii de la .hwniere et de 1'air sur les spores de Ja bacteridie du charbon. Ann. de 1'Inst. Pasteur, T. I, 1887, pp. 445- 452. ('88). ENGELMANN, TH. W. Die Ptirptirbakterien und ihre Beziehungen zum Lichte. Bat. Zeitung, iSSS, Jahrgang XLVI, col. 661, 677, 693, and 709. ('88). ENGELMANN, TH. W. Ueber Baeteriopurpurin und seine physiologische Bedeutung. Pfliiger's Archiv,, "1888, Bd. XLII, pp. 183-186. ('89). RAUM, JOHANNES. Der gegenwartige Stand unscrer Kenntnisse iiber den Einfluss des Liclites anf Bacterien und auf den thier- ischen Organismtis. Z«it. f. Hyg., Bd. vi, 1889, pp. 312-368. This paper contains a bibliography of 7 pages. ('Mo). PANSINI, S. Dell'azirine della luce solare sui microorganismi. Revista d'igiene practica e speriinentale, Napoli, 1889, pp. 69-101. Re- view in Ann. de Micr.. 1890, p. 516. ('90"). SAVKRIU. I/intluenza della temperatura sull' a/rione microbicida della luce. Ann. dell" Inst. d'ig. di Roma. Not seen. Cyo). JANOWSKI, TH. Zur Biologic der Typhus- -badllen. Centralb. f. Bakt., vm Bd., 1890, pp. 167-172, 193-199, 230-234, and 262-266. Discusses the action of sunlight. ('in). GEISLER, F. K. On the action of light on bac- teria. (Russian.) Wratsch, 1891, No. 36, Pp. 793-797 Not seen. ('92). MOMONT. See xxxv. ('92). KOTLJAR, E. Zur Frage iiber den Einfluss des Lichtes auf Bakterien. Wratsch. 1892, Nos. 39 and 40. Rev. in Centralb. f. Bakt., xn Bd., 1892. p. 836. Also in Ann. de 1'Inst. Pasteur, T. vn, 1893, p. 430. 244 BACTERIA IN RELATION TO PLANT DISEASES. ('92). CHMELEWSKY. Zur Frage fiber die Wirkung des Sonnen- und elektrischen Lichtes auf die Eiterbakterien. Wratsdi, 1892, No. 20. Reviewed in Centralb. f. Bakt., Bd. xn, pp. 174-175, 1892. ('92). GEISLER, THEODOR. Zur Frage fiber die Wir- kung des Lichtes auf Bakterien. Centralb. f. Bakt., xi Bd., 1892, pp. 161-173. ('92). BUCHNER, H. Ueber den Einfluss des Lichtes auf Bakterien. Centralb. f. Bakt., xi Bd., 1892, pp. 781-783. Deals with question of effect of light cm bacteria sus- pended in water. " The result of all these experiments points to the conclusion that light exerts a powerful dis- infecting influence upon the named bacterial sorts when these are suspended in water." The experiments were made on typhoid bacilli, B. coli cominunis, B. pyocyan- eus, cholera vibrios, and various bacteria of decay. ('92). BUCHNER, H. Ueber den Einfluss des Lichtes auf Bakterien. iiMitth. Centralb. f. Bakt., Bd. xii, 1892, Heft 7-8, pp. 217-219, i fig. Author obtained bacterial letters and figures on agar and gelatin plates by covering a part and exposing to sunshine. Light passed through water still possesses active bactericidal powers. ('93). BUCHNER, H. Ueber den Einfluss des Lichtes auf Bakterien und fiber die Selbstreinigung der Flfisse. Arch. f. Hyg., Bd. xvn, 1893, pp. 179-204. ('93). RICHARDSON, ARTHUR. The action of light in preventing putrefactive decomposition; and in the formation of hydrogen peroxide in organic liquids. Jour. Ghem. Soc., Trans- actions, London, 1893, vol. LXIII, pp. 1,109- 1,130. ('94). D'ARCY, R. P., AND HARDY, W. B. Note on the oxidizing powers of different regions of the spectrum in relation to the bactericidal action of light and air. Jour, of Physio!., i894-'95, vol. xvn, pp. 39O-393. ('.14). WARD, H. MARSHALL. The Action of light on bacteria. Phil. Trans. Royal Soc., Lond., vol. 185 (1894), pp. 961-986. Also a sepa- rate. 410., 25 pp., i plate. See also Revue Sci., 1894. ('i)il. FISCHER, BERNHARD. Die Bakterien des Meeres naoh den Untersuchungen der Plank- tonexpedition unter gleichzeitiger Beriick- sichtigung einiger alterer und neuerer Un- tersuchungen. Centralb. f. Bakt., Bd. xv. 1894. pp. 657-666. Shows that sunlight tends to reduce number of bacteria in upper layers of the sea. There are always more bac- teria iu upper layers of the sea at suurise than in the afternoon. There are also in the daytime a great many more at a depth of 10 meters than near the surface. The depth to which the bactericidal action of the sunlight penetrates depends on its intensity, duration of action, clearness of the water, etc. It probably reaches down several meters. Cultures of various water bacteria were killed in a short time when exposed to the midday sun in August, the sun's rays being first passed through one-half meter of sea watt-i . i '114). DIKUDONNE, A. Beitrage zur Beurteilung der Eimvirkung des Lichtes auf Bakterien. Arbeiten a. d. Kaiserl. Gesundheitsamte, Bd. ix, 1894. Heft 2, pp. 405-413. Rev. in Cen- tralb. f. Bakt., xvn P.fl.. 1805. pp. 646-647. The red end of the spectrum (between the lines D and El has no injurious effect. In the green (between lines K and F) there is a distinct inhibiting action. In the lihi< violet and ultra-violet the bactericidal action is most marked. The action of the light is directly on the bac- teria. Bacteria inoculated in plates already exposed tc the light grew just as well as in the control plates. Light, which has had the heat rays removed by filtration through alum solution, possesses the same germicidal action. ('94). DIEUDONNE, A. Ueber die Bedeutung des Wasserstoffsuperoxyds fur die bakterien- todtende Kraft des Lichtes. Arbeiten aus dem kais. Gesundheits-Arnt., Bd. ix, 1894, PP. 537-540. Ascribes bactericidal effect of light in great part to the formation of hydrogen peroxide in the culture-medium. ('94). ENGELMANN. See xix. ('94). D'ARSONVAL AND CHARRIN. See xxxni. ('96). BECK, M., u. SCHULTZ, P. Ueber die Ein- wirkung sogen. monochromatischen Lichtes auf die Bacterienentwicklung. Zeitschr. f. Hyg., Bd. xxin, 1896, pp. 490-496. ('99). KEDZIOR, LAURENZ. Ueber den Einfluss des Sonnenliohtes aiif Bakterien. Arch. f. Hyg., Bd. xxxvi, 1899. pp. 323-334. Rev. in Cen- tralb. f. Bakt.. Bd. xxvn, 1900, pp. 203 and 759- Sunlight also destroys in an atmosphere of hydrogen, although less actively. ('oi). SIMONCINI, G. B., E VIOLA, D. L'influenze dell'innaffiamento sul contcnutp batterico delle polveri di strada. Ann. d'igicne sper, Roma, vol. \i, IQOI, pp. 373-392. Bibliog. of 21 titles. The bactericidal action of light was greater on the moistened dust of the street than on the dry dust. XXX. Effect of Electricity. ('91). SPILKER, W., UND GOTTSTEIN, A. Ueber die Vernichtung von Mikroorganismen durch die Induktionselektricitat. Centralb. f. Bakt, ix Bd., 1891, pp. 77-88. ('91). FERMI, CLAUDIO. Lleber die Reinigung der Abwasser durch Elektricitat. Arch, fur Hyg., Bd. xiii, 1891, pp. 207-228. Cii.i). KRUECER, S. LTeber den Einfluss des constan- ten elektrisohen Stromes auf Wachsthum der Bakterien. Zeitschr. f. Win. med., Bd. xxn, 1893. pp. 191-207. Krueger's conclusions are : The inhibition or destruc- tion of the bacteria is due to the liberation of ions in the Quid. ('93). BUKCI, E., E FRASCANI, V. Contribute a'llo studio dell'azione battericida della corrente continua. — Atti della Soc. Tosc. di Scienze nat. Pisa. Mem., vol. xn, 1893, pp. 99-119. ('94'). D'ARSONVAL AND CHARRIN. See xxxni. ('96). FRIEDENTHAL, H. Ueber den Einfluss des elektrischen Stromes auf Bakterien. Krit- isches Referat. Centralb. f. Bakt., xix Bd., 1896, pp. 319-324. ('96). GOTTSTEIN, A. Ueber den Einfluss des elek- trischen Stromes auf Bakterien. Centralb. f. Bakt., xix Bd., 1896, pp. 602-605. I'M'' i. FRIEDENTHAL. H. Ueber den Einfluss des Induktionselektrizitat auf Bakterien. Krit- isches Referat. Centralb. f. Bakt., xx Bd , 1896, pp. 505-508. ('96). MARMIER, L. A. Les toxines et 1'electricite. Ann. de 1'Inst. Pasteur, T. x, 1896. pp. 469- 480. ('99). THIEI.K. HERMANN, UND WOLF, KURT. Ueber die Eimvirkung des elektrischen Stromes auf Bakterien. Centralb. f. Bakt., xxv Bd., iSoo, PP. 650-655, with I fig. Results all negative. i 'mi). KRAUSE. See xv. ELECTRICITY; KOKNTHKN KAN'S, KTC. ; HIGH PRESSURE. 245 Coi). STREBEL, UI'KMAXX. Untersuchungen iiber die kiktericide Wirkung des Hochspannungs- funkenliclUcs neb.st Angabe einer Methode /iir licsseren Ausniitzung der baktericiden Kraft des Voltabogenlichtes. D. med. Wochenschr.. Berlin, Bd. xxvn, 1901, pp. 69-72, pp. 87-89. ('oi). ULLMANN, JOHANNES. Ueber die Einiwirkung elcktrischen Bogculichts anf Mikroorgan- iMiien in Gegenwart von fluoreszierenden Si "(Ten. Diss. Miinchen (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 quelconque sur les bacteries? Ann. de micro., T. vin, 1896, pp. 514-515- Author finds that the Roentgen rays have uo effect upon bacteria. ('96). MIXCK, F. Zur Frage tiber die Einwirkung der Rontgen'schen Strahlen auf Bakterien und ihre eventuelle therapeutische Verwend- barkeit. Miinchener mediz. Woohenschrift. 1.896, Bd. XLIII, pp. 101-102 and p. 202. Author obtained only negative results. ('97). POTT, FRANCIS. Concerning the action of X- irays 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 1'aetion des rayons X sur Je Pyocyaneus et la bacteridie char- bonneuse. C. R. des sc. et mem. de la soc. de biol., T. iv, me 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. rv, loe serie, 1897, pp. 803-804. The bacteria are much less sensitive than higher or- ganisms. RIEDER, HERMANN. Wirkung der Rontgen- strahlen auf Bakterien. Miinch. med. Wochenschr., 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. Voltohm's apparatus was used. The phot,, graphs show the center of the agar Petri-dish cultures cleared of bacterial colonies. ('98). WOLFENDEN, NORRIS, 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 prodigiosus on 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 Haul. Miinch. med. Wochenschr., 45 Jahrg., 1898, pp. 773- 774- Coi). CASPARI, W. Ueber die bacterienschadigende Wirktmg der Becquerelstrahlen. Naoh in Gemeinschaft mil Priv. Doc. Dr. Aschkinass ausgefiihrten Versuchen. Arch. ital. biol., Turin, T. xxxvi, 1901, p. 130. ('02). RIEDER, HERMANN. Nochmals die bakterien- todtende Wirkung der Rontgenstrahlen. Miinchener med. Wochenschr., Bd. xux, 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 cerevisiie 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 CHARKIN. Pression et microbes. La scmaine medicale, 1893, T. xm, 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 t<> a pressure of 50 atmospheres under carbon dioxide. All were dead inside of 24 hours. Even two hours' exposure interfered with the reproductive function, i. e., lessened the number of organisms capable of producing colonies, nml 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 pigment, and generally no colonies when sown upon agar, but in one case there were a few. ('94). D'ARSONVAL AND CHARRIN. See xxxm. ('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 j.oooto 3,000 atmospheres were tried with, out destroying the bacteria. Certain functions, however- were destroyed, e. (j., pathogenicity. ('97). MALFITANO, G. Sul comportamento dei micro- organism! all'azione dei gasi compressi. Boll, della Soc. medico-chirurgica di Pavia, 1897. Rev. in Centralb. f. Bakt., xxin 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 Entvvicklung 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 Einfiuss 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 Abt., 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). CHAMEERLAND, CH. Resistance des germes des certains organismes a la temperature -de 100 degres ; conditions de leur developpe- ment. C. R. des se. de 1'Acad. des sci., T. LXXXVIII, 1879, pp. 659-661. ('82). LEBEDEFF, A. Contribution a 1'etude de 1'aotion de la chaleur et de la dessieation sur la virulence des liquides septique et sur les I'organismes inferieurs. Archives de Physiol. normale et Path., Ser. II, 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). ESMARCH,E. Der Henneberg'sche Desinfector. Zeitschr. f. Hyg., Bd. n, 1887, pp. 342-368. ('88). GLOBIG. Ueber einen Kartoffel-Bacillii^ mit ungewdhnlich iwiderstandsfahigen Sporen. Zeitschr. f. Hyg., Bd. Ill, 1888, pp. 322-332. ('XX i. GRUBER, MAX. Notiz iiber die Widerstand- fahigkeit der Sporen von Bacillus subtilis gegen Wasserdampf von 100° C. Centralb. f. Bakt., 1888, Bd. in, pp. 576-577. Iti six tubes of hay infusion inoculated with spores of H. suhtiiis, sealed by heating ueck in flame, and then steamed l/2 hour, there was an abundant growth of the hay bacillus in 36 hours at 37° C. Subsequently 21 sam- ples of spores, dried on silk threads and exposed to streaming steam tor 2% hours, in Thursfield's apparatus, grew readily ; iu 24 hours, at 35- C., there was a must lux- uriant vegetation. ('88). FISCHER. B. Bakterienwachstum bei o° C. Centralb. f. Bakt., Bd. iv, 1888, pp. 89-92. ('90). LUSTIG, ALEXANDER. Bin rorher Bacillus im Flusswasser. Centralb. f. Bakt., Bd. vm, 1890, pp. 33-40. I.ustig isolated a motile bacillus from river water, which grew from room-temperature (probably 15° C } to 60' C. i ''Ut. FORSTER, J. Ueber die Entwickelung von Bak- terien bei niederen Temperaturen. Centralb. f. Bakt., xn Bd., 1892, pp. 431-436. The kinds of bacteria able to grow at o° are not very numerous, but seem to be widely distributed, especially in water and on the surface and iu the intestinal tract of fresh-water fish and salt-water fish. Co,0. PICTET, RAOUL. De 1'e.mploi methodique des basses temperatures en biologic. Archiv. d. sci. phys. et nat., 3e Periode, T. xxx, pp. 293-3i4, Geneve, 1893. Experiments with higher animals and plants, infusoria, microbes, diatoms. With these two latter, excessive and prolonged cold gave negative results. They were sub- lected to a temperature of minus 200°. ('93). PHYSALIX. Influence de la chaleur ^ur la prp- priete sporogene du Bacillus anthracis. Abolition persistante de cette fonction par heredite des characteres acquis. Arch, de physiol. nonmale et path., Paris, 1893, T. v, ser. 5, pp. 217-225. ('94). D'ARSONVAL ET CHARRIN. Influence des agents cosmiques (electricite, pression. lumiere, froid, ozone, etc.) sur 1'evolution de la cell- ule bacterienne. Arch, de physiol. normale et path., 1894, T. VI, series 5, pp. 33S~342- ('94). WALDO AND WALSH. See XLVIII. ('94). HAVEMANN. Ueber das Wachsthum von Mikroorganismen bei Eisschranktemperatur. (Liang. Diss.) 8vo., 21 pp., Rostock, 1894. Not seen. ('95). STERNBERG, GEORGE M. What shall be the methods followed in determining the rela- tion of bacteria to temperature? Jour. Am. Public Health Asso. Ann. vol. xx, 1895. pp. 4H-4I4. ('95). KLEPZOFF, CONST. Zur Frage iiber den Ein- flnss niederer Temperaturen auf die vegeta- tiven Formen des Bacillus anthracis. Cen- tralb. f. Bakt., xvii Bd.. 1895, pp. 289-295. Exposure to intense cold (average — 24° C.) for 12 days killed the anthrax organism in Hooil and various organs. Exposure for 25 days (at — i° to — 24° ( ' , average — 10.40° C.) killed agar cultures. The colonies in agar plates be- came less and less numerous as time passed No spores were present. Long exposure reduced the virulence. ('95). MIOUEL, P., ET LATTRAYE. De la resistance des spores des bacteries aux temperatures humides egales et superieures a 100°. Ann. de micr., T. vn, 1895, pp. 110-122, 158-170, and 205-218. ('99). DANNAPPEL, MAX. In wie weit ist die hohcre Widerstandsfahigkcit der Bakteriensporcn ein allgemeines Charakteristrikiun dersellim gegeniiber den vegetativen Spaltpilzformeu ? 8vo., pp. 27, Konigsberg, i. Pr., 1899, von E. Karg u. R. Manncck. Some spores are said to show only a slight resistance to steam at 99° C. Of 25 species obtained from soils, decay- ing mixtures, milk, butter, etc., and said to be sporifer- ous, all but three were destroyed l.y exposure t" steam at 99° C. for 10 minutes and all but S by exposure for ,s minutes while 4 were kilk-d by exposure for as short a time as 15, seconds, and two others by exposure for i minute. The names of these organisms are not given, so that the ex- periments cannot be duplicated, and in most cases it is not stated that the sporiferous nature of these bacteria was settled definitely by seeing the spores germinate. It is possible, therefore, that some of the extremely sensi- tive forms were not actually spore-bearing, but only gave microscopic appearances, which were interpreted as such. It is possible, also, that the spores were tested before they were fully matured. Only twopf the very-sensitive forms were examined critically, owing, it is said, to lack of time, and of one of these sensitive forms it is said : "A direct observation of the germination was not undertaken because the spore nature of the culture appeared unques- tionable." Even heating for i to 3 minutes at 75° to So0 C destroyed this organism. Germination of the other was observed. The maximum temperature which could be endured in this case, for i minute, was 75° C. Both were green spores. Both were double stained by Moeller's method. ('99). RAVENEL, M. P. The resistance of bacteria to cold. New York Medical News, vol. LXLIV, 1899. Also a separate, 5 pp. Rev. in Cen- tralb. f. Bakt., xxviii Bd., 1900, p. 751. Tests in liquid air : B. diphtherias was alive at end of 30 minutes, B. typhi and B . prodigiosus at the end of 60 minutes, B. anthracis after 3 hours. ACTION OF 11KAT AND (dl.l); Til KRMOPIIILIC BACTERIA. 247 ("99). S\ T ill . Tlir Ilirnnal death point of tnVrcle bacilli in milk anil some other tlniils. Journal of Experimental Med., vol. iv. (899, i>i> 317-233 Uev in Central!), f. ]'.-ik; . xxviii Bd., njoo, p. 409. \\ In ii embedded in the film ou the surface of milk, Dr. Smith hmiul the tubercle organism resisted a tempera- ture ol 6ou C. for an hour. ('99). KASAX>KY, M. \Y Die Einwirknng der Win- terkalte atif die Pest- mid Dip'htheriebacillen. t'i-iitralli. f. P.akt.. xxv Bd., 1899, pp. 122-124. Thtse organisms withstood exposure for 6 months to severe temperatures. They \vrie fro/.en all of the time for the first five months. From December 4 to 28, aud again from February 13 to Marcu 9. the maximum tem- perature was — 10° to — 23 4" C , and the minimum was - 14° to- 33.8° C. ('99) l.i \ IN. See xi.ii. ('99). MIKOXESCO, THEODOR G. Ueber eine besondere Art der Beeinflussiing von Mikroorganis- men dtirch die Temperatttr. Hygien. Rund- schau, Jahrg. ix, 1859, pp. 961-964. Rev. in Centralb. f. Baku xxvn Bd., 1900, p. 86. (~oo). MEYKR, J. Ueber Einwirkung flussiger Luft auf Babterien. Centralb. f. Bakt., xxvm Bd., 1900, pp. 594-595- Anthrax spores and Staphylococcus pyog. aureus were tested. The exposure to the liquid air varied from 5 seconds to 15 minutes. Neither organism was killed. The temperature of licjuid air is - 190° to— 220° C .accord- ing to Spiess, and — 182° to — 192° C. according to Mac- fadyen I'oo). SEDGWICK, \V. T., AND WINSLOW, C. E. A. Experimental and statistical studies on the influence of cold upon the bacillus of typhoid fever, and its distribution. Jour. Bost. Soc. Med. Sci., vol. iv, Xo. /, 1900, pp. 181-182. See also Centralb. f. Bakt., xxvn Bd., 1900, p. 6X4. 30 to 60 per cent of the bacilli were destroyed in water during the first hour of freezing. After exposure for two weeks 99 per cent were destroyed. " The last two or three germs per thousand appear to be very resistant, some remaining a ter twelve weeks of freezing. The four races used showed constant individual differences in their sus- ceptibility to cold. Alternate Ireezinga-d thawing was tested and found only slightly more destructive than con- tinuous freezing." As several races of typhoid organism were tested, we may infer tliat ice is not very likely to communicate typhoid fever. ('oo). PARK. \\M. 1 IAI.I.HCK. A few experiments upon the effects of low temperature and freezing on typhoid bacilli. Jour. Bost. Soc. Med. Sci., vol. iv, Xo. 8, 1900, pp. 213-216. Cultures were used from twenty different cases of typhoid fever. They behaved when frozen much ns Sedg- wick and Winslow's. Ou the average, at the end of twelve weeks' freezing only 0.05 of one per cent remained alive, i e., 1,250 per cubic centimeter as against 2,560,410 per cubic centimeter at the beginning. "At twelve weeks ihe bacilli in the ice from nine sources are all dead. Two more show no growth in i cc. The others contain from So to n,coo in each cc. of ice. Only one, however, contains over 1,000 i, culture 9) When typhoid bacilli are in feces, freezing does not exert so much of an effect Thin typhoid and colon bacilli, originally 37,000 to a loopful of feces, wi re still 12,000 at the end of five weeks' exposure to a temperature ranging daily between zero and 28' F , and typhoid bacilli as well as colon were still abundant in the fecep at nine weeks It is a difficult matter to say for just how loug a period ice made from infected water remains dan- geruus. The bacilli, even when few in number, are often vigorous aud fully virulent, aud, so far as I am ;i\\ .ire, u t are ignorant as to the number of bacilli required to start infection in man. The longer the infected ice remains frozen the less the number of pathogenic bacteria which remain alive in it." ('or). PARK, W. H. Duration of life of typhoid bacilli, derived from I \venty different sources, in ice. Abstract of paper read at 2d meeting Soc. Am. Bacteriologists, Dec., 1900, Centralb. f. I'.akt , i Ah:., Bd. xxix, 1901, pp. 444-445. This describes the completion of an experimentalready reported upon in part (see above). At the end of the twenty-second week of exposure the bacilli were dead in all the cultures of each one of the twenty races tested by freezing. ( '01 ) . D'ARSONVAL. La pression osmotique et son role de defense contre le froid dans la cellule vivanite. C. R. des se. de 1'Acad. des =ci., Paris, 1901, T. cxxxm, pp. 84-86. The fluid in the bacteria is probably not solidified, if the cell is not ruptured, owing to the enormous osmotic pressure in those small organisms. By lowering the os- motic tension the author thinks that any cell may be killed by cold. ('02). ScHMiDT-XnasEN, SIGVAL. Ueber einige psy- ch rophile Mikroorganismen und ihr Vor- kommen. Centralb. Bakt., Abt. 2, Bd. ix, 1902, pp. 145-147. ('O2). MACFADYENj Al.I.EX, AND ROWLAND, Svi'NKV. On -the suspension of life at low tempera- tures. Abstract of paper read before Sec- tion K of the British Association, Belfast, 1902. Annals of Botany, vol. xvi, 1902, pp. 589-590. Various bacterial organisms were exposed from 20 hours to 7 days at —190° C. " These exposures did not produce any appreciable impairment in the vitality of the organ- isms, etc." Also 10 hours at —252° C. the temperature of liquid hydrogen had no appreciable effect on the vitality of the micro-organisms tested. Bacillus typhosus, B.coli- communis, Staphylococcus pyogeues aureus and a Sach- aromyceie grew after exposure to liquid air for six mouths. " In no instance could any impairment of the vitality of the organisms be detected." The objection to these statements is that quantitative determiuatious appear ii"t to have been made, at least there is no mention ot any. The writer of this review obiaiued a decided diminution of the number of viable bacteria in several species by exposure to liquid air for 20 hours. {'02). MACFADYEN, ALLEN. On the influence of tin- prolonged action of the temperature of liquid air on micro-organisms, and on the rfiVct of mechanical tritnration at the tem- perature "f liquid air on photogenic bacteria. I, Minloti. Proc. R. Soc., vol. LXXI. No. 4'^. Oct.. 1902, pp. 76-77. " The above experiments show that a prolonged expo- sure of six months to a temperature of about — 190° C. IKIS no appreciable effect on the vitality of micro-organisms The organisms tested were B. 'tyf-homs, It.^coli cmn- inu'iis. tjtaphyfococcus pyntienat aiff «j», and a yeast. The triturated bacteria k»t their luminosity. ('05) SMITH \\n SWINGLE. See p. 83. XXXIV. Thermophilic Bacteria. (>p) \II.MU. P. Title?* Bull. (lr la statistiquc munuipalr crvatoire de Montsouris, pour 1881, p \< > \ 248 BACTERIA IN RELATION TO PLANT DISEASES. i 'Si 1 1. CKRTES, A., ET GAFRIGOU. De la presence con- .-tante de micro-organismes dans les eaux de Luchon, recueillies an 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, 18X6, pp. 703-706. ('88). GLOBIG. Ueber Bakterien-Wachsthum bri 50 bis 70°. Zeitschr. f. Hyg., Bd. Ill, 1888, pp. _'. T. vi, pp. 21-31. SWAN, AI.I.EN P. On the resisting vitality "f tin- spores i.f Bacillu-- megateririm to the condition of dryness. Annals of Botany, vol. vn, p. 153-154, 1893. KKSISTAM'K Id HUY A I U ; ACTION OF ACIDS AND AI.KAI.I KS, ETC. _>4<) ('94). WALUCZEK, HEINRICH. Die Resistenz des Bacterium coli commune gegen Eintrock- inin.sf. Centralb. f. Bakt.. Bd. xv, 1894, pp. 949-9SO. 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 on cover-glasses, while Bact. hyacinth! lived under similar conditions for more than a month. Jones fuund his Bacillus carotovorus to be even more sensitive than B. tracheiphilus. See following citations. SMITH. KKWIX F. Bacillus tracheiphilus, etc. Centralb. f. Bakt., 2 Abt, Bd. I, p. 37°. ('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. (Hi). TUXES. L. R. A soft rot of the carrot, etc. 1 3th Ann. Rep. Vt. Agric. Exp. Sta. for 1900. Burlington, Vt., 1901. See p. 328 for refer- ence to this subject, foi). SMITH. ERWIX F. The cultural characters of Ps. •hyacinthi, etc. Bull. 28, Div. Veg. Phys. and Path., U. S. Dep. Agr., Washington, D. C,. TOOT. p. 145. XXXVI. Action of Acids and Alkalies. ('86). ABBOTT, A. C. The germicidal value of some of the vegetable acids. The Medical News, Phila., 1886, 9 Jan., pp. 33-34- uj ) DEI.ERUECK, M. Ueber das Verhalteu der Cholerabacillen auf frischen Friichten, einigen Genuss- und Nahrungsmitteln. Son- derabdruck aus den Veroffentlichungen des Kaiserlichen Gesundheitsamte, 1892, Nn. 4-'. 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 o.n, (certain pears) to 2.65 (red currants^. In the feebly acid fruits, the cholera bacilli were dead inside of 3 to 7 days ; in the the tarter fruits they retained their vitality only for a period measured by hours. Usually they were dead in from i to 6 hours. ('92). SCHI.UETER, G. Das Wachstum der Bakterien auf saurem Nahrboden. Centralb. f. Bakt., Bd. xi, 1892, pp. 589-598. 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 % per cent alum. Six grew abundantly in gelatin acidified \vith citric acid.so that 8 cc. of the'gelatin required for its neutralization 4CC. 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). HES>E. YV. Uebor den Einfluss der Alka- lescenz des \ahrbodens auf das Wachsthum der Bakter.ien. Zeitschr. f. Hyg., Bd. xv. 1893. pp. 183-191. ('93)- VoGES, O. Ueber das Wachstum der Cholern- bacillen auf Kartoffeln. Centralb. f. Bakt.. Bd. xni, 189,?. pp. 543-550. Organism would not grow on potato as ordinarily pre- pared.but grew well at 37° C. (and more slowly -it zo ; mi the addition of a 2 to 3 percent solution of sodium .linn ide. Nearly as good results were obtained with ', to J.J per cent sodium carbonate solution. Growth was also obtained on potato with ^ to % per cent sodium hydrate solution. ('97). DEELEMAN, M. Der Einfluss der Reaktion des Nahrbodens auf das Bakterienwachstum. Arbeit, aus dem KaiserJ. 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- enzierung der Mikroorganismen. Centralb. f. Balot, 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. XXXVII. Agglutination and Precipitation. ('96). WIDAL, FERNAND. Sero-diagnostic de la fievre typhoide. Bull, et mem. de la soc. med. des hop. de Paris, 26 juin, 1896, pp. 561-566. ('97). WIDAL, F, ET SICARD, A. Etudes sur le sero- di. ignostic et sur la reaction agglutinante chez les typhiques. Ann. de 1'Inst. Pasteur, T. xi, 1897, pp. 353-432. ('97). FLEXNER, S. A recently discovered property of the blood serum in animals immune from certain diseases, and its application to the diagnosis of those diMMM-s in human beings. Science (n. s.), vol. v, pp. 193-194, 1897. ('97). MALVOZ, E. Recherches sur ['agglutination du Bacillus typhosus par des substances chiniique. Ann. de 1'Inst. Pasteur, T. xi, 1897, pp. 582-590. ('98). NICOLLE, CHARLES. Recherches sur la sub- stance agglutinee. Ann. de 1'Inst. Pasteur, T. xn, 1898, pp. 161-101 ('99). BORDET, JULES. Le mechanisme de 1'agglutina- tion. Ann. de 1'Inst. Pasteur, T. xni, 1899, pp. 225-250. ('99). KRAUS, R. Ein Beitrag zur Kenntnlss des Mechanismus der agglutination. Wiener Klin. Wochenschr. 1899, Jahrg. xn, pp. 1-4. ('99). GRI'BER. Zur Theorie der Agglutination. Munch, med. Wochenschr, 1899, No. 41. Rev. in Centralb. f. Bakt, xxvn Bd., 1900, pp. 285-286. ,, SAHRAZES ET BRENGUES. Agglutinines chim- iques. C. R. de la Soc. de biol, 1899, No. 35- P- 93°- RCV- i'1 Centralb. f. Bakt, xxvn IM, icn»>. p. 756. Coo). ZIKES. Ueber das Ausschleudern von Mikro- orgatiismen unter Zuhilfenahme von Fal- lungsmitteln. Oesterr. Chemiker-Zeitung, 1900. \n. 2. Rev. in Centralb. f. Bakt, xxvil Bd., 1900, p. 628. ("oo). SMITH, R. GREIG. The flocculation of bacteria. The mechanism of agglutination. Proceed- ings of Linn. Soc. of New South Wales, 1900, Pant i. pp. 65-74, "5-83. Also a sepa- rate (issued Aug. 8. 1900). ('oo). DURHAM. HERBERT E. Some theoretical cpn- siderations upon the nature of agglutinins, together with further observations upon Bacillus tvphi abdominalis. Bacillus enteri- tidis. Bacillus coli communis. Bacillus lad is aerogenes, and some other bacilli of allied character. Jour, of Exp. Med, vol. v, pp. 353-388. 25° BACTERIA IN RELATION TO PLANT DISEASES. • >i i \\ II.MIN, 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. Coi). DOUGH, CHARLES WRIGHT. A short nn-tlmd fur the Widal test. Jour, of Applied Micro., vol. iv, 1901, p. 1,565. Also a separate. i'o_'). Xr.rFELD, F. Ueber die Agglutination der Pneumokokken und iiber die Theorieen der AuLjlutination. Zeitschr. f. Hyg., 1902, Bd. XL. pp. 54-72. ('02). EISENBERG, PHILIPP, UND VOLK, RICHARD. Untersuclumgen iiber Agglutination. Zeitsch. f. Hyg., 1902, Bd. XL, pp. I55-I95- Bibliog. of 56 titles. ('02). Joos, A. Untersuchungen iiber den Mechan- isnius der Agglutination. Zeitsch. f. Hyg.. 1902, Bd. XL, pp. 203-230. ('02). SMITH, R. GRE»O. 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. xvm. No. 444, r9°3, PP- 3-15- •74). ('75). XXXVIII. Antiseptics and Germicides. (See also XXXVI. i 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. DAVAINE, C. Recherches relatives a Faction des substances antiseptiques sur le virus de la septicemie. Gaz. med. de Paris. 1874, p. 44. Reprinted in 1'Oeuvre de Davaine, Paris, 1889. I.EUIX, L. Das Thymol ein Antisepticum und 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. POLLI, G. Sulle proprieta anti fermentative dell'acido boracico e sue applicazioni alia terapia. Mem. 1st. Lomb., vol. xni, pp. 453- 468. Journ. de Pharm., et de chimie, T. xxvi, 1877, 4 se., pp. 77-79. BOVET, V. Ueber die antiseptischen Eigen- schaften der Pyrogallussaure. Journ f. prakt. Chem. Neue Folge, Bd. xix, pp. 445-461, 1879. PAVESI, C. Del solfato di potassa, e special- mente della sua proprieta antisettica. anti- fermentativa. Polli. Annali, vol LX.XI. serie 33, 1880, pp. no-i 15. ENDEMANN, H. Boracic acid as a preservative. Chem. News, vol. XLI, pp. 152-153, 1880. SCHWARTZ, NICOLAI. Ueber das Verhalten einiger Antiseptica zu Tabacksinfusbacte- rien. Pharm. Zeitschr. f. Russland, Bd, xix, 1880, pp. 610-625, 641-658, 673-1 iS^ Tested chloroform and found it of little worth. Ex- periments were made with 40 substances. The tabular summary is on pp. 68^-'iSs. Picric acid heads the list for efficiency. ('So). REGNARD, PAUL. Influence de I'eau oxygenee sur la fermentation. Gaz. med. de Paris, T. ii, 6 ser., 1880, p. 358. ('79). ('So). C8o). CSo). C'Si). CHAPPUIS, E. Action de 1'ozone sur les germcs contenus dans 1'air. Bull, de la Soc. chim. de Paris, I sem., n. s., T. xxxv, Paris, i8Si. p. 290. C8i). BARNES, J. B. The antiseptic properties of cinnamic acid. Pharmaceut. Jour, and Transactions, vol. xn, pp. 477- 478, iSSi. ('Si). 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. 2,83=;- 2,838. C8i). GOSSELIN, L., ET BERGERON, A. Recherches sur la valeur antiseptique de certaines substances et en particulier de la solution akoolique de Gaultheria. Arch. gen. de med., Paris, 1881, vol. i, vni, se., T. 7 (misprinted 6), 147 vol. de la collection, pp. 16-29. ('82). BERT, P., ET REGNARD, P. Action de 1'eaii oxyge-nee 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. Sur 1'action desinfectante et anti- M-ptique du cuivre. C. R. des se. de 1'acad. 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" death from either disease in the Societe de Bonaccord (copper, bronze and brass workers) since its establishment in 1819. ('82). SCHIEFFERDECKER, P. Ueber eine neue Injcc- tionsmasse zur Conservirung der Leichen fiir den Praparirsaal. Arch. f. Anat. u. Entwickelungsgesch.. 1882, pp. 197-198. (>\2). VULPIAN. Etudes expi-rimentales relatives a 1'action que pent exercer le permanganate de potasse sur ,les venins, les virus et les maladies zymotiques. C. R. des se. de 1'Acad. des sci., T. xciv, 1882, pp. 613-617. Jour, de Pharm. et de Chimie, T. vi. 5 serie, 1882, pp. 100-104. C86). UNNA, P. G. Ichthyol und Resorcin als Rep- rasentatcn der Gruppe reduzicrender Heil- mittel. Hamburg, 1886. Unna's Derma- tologische Studien, 2 Heft, pp. 1-85. ('88). XUTTALL. GEO. Experimente iiber die bak- terienfeindlichen Einfliisse des thierisclicn Korpers. Zeitschr. f. Hyg., Bd. iv, 1888, pp. 35.V394, i Plate. ('SSi. SALKOWSKI, E. Ueber die antiseptische Wirk- ung des Chloroformwassers. Deutsche med- ioin. Wochenschrift, 1888, Bd. xiv, pp. 309- 3". ( SS). L.OEW, O. Physiologische notizen iiber For- maldehyd. Miinchen. med. Wochenschr., 1888. Bd. xxxv. pp. 412-413. Physiol. Ges. Miinchen d-6). 1888, pp. 39-41. Rev. in Ch. Centralb., 1889. LX Jahrg, Bd. i, p. iio. ('XX). BKHKIXI-,. Ueber Quecksilbersublimat in eiweisshaltigen Fliissigkeiten. Centralb. f. Bakt.. 1888, Bd. in. pp. 27-30 and 64-0(1 ('89). FRAEXKEL, CARL. Die Einwkkung der Kohlen- saure auf die Lebensthiitigkeit der Mikro- organismen. Zeitsch. f. Hyg., Bd. v, 1889, PP- 332-362. ('90) \i IEHOEFEE. I'eber die Desinfectionskraft Min Wasserstoffsuperoxyd auf Wasser. Cen- tralb. f. Bnkt., 1890, Bd. vm, pp. 129-137. Cgo). KIRCH NEK, M. Untersuchungen iiber der Ein- \virkung des Chloroforms auf die Bakterien. Zeitschr. f. Hyg., Bd. vm, pp. 465-488, 1890. ANTISEPTICS AND GERMICIDES. 251 ('90). SONNTAG, HERMANN. Ueber die Bedeutung dcs Ozons als Desinficiens. Zeitschr. fiii Hyg., 1890, Bd. vin, pp. 95-136. ('91). TIZZONI, GUIDO, y. CATTANI, 0. Ueher die Widerstandsfdhigkeit der Tetanusbacillen •gegen physikalische mid chemische Einwirk- ungen. Archiv. f. cxper. Path. ti. Pharm., 1891, Bd. xxvjn, pj). 41-60. I'oi). FROELICH, O. Ueber das Ozon, dessen Her- stelkmg auf elektrischem Wege und dessen teohnisohe Anwendimgen, insbesondere in der Gesundheitstechnik. Gesundsheits- Ingenicur, 1891, No. 16, pp. 543-551. ('91). FROELICH, O. Ueber das Ozon, dessen Her- stellung auf elektrischem Wege und dessen technische Anwendungen. Electrotechnisohe Zeitschr., 1891, 12 Jahrg., pp. 340-344. Contains short paragraph on physiological action of ozone. Bacteria living in water are killed " summtlich." No experiments with pathogenic bacteria. It is still a question whether bacteria in the air are killed. (''iii. GERLACH. V.\i.. Ueber Lysol. Zeitschr. f. Hyg.. Bd. x, iRgi. pp. 167-196. Also a sepa- rate. Lysol is more active than carbolic acid or creolin. The hands may be disinfected in a i per cent, solution without soap. Surgical instruments may be sterilized in l/4 per cent solution without the least injury. Walls may be dis- infected in 33 per cent solution. It is to man the 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 stir la fermentation lactique. C. R. des se. de 1'Acad. dcs sci., T. c.xiv, 189.2. PP- L494-I.496. ('92). i 'HI. MUELLER. Ueber die Einwirkung des Ozons auf Bakterien. Arbeiten aus dem Kaiserl. Gesundheitsamte, Bd. vin, 1892, Heft 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. ( '<).!>. DELBRUECK. See xxxvi. ('92). HAMMER, HANS. Ueber die desinficirende \V irk ting der Kresole und die Herstellung neutraler 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- prietes des vapeurs du formal on aldehyde formique. C. R. des se. de 1'Acad. des sci., 1892, T. cxv, pp. 290-292. ('92). HANKIN, E. L' act ion bactericide des eaux de la Jumna et du Gauge stir le microbe du cholera. Ann. de 1'Inst. Pasteur, T. x, pp. 5II-523- Cholera does not descend the rivers in India. Bacteria are much rarer in these rivers than ill European rivers. The filtered, unboiled river water has a decided bacteri- cidal action on the cholera organism. When boiled the water lost its germicidal property. ('93). IJE CHRISTMAS, J. Sur la valeur antiseptique de I'ozone. Ann. de 1'Inst. Pasteur, T. vn, 1893, pp. 776-780. I'M,; i. SCHILD. See xvin. ('<).;>. VOCES. See xxxvi. (V.U. GREEN. Ueber den \Verth der Kupfersalzc als Desinfektionsmittel. Zeitschr. f. lid. xni, 1893, ]ip. 405-511. States that copper salts have considerable value as germicides, especially the soluble on"S. Cuprum bichlo- ratuin 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 better than copper sulphate. ('93). LOEW, OSKAR. Ein naitiirliches System der Giftwirknn.ge.n. Miinchen, 1893, Wolff und Liineburg, pp. vin, 136. Rev. in Central!), f. Bakt, 1893, Bd. xiv, p. 234. ('93). GRUBER, MAX. Ueber die Loslichkeit der Kre- sole in Wasser und iiber die Verwendung ihrer wiissrigen Losungen zur Desinfektiini. Arch. f. Hyg., B'd. xvn, 1893, pp. 618-625. ('94). DIEUDONNE. See x.xix. ('94). D'ARSONVAL ET CHARRIN. See xxxni. ( ''14). ABEL. See xvm. ('94). MIQUEL. De la desinfection des poussieres seches des appartements; and Contribution nouvelle a 1'etude de la desinfection pair les vapeurs d'aldehyde formique. Ann. de tnicr., 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. vin, 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- serstoffsuperoxydes auf einige pathogene Mikroorganismen. St. Petersb. med. Woch- enschr., 1894, No. 6. Rev. in Centralb. f. Bakt., Bd. xvi. 1X1)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 lo minutes ; iu 1:1000, after i hour. Anthrax spores, iu 14 per cent solution were killed in less than 3 minutes; a 2 per cent solution killed them in less than one hour; a I per cent solution did not kill in I hour. Staphylococcus pyogeu. aureus, from cul- tures i day old. was killed in 1:100 iu less than 10 min- utes; in ! :2oo it required more than 15 minutes. Diplo- coccus pueumoiiise does not grow iu bouillon to which hydrogen peroxide has been added iu i:io,oooto 1:18.000. Solutions of 1:200 destroyed a one-day old culture iu 15 minutes. i '04 1. WALLICZEK. HEINRICH. Die baktericiden Eigenschatten 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. anthracis, and Staphylococcus aureus. ('95). BURCKHARD, G. Zwei Bei'triige zur Kenntnis der Formal in wirkung. Centralb. f. Bakt., xvm Bd., 1895, pp. 257-264. Twenty titles are cited at the end of this paper. ('95). VAN ERMENGE.M. K. De la sterilization des eaux par 1'ozone. Ann. de 1'Inst. Pasteur. T. ix, 1895, pp. 6/V7O9. Rev. in Centralb. f. Bakt., Bd. xix, 1896, pp. 836-838, 2 figs. Van Ermeugem's report is favorable. ('95). SCHEPILEWSKV, EUGEN. Formaldehyd 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 goud- ron et de viraaigre de fools. 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, aud 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 i 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 biol., Paris, 10 se., T. n, 1895, pp. 500-502. The writer's experiments were negative, and he is very skeptical as to germicidal power of ozoue. ( 'c>5 ). FISCHER. See XL. ('96). WALTER, K. Zur Bedeutimg des Formalins. bezw. Formaldehyde als Desinfektions- Tnittel. Zeitschr. f. Hyg., Bd. xxi, 1896, pp. 421-451. Rev. in Centralb. f. Bakt., xx Bd., 1896, p. 280. ('97). IwANOFF, 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. Dosinfektioiiswirkung und Eiweissfalhmg ohemischer Korper. CYn- tralb. f. Bakt., xxi Bd., 1897, pp. 798-802. (-97). FuERBRINGER UND FREYHAN. Neue Ullter- suchungen iiber 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 maybe 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 Verfaihren zur Her- stellung keimfreien Trinkwassers. Deutsche med. Wochenschr., Bd. xxin, 1807, No. 10, PP- I4S-U6. Bromide treatment. i''iX). 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 autiseptic substances are less active thau water solutions. CoX). SCHULTZ, N. De ['action des antiseptiques sur le bac. pestis hominis et de la disinfection d'effects et de locaux contamines par la peste bubonique. Arch, des sci. biol. publiees par 1'inst. imper. de med. exper. a St. Peters- bourg, T. vi, 1898, pp. 397-426, I plate. The appearance of the bacteria subjected to the anti- septics, as shown on the plate, strongly Mii^ests 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 substances developed in the media as the result of bacterial occupation. I'nS). FLUEGGE, C. Die Wohmingsdesinfektion durch Formaldeliyd. Zeitschr. f. Hyg., Bd. xxix, 1898, pp. 276-308. ('i)S). FERMI. See xxxvi. ('98). POPOFF, S. P. Vergleichende Studien iiber die desinfizierende Wirkung reiuer Sublimat- Insungen 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 pheuol, 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-CHAMPIONNIERE. Sur la valeur anti- septique de 1'eau oxygenee. Bull, de 1'acad. de med., i8q8, T. XL, serie 3, Paris, pp. 599- 617. i ''ii). MARMIER ET ABRAHAM. La sterilisation indus- trk-lle des eaux potables par 1'ozone. Rev. d'hyg. et de Police Sanitafre, 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 de-struction, aud 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. i Vol. ST. \IU.EK, EUUARD. Ueber die Einwirkung von K.ich^alz auf Bakterien, die bei deu soge- uanuten Fleisehvergiftungen eiue Rolle spieleu. Arch. f. Hyg., Bd. xxxv, 1899, pp. 40-82. Rev. in Centralb. f. Bakt., xxvi Bd., 1899, p. 411. B. coli, B. morbificans bovis, aud B. enteritidis grew in bouillon with 7per cent sodium chloride. During the first 2 to 3 days they were injured, but after that they made a luxuriant growth. The toleration limit for B. coli aud B. enteritidis is between 7 and 8 per cent of NaCl. aud that of B. morbificans hovis between S and lo per cent. The writer of this abstract found certain plant bacteria much more sensitive to salt, e. g. P*. hyacinthi was restrained by i. 5 per cent. ('99"). BLISS, C. L., AND NOVY, FR. G. Action of formaldehyde on enzymes and on certain proteids. Tlie Jour. Exp. Medicine, vol. iv, 1899, pp. 47-80. ('99). WEYL, TH. Keimfreies Triukwasser mittels Ozon. Cenitralb. f. Bakt., xxvi Bd., 1899, pp. 15-32, 'with i fig. "Ozone is a specific bacterial poison." It is recom- mended for sterilizing drinking water. ('•I'M. KOCH, E., AND FUCHS, G. Ueber den anti- Inkteriellen Wert des Acrolein. Centralb. f. Bakt., xxvi Bd., 1899, pp. 560-563. Acrolein is a substance related to formaldehyd. In 0.25 to 0.5 per cent solutions it proved more effective ihau formaldehyd on a number of uoii-sporiferous organisms. ('99). CALMETTE, A. Rapport sur la sterilization in- dustrielle des caux potables par 1'ozone. Ann. de 1'Inst. Pasteur, 1899, T. xin, pp. 344-357- A favorable report on sterilization of water by ozoue by a committee, of which Calmette was secretary. They recommended the system of Marmier aud Abraham for the city of Lille. ('99). Roux ET CALMETTE. Sterilization of water by ozone. Rapport presente a la imunicipalite de Lille, fevrier, 1899. ANTISEPTICS AND GERMICIDES; CHEMOTROPISM , ETC. 253 ('99). MARPMANN. Die luktcricide Wirkung des Fhiornatriums unil der Nachweis desselben in Nahrungsmitteln. Ceiutralh. f. Bakt., Bd. xxv, 1899, pp. 309-311. Considers sodium fluoride a valuable disinfectant for all bacteria in nutrient media. Sodium fluoride is said to be scarcely more harmful to man than commou salt. In beer wort in doses of I gram per litre it inhibits the growth of bacteria without lessening that of the yeast. (,'oo). OTTO. Sterilization of water by ozone. Bulle- tin de la Spciete des ingenieurs civils de France, fevrier, 1900. ('oi). MAYER, EUGEN, UND WOLPERT, HEINRICH. Beitrage ztir WolHingsdesinfektion durch Formaldehyd : I. Die zweckmassigste Form des Verdampfungsapparats. II. Einfluss der Temperatur atif die Desinfektionswirkung. in. Verstiirkung der Desinfektionswirkung durch kiinstliche Luftmisclumg (Vorl. Mitt.). Hyg. Rdsch., Berlin, xi Jahrg., 1901, pp. 153-158. ("oi). SMITH, ERV.IN F. Growth of bacteria in the presence of chloroform and thymol. Science, n. s., vol. xin, p. 327, March I, 1901. See also Jour. Boston Soc. Mied. Sci., vol. v, p. 375, and Centralb. f. Bakt., 1901, I Abt., Bd. xxix, pp. 445-446. ('oi). HESS, Orro. Der Formaldehyd. Seine Dar- stellung, Eigenschaften, und seine Ver- wendimg als Konservierungs-therapeutisches und Desinfektionsmittel mit besonderer Beriicksichtigung der Wohmmgsdesinfec- tion. Marburg, N. G. Elwert, 2 Aufl., 1901, pp. IV, 129. ('02). ROLLY. Zur Analyse der Borax- und Borsaure- wirkung bei Faulnissvorgangeii, nefost Studien iiber Alkali- und Saureproduktion der Faulnissbakterien. Arch. f. Hyg., Bd. XLI, 1902, pp. 348-405. ('02). PRESCOTT, SAMUEL C. Antiseptics and their use in the preservation of food. Technology Quarterly. Vol. xv, 1902. pp. 335-342. (!02). HlI.L, HlBBERT W., AND RlCKARDS, BlJRT R. Notes on formaldehyd. Proc. Am. Pub. Health Asso., 3oth Ann. Meeting, New Or- leans, Dec., 1902. Also a separate, pp. 12. ('02). SCHUEDER. Entgegmmg auf die Schumburg' sche Arbeit : "Das Wasserreinigungsver- fahren mit Brom" und die Arbeit von A. Pfuhl: "Zu den Schiider'schen Priifungsver- suchen des Bromverfahrens nacli Scluim- burg." Zeitsch. f. Hyg., Bd. xxxix, 1902, PP. 532-539. ('02). ScHUim-Kf,, WII.IIEI.M. Zu der Schiider'schen Entgegmmg beziiglich des Bromverfahrens zur Trinkwasser-Reinigung. Zeitsch. f. Hyg., Bd. xi., 1902, pp. 199-202. ('02). GREEN, A. B. The disinfectant action of chloroform and various other substances on the specific and extraneous micro-organisms of vaccine. Rep. Med. Off. Loc. Gov., Lon- don, 1902, p. 639-663. ('02). ENGELS, EUCEN. Das Schumburg'sche Ver- fuhren der Trinkwasserreinigung mitteU Brom. Centralb. f. Bakt., Abt. I, Bd. xxxi, Originale, 1902, pp. 651-670. ('02). KONRADI, DANIEL. Ueber die baktericide \\'irkung der Seifen. Arch. f. Hyg., Bd. xr.iv. 1902. pp. 101-112. Recommends a resorcin soap. Co2). MAYER, EUGEN, UND WOLPERT, HEINRICH. Ueber die Verfalin-n und Apparate zur Entwickhmg von Formaldehyd fur die Zwecke der Wohnungsdesinfektion. Arch, f. Hyg., Bd. XLIII, 1902, pp. 157-169. ('02). COHN, ERNST. Ueber den antiseptischen Wert des Argentum colloidale Crede und seine Wirkung bei Infektion. Diss. Konigsberg i. Pr. (Druck v. M. Hiller), 1902, p. 57. ('03). RICKARDS, BURT RANSOM. A comparison of some of the more common liquid disin- fectants. Jour. Mass. Asso. of Boards of Health, vol. xm, No. 3, Oct., 1903, pp. 70-76. ('03). FREER, PAUL C.. AND NOVY, FREDR. G. On the organic peroxides. Vaughan Quarter Cen- tury I'..i.ik. )>i». 63-127. Ann Arbor, 1903. " Acetyl and benzoyl hydrogen peroxides are extremely germicidal, and easily rank with the most active disin- fectants. * Hydrogen peroxide is cousiderably weaker than these organic peroxides. The activity of the peracids and. of hydrogen ]M-ro\iil<- is not due to active oxygen, but is probably due to the acid ions." ('03). RIDEAL, SAMUEL. Disinfection and the preser- vation of food, together with an account of the chemical substances used as antiseptics and preservatives. 3d ed., London, Sanitary Pub. Co., Ltd.; New York, John Wiley & Sons, 1903, pp. 494. ('04). KONRADI, DANIEL. Weitere Untersuchungen ueber die bakterizide Wirkung der Seifen. Centralb. f. Bakt., I Abt., Originale, xxxvi Bd., 1904, pp. 151-160. St. Laceleau soap is actively bactericidal, ('05). KRAEMER, HENRY. The Oligodynamic Action of Copper Foil on Certain Intestinal Organisms. Proc. Am. Phil. Soc.. vol. XLIX, pp. 51-65. Phila., 1905. Also a separate. XXXIX. Chemotropism, Thermotropism, Geotrop- ism, Contact-Irritation, Etc. S4 i . PKEFFER, W. Lokomotorisohe Richtungs- bewegungen durch chemisdie Reize. Unter- suchungen aus dem toot. Institut Tubingen, i Bd., 1884, pp. 363-482. The chapter on Spaltpilze begins on p. 449. DUBOIS, R. Influence du magnotisme sur 1'orientation des colonies microbiennes. C. R. des se. et mem. de la soc. de toiol., Paris, 1886, 8 se.. T. in, pp. 127-128. _ PFEFEER, W. Ueber chemotaktische Bewe- gungen von Bakterien, Flagellaten und Vol- vocineen. Untersuch. a. d. bot. Inst. zu Tuhincten, 1888, Bd. n, Heft 3, pp. 582-661. ALI-COHEN, CH. H. Die Chemotaxis als Hulfs- mittel der bakteriologischen ForsChung. Centralb. f. Bakt., vni Bd., 1890, pp. 161-167. BOYCE AND EVANS. Upon the action of gravity mi Bacterium Zopfii. Communication made to the Royal Society, Feb., 1893. Rev. in Centralb. f. Bakt.. Bd. xv, 1894, pp. 568-569. ROTH, A. Ueber das Verhalten beweglicher Mikroorganismen in stromenden Fliissig- keiten. Deutsch. med. Wochenschr., 1893, No. 15, pp. 351-352. In streaming fluids this author observed in motile bac- teria a decided tendency to move against the current. ('94). MIYOSHI. MAXAI-.U. Ueber Chemotropismus der Pilze. Botanische Zeitung, 1894, Hft. I, col. 1-28. with i table. Deals only with fungi. ('86). ('88). {'90) ('93). ('93). 254 BACTERIA IN RELATION TO PLANT DISEASES. C,)4). BEYERINCK, \l \\ Ueber Tlurmotaxis bei I'.akuiinni Znpfii. Ccntralb. f. Bakt., Bd. xv. 1804, p. 799. K.t.i- ti> UK ! tiii)i;i\i'- UK- movements of the thie.;ids of liact. /.tiplii, which lioy-c and Evans supposed to he due to gcotiopisui . .'.H i I . ,i IGS, II. S, AND CKIISHY, J. H. Studies i in reactions to stimuli in unicellular or- ganisms, vn. The manner in which bac- teria react to stimuli, especially to chemical stimuli. Am. Jour, of Physiol., vol. vi, 1901, PP- 31-37. Also a separate. ('oil. KOTHKKT, \V. Beobachtungen und Betrach- tu'.igcn iiber tactische Reizerscheinungen. Flora oder Allgemeine Botanische Zeitung, ss Bd., moi. lift, in, pp. 371-421. ('02). CI.ARK, JUDSON F. On the toxic properties of some copper compounds with special refer- ence to Bordeaux mixture. Botanical Ga- zette, vol. xxxiii. 1902, pp. 26-48, 7 figs. Controverts Miyoshi on chemotropism. ('03). ROTIIEKT, \V I'eher die Wirkung des Aethers tmd Chloroforms auf die Reizbewegungen der Mikroorganismen. Jahrb. f. Wiss. Bot., Bd. xxxix, 1903, pp. 1-70. The variability in sensitiveness of the same organism at different tiiiies was most disturbing. " Material which to-day is strikingly chemotactic or phototactic, may be to-morrow unusable. Especially striking and perplexing was the behavior of tipirillitin iiiulnlit, this classical object for chemotaxis and osmotaxis, with uhich Pfeffer made his celebrated investigations, and with which in former years I confirmed the experiments ul Pfeffer." XL. Osmotic Piessures. ("91 ) WLADIMIKOI !••, AI.KXANDER. Osmotische Ver- suche an lebenden Bakterien. Zeitschr. f. physikalische Chem., Bd. vn, 1891. pp. 529- 543- ('i)il. WLADIMIROFF, ALEXANDER. Biologische Studien an Bakterien. I. Uber das Verhalten beweglicher Bakterien in Losungen von N'eutralsalzen. Zeitschr. f. Hyg., 1891, Bd. x, pp. 89-110. t 'in). FISCHER, ALFRED. Die Plasmolyse der Bak- terien. Ber. iiber die Verhandlungen d. K. sachs. Ges. d. Wissenschaften. Math em. - phys. Classe. Leipzig, 1891, Bd. xun, PP 52-74, I plate. Plasmolysis was either definitely established or rvn- di n ri extremely presumptive (smaller forms) for lykinds nt !>;uUii;i. The concentration •which induced it varied in IMO-I cases from 0.5 to 5 per cent. In a few cases the It -a->t concentration which would induce plasmolysis was n»t determined. Generally strengths of I to 2 percent -'"hum chloride were sufficient. "Die untere Grenze ist I'lir alle Bacterien bei I per cent oder 0.75 per cent NaCl." I'iM'iiF.R, AI.I'RF.I. N'eue r.oilrachtiin^en ill" r nolysedei l'.,ikierien in Untersuchungen iiber Bakterien. Jahrb. f. wissensch. Bot., Berlin, 1895, Bd. xxvu. pp. 1-34. uNVAi.. See xxxm. XLI. Chemical Analysis of Bacteria. ('79). NKNCKI, M., UND SCHAFFER. F. Ueber die chcmische Zusammensetzung der Faulniss- baktcrien. Jour. f. Praktische Chemie. netie Folge, Bd. xx, 1879, pp. 443-466, I fig., I plate. Also a separate. ('Si). SCHAFFER, F. Zur kenntniss des Mykoproteins. Journal f. Prak. Chemie, neue Folge, Bd. xxm, 1881, pp. 302-304. ('86). BROWN, ADRIAN J. On an acetic ferment which forms cellulose. Journal Chem. Soc. Trans.. London, vol. XLIX, pp. 432-439. ('87). BROWN, ADRIAN J. Note on the cellulose formed by Bacterium xylinum. Journ. Chem. Soc., London, Trans., vol. LI, 1887, P- 643. ('87). VINCENZI, LIVIO. Ueber die chemischen Be- standteile der Spaltpilze. Zeitschr. f. physiolog. Chemie, 1887, Bd. xi, pp. 181-183. ('88). HAMMERSCHLAG. Ueber bacteriologisch- chemische Untersuchung der Tuberkel- bacillen. Verhandlungen der Schweizeri- schen Naturf. Gesselsch. in Solathurn, Au- gust, 1888, 71 Jahresversammlung, pp. 85-86. ('93). CRAMER, E. Die Zusammensetzung der Bak- terien in ihrer Abhangigkeit von dem Nahr- .material. Arch. f. Hyg., Bd. xvi, Heft 2, 1893, pp. 151-195. ('93). NISHIMURA, TOYOSAKU. Untersuchuiig iiber die chemische Zusammensetzung eines Wasserbacillus. Arch. f. Hyg., Bd. xvm, 1893, PP- 318-333. ('93). DREYFUSS, ISIDOR. Ueber das Vorkommen von Cellulose in Bacillen, Schimmel- und anderen Pilzen. Zeitschr. f. physiol. chem., Bd. xvm, 1893. pp. 358-379- The conclusion is that cellulose occurs in hay bacilli and in pns bacilli. ('95). CRAMER, E. Die Zusammensetzung der Cholerabacillen. Arch. f. Hyg.. Bd. xxn, 1895, pp. 167-190. ('98). DE SCHWEINITZ, E. A., AND DORSET, MARION. The mineral constituents of the tubercle bacilli. Centralb. f. Bakt., Bd. xxm, 1898, PP 993-995- ('02). KRESLING, K. I. De la substance grasse des bacilles de la tuberculose. Arch, des sci. biol., pnbliees par 1'inst. imp. de med. exper. a St. Petersbotirg, T. ix, 1902, pp. 359-376. XLI I. Distribution of Bacteria -Geographical and Attitudinal. (Deserts, mountains, arctic regions, sea air, depths of the sea, deep wells, surface soils, air at the earth's surface, and at higher levels.) ('81). MIOUEL, P. Stir le dosage des bacteries dans les poussierc^ rt dans le sol. Bull. Soc. Bot. de France, 1881. T. xxvin, ser. 3, pp. 44-51. ('82). TYNDALL, JOHN. Essays on the floatinK-in.it ter of the air in relation to putrefaction and infection. New York, D. Appleton & Co., 1882, pp. xix, 338. ('83). MicjuEL, P. Les organismes vivants de 1'at- iii.. sphere, pp. vni. 310. Paris. 1883. Gau- thier-Villars. IHSTK! lll'TIOX OF i:.\(TIUIA GEOGRAPHICAL AND ALTITUDINAL. 255 ('83). Mii.irici.. P. Nouvellcs recherolics sur lr> bac- teries atmospheriques cffootuOr< :i 1'observa i. iiiv ilr M-Mitsouris. Ann. dc 1'observatoirc de :\limt-nuris ji.mr 1'an i8---ertation. 78 pp., 2 Taf., Leipzig, iSSii Rev. in Centralb. f. Bakt., 1887. Bd. i. p|. S-io. I'Sj). PETRI, R. J. Zusammenfassender Bericbt iiber Nachweis nnd Bestimmnnt; der pflanzliclu-n Microorganismen in drr Luft. Centralli f Bakt., Bd. IT, 1887, pp. 113-118 und 151-158. ( '87). MAC.GIORA, A. Ricercbe quantitative sui micro- organisini del suolo eon speciale riguardo, all'inquinazione del niedcsimo. Giornale della R. Acrademia de medicina di Torino, 1887, vol. xxxv, Series 3, pp. 153-172. ('87). FRAENKEL, CARL. Untersuchungen uber das Vorkommen von Mikroorganismen in ver- schiedenen Bodenschichten. Zeitschr. f. Hyg., 1887, Bd. II, pp. 521-582. ('89). REIMERS, JOHN. Ueber den Gehalt des Bodens an Bacterien. Zeitschr. f. Hyg., 1889, Bd. vn, pp. 307-346. ('89). REIMERS, JOHN. Ueber den Gehalt des Boden? an Bacterien. Inaug. Dissert. Jena, 1889, 8yo., 44 pp., Leipzig, Veil u. Comp. Re- viewed in Centralb. f. Bakt., I Abt. Bd. x, 1891, p. 489. " Die Zone dieser pldtzHcIu-ii Keimvermiiiderung liegt im Jeneuser Boden — wie im Berliner — zwischeu i uud 2 metres." ('90). KRAMER. See m. ('91). MANFREDI. See XLIII. (*93). CRISTIANI, H. Analyse bacteriologique de 1'air des hauteurs puise pendant un voyage en ballon. Ann. de 1'Inst. Pasteur, T. vii. pp. 665-671. At elevations above the soil of i.oco meters and upward the cultures remained sterile although in each case 10 litres of air was allowed to bubble through the culture media. Even at much lower levels the majority of colonies are believed to have come from the earth Indi- rectly, i. e., by way of the balloon. ('93). DUCLAUX, E. La distribution de la matiere organique et des microbes dans le sol. Revue critique. Ann. de 1'Inst. Paseur, T. vn, 1893, pp. 823-833. ('97). MORITZ, OTTO, VXD NEUMANN, R. O. Ueber emige liakteriologischc Wassernntersuchun- gen im Atlantischen Ozean. Centralb. I. Bakt. 2 Abt., Bd. xin, 1904, pp. 481-489. ('99). LEVIN. Les microbes dans les regions arc- tiques. Ann. de 1'Inst. Pasteur, T. xin, 1899, pp. 558-567. The arctic air was tested in twenty places, approxim- ately the same amount being filtered at each place. A total of 21,600 litres of this filtered air yielded three bac- terial colonies and a few mold spores. The surface waters of the arctic also contain few bacteria. Ninety samples of water were taken from the sea at great depths i i.ooo to 3,000 metres). These samples also contained bacteria of several kinds but in small numbers. The temperature at this depth is below zero centigrade. "Tout un moude de bacteries existe a line temperature qui descend jus- qu'a 2° an dessous de z£ro." ('01). GAZERT, HANS. Bakteriologische Aufgaben der deiitscben Stidpolar-Expedition. Peter- m.-mn-. geogr. Mitteil., Bd. XLVII, 1901, pp. 153-155. I'oi). BELLI, C. M. Chcmi>cne. mikroskopische und bakteriolo.ui-die I'ntersuchungen 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, (ier semest.), 1902, pp. 359-362, av. fig. C. R. des se. de 1'Acad. des sci., T. cxxxiv, 1902, pp. 673-676. BACTERIA IN RELATION TO PLANT DISEASES. XLI1I. Soil-Organisms; Putrefactive Organisms. i '82). I 'Si i I I 'Sd I. (>>7). ('89). ('91). ('91). ('93). ('96). ('99). Coi). ('02). ('02). ('<)_•). ('04). TVM.M.I.. See xi. ii. AliA.MIiTZ. See M.II. l!i;|-.MEK. Sec M.II. FRAK\KI:I.. See xi.ii. REIMERS. See xui. BERTHELOT, M., ET ANDRE, G. Sur 1'odeur propre de la terre. C. R. de se. 1'Acad. des sci., Paris, 1891, T. cxn, 598. MANI;RF.I>I, LUIGI. Sulla cnntaminazione della superficie stradale nelle grandi citta dal punto di vista dell'igiene e deH'ingegneria sanitaria. Recerdie e studi fatti con speciale riguardo alia citta di Napoli. Atti della R. Accad. delle sci. fis. e mat. di Napoli. 23 serie, vol. iv, 1891, appendice. No. 4, pp. 1-79. DUCLAUX. See xi,n. UUCI.AUX. See xxvin. LEVIN. See xui. 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. IOOJ, pp. 107-117. Also a separate (issued Aug. 13, 1901). Disintegration uot due to the bacteria. KATAYAMA, T. On the general occurrence of Bacillus methyliens in the soil. Bull, of the College of Agr., Tokyo Imperial Univ., vol. v, No. 2, 1902, pp. 255-258. Also a separate. CHESTER, FREDERICK D. The bacteriological analysis of soils. Proc. J.vl Ann. meeting of Soc. for Prom. Agric. Sci., 1902, pp. 173- 182. Also a separate. CHESTER, FREDERICK D. Bacteria of the soil in their relation to agriculture. Bulletin No. 98, De.pt. of Agric. of Pennsylvania, 1902, pp. 88, with plates. A bibliography of 105 titles. REMY, TH. Bodenhakteriologische Studien. Centralb. Bakt. Abt. 2, Bd. vm, 1902, pp. 657-662, pp. 699-705, pp. 728-735, pp. 761-769. CHESTER, FREDERICK D. Observations on an Important Group of Soil Bacteria. Organ- i ins related to Bacillus subtilis. Fifteenth Annual Report of the Delaware College \ 141-1. Rxp. 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. C'6l). PASTEUR. Acetic fermentation due to bac- teria. Ann. scient. de 1'Rcole normale supe- rietire, iSin. Not seen. PASTEI K, 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, iS()S. Gauthier-Villars, Imprimeur-Librairc. Yicti >r MasMin el l-'ils, I.ibrain-s, pp. vm, i m ('86). I'.KIWN. See xu. CS6). (93). (93). C94). C95). C97). ('98). Coo). lli«n\N, A. J. The chemical actions of pure cultivations of Bacterium aceti. Jour. Chem. Soc. Trans., iSS6, vol. xux, London, pp 172-187. BROWN, A. J. Further notes on the chemical action of Bacterium aceti. Jour. Chem. Soc., London, 1887, vol. u, Transactions, pp. 638- 642. HANSEN, EMIL CHR. Botanische Unter- suchungen iiber Essigsaurebakterien. Ber. d. deutsch. bot. Gesellsch., Bd. xi, 1893, pp. (6g)-(73). General Versammlungs-Heft. LAFAR, F. Physiologische studien iiber Essig- garung tmd Schnell-Essigifabrikation. Cen- tralb. f. Bakt., 1893, Bd. xni, pp. 684-607. Bibliography of 13 titles. HANSEN, E. C. Recherches sur les bacteries acetifiantes. Compt. rend. d. trav. du Lab. Carlsberg, T. ill, 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- 150. LAFAR. See in. B.EYERINCK, M. W. Ueber die Arten der Es- sigbakterien. Centralb. f. Bakt., 2 Abt., Bd. IV, 1898, pp. 209-216. Tl \NSEN, EMIL CH. Recherches sur les bac- teries acetifiautes. (Troisieme inemoire.) I'. R. des travaux du laboratoire de Carls- iberg, 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.) ('871. ALVAREZ, E. Sur un nouveau 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. I. The biology of ensilage. Bull. Ag. Exp. St. Univ. of 111., 1889, No. vn, pp. 177-194. Coi). ALBERT, FRIEDRICH. Lhitersuchungen iiber Griinpressfutter. Jahrb. d. deutsch. Land- wirtsch. -Gesellsch., Bd. vi, Tl. I, pp. 149- 250, Berlin, 1891. This author says bacteria exert a preponderant influ- ence ou the course of ttie fermentation. ('91). SUCHSLAND, EMIL. Ueber Tabaks fermenta- tion. Ber. d. deutsch. bot. Gesselsch., Bd. ix, Berlin, 1891, pp. 79-81. (.'94). VAN LOOKEREN-CAMPAGNE, C. J. Bericht iiber Indigo-Untersuchungen, ausgefiihrt an der Versuchs-Station zn Klatten auf Java. D. landw. Vers.-Stat., 1894, Bd. xi.ni, pp. 401- 426. SILAGK-15ACTKRIA. FERMENTATION OF TOBACCO, INDIGO, FLAX, ETC. 257 MI-;) \\i\m, K \I»KY, Si:K<;ir>. Sur Ic rouissage du lin et son .-i.^i'iit microliitMi. (,'. K. ties se. de 1' Vad. drs s,-i , Rm>, ]S(|;, T. CXXI, pp. 742-745- A resume of tlie priucipal results of work done by Fribes in \V]!IML;I :ieg. Phys. & Path.. 1900. Report 65. 57 PP- Coo). BEI.TERINCK. Verdere onderzoekingen over de indigovorming uit weedl (Isatis tinctorial Proc. K. Akad. Wetensch. Amsterdam, Deel ix, June 30, 19011, pp. 74-9°- Abstr. in Bot. Zeitung. 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 in the protoplasm ; isatase occurs in the chromatophores. In the living cell the author thinks reactions are prevented bv the acidity of the cell-sap. He says : " The action of is'atase upon isatan is possible only in neutral or ampho- teric aud very weakly acid solutions." A temperature of 48° to 50° C. is the optimum for this action." Coi). L"E\v. 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. ('oil. RUSSELL, H. L.. AND BABCOCK, S. M. Concern- ing the theories of silage formation. Science, n. s., vol. xin, p. 328, 1901. The conclusion reached is that bacteria do not play any very considerable r61e in the fermentation of silage. Coi). PREYER, AXEL. Ueber Kakaofermentation. Tropenpflanzer, Berlin, Bd. v, 1901, pp. 157- 173. Coi). SCHULTE IM HOPE, A. Zur Kakao-Fermenta- tion. Tropenpflanzer, Berlin, 1901, Bd. v, pp. 225-227. 258 BACTERIA IN RELATION TO PLANT DISEASES. (02). 1! \I:O«-K, S. M., UND RUSSELL, H. L. Die hei cli-r Herstellung von Gar flitter (Silage) wirkenden Ursachen. Centralli. f. Bakt., A.bt. 2, Bd. IN. igo2, pp. 81-88. ('02). H.U-MAN. L. fitude microbiologique et chimique du rotiissage aerobic du lin. Ann. de I'lnst. Pasteur, T. xvi, 1902, pp. 379-385. t'oji. BAIL, OSKAR. I'ntfrsudiung einiger bei der Verwesung pflanzlicher Stoffe thatiger Sprosspilze. Central!), f. Bakt., Abt. 2, Bd. vni, 1902, pp. 567-584. ('03). \YEHMER, C. Die Sauerkrautgarung. Cen- tralb. f. Bakt., 2 Abt., 1903, x Bd., pp. 625-629. ('04). STOERMER, K. Ueber die Wasserroste des Flat-lises. Central-Matt f. Bakt.. 2 Abt., Bd. xin, 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). OMELIANSKI, W. Die histologischen und chemischen Veranderungen der Leinstengel unter Einwirknrg der Mikroben der Pektin- un-d Cellulosegarung. Centralb. f. Bakt., 2 Abt., xn Bd., IQO4, pp. 33-43, I plate. ('04). WEHMER, C. Die Sauerkrautgaernng. Be- richt des V. Internationalen Kongresses f. angewandte Chemie zn Berlin, 1903. Sekt. VI, Bd. in, Berlin, 1904, p. 712. Also a separate, 5 pp. i ihlaiiis results decidedly different from Conrad. The ordinary sauerkraut fermentation i* a mixed fermeuta- lioii. Gas-developmt-ut is due to a bottom yeast ; acid production to a non-liquefying, non-motile, non-gas- forming;, lactic acid bacterium. XLVI. Bacteria in Water and Ice; Dung-Bacteria. (See also XXXIII and XXXIV.) ( '71 1 1. \V ARM INC. See xn. I'Su). FRAENKEL, CAUL. Ueber den Bakteriengehalt des Eises. Zeitschr. f. Hyg., Bd. I, 1886, pp. 302-314. ('87). MACE. -Stir 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 I'lnst. Pasteur, T. I, 1887, pp. 409-410. Dr. Prudden tested the resistance of various bacteria to prolonged cold, in blocks of ice and to repeated freezings and thawings. Proteus vnlgaris and Bacillus prodigiosus did not grow after 51 days freezing. A slender liquefying bacillus from Croton water was killed in seven days. The following withstood freezing: Staphylococcus pyo- geues (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 thawiugs were more filial to the typhoid bacillus than a constant low tem- perature. Five freezings and thawings at intervals of Ihree days destroyed this bacillus. ('87). KnRDONi-UFFREDUzzi, GuiDp. Die biologische I'ntLTsuchung des Eises in seiner Beziehung zur oftentlichen Gesundheitspflege. Cen- tralb. f. Bakt., 1887, Bd. II, pp. 489-497. ('88). SCHMEI.CK, L. Eine Gletscherbakterie. Cen- tralb. f. Bakt., 1888, Bd. iv, pp. 545-547. A green fluorescent organism was thecommouest form. This was a short rod which liquefied gelatin. ('91). VIRON. I,. Du role des Sohizophytes dans les reactions qui se passent dans les eaux dis- tillees. Jour, de Pharm. et de Chim., 1891, T. xxm, series 5, pp. 586-593. ('91). NORDTMEYER. See xvn. ('94). LASER. See xx. ('94). HOUSTON. See xvn. ('94). FRANKLAND, P., AND MRS. P. Micro-organ- isms in water. London, 1884, pp. xi, 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 cpli 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. S. Bakt., xvni 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 799-817. ('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 wiiter. i'<7). KERX. HEINRICH. Beitrag zur Kenntniss der im Darme und Magen der Vtigel vorkom- menden Bacterien. Arb. a. d. bact. Inst. d. tech. Hochschule zu Karlsruhe, Bd. i, Heft iv, 1897, pp. 379-53^. Many bacteria are described at length. ('9^). WARD. 11. 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 (Jakschl; (2\ A colorless capsuled coccus or bacterium ; (3) rose-pink Micrococcus : type of M. carneus (Zimm.) ; (4) A pseudo-bacillus. ('99). KASANSKV. Sec xxxm. ('C9>. FULLER, GEORGE W., AND JOHNSON, GEORGE A. ( in tlie differentiation and classification of water bacteria. Jour. Exp. Med., vol. iv, 1899, pp. 609-626: also a separate. ('oo). KOHLERUGGE, J. II. F. Vibrion-Studien. I. Die Ubiquitat choleraahnlicher Wasser- vibrionen. Centralli. f. Bakt.. xxvm Bd., 1900, pp. 721-726. II. Panmorphismus und erbliche Variationen. Ibid, pp. 833-842. ('oo). SEDGWICK AND WINSLOW. See xxxm. Coi). PARK .See xxxm. ('oij. HORROCKS, W. H. An introduction to the bac- teriological examination of water. London, J. & A. Churchill, 1901, pp. x, 300, 5 plates. MILK-H.UTKKIA. 1IUTTER-BACTERI A, CI I KF.SK-1'..U TKRI A, M KAT-BACTERI A. 259 I'oi). GAGE, STEPHEN DE M. Bacteriological Studies at the Lawrence Experiment Station, with special reference to the determination of B. coli. ,13d An. Rep. St. Bd. of Health of Mass., for 1901. pp. 307-420. Also a sepa- rate, pp. 26. ('01). MIGULA, W. Compendium tier bakteriologis- chen Wasseruntersuchung nebst vollstaen- diger Uebersicht der Trinkwasserbakterien. O. Nemnich, Wiesbaden, 1901, pp. VH, 440, with 2 plates. ('02). ('03)- ('03). ('03). ('03). ('04). ('04). MACFADYEN. See xxxui. 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) mit Chemischen Mitteln. Berlin, Mitt. d. Landw. Ges., Bd. xvm, 1903. pp. 99-101. SCHUEDER. See xvm. WINSLOW AND NIBECKER. See xvn. GAGE AND ADAMS. See xvi. STOKES. See xvm. XLVII. Milk-Bacteria; Butter-Bacteria; Cheese- Bacteria; Meat-Bacteria. ('81). JALAN DE LA CROIX. See xxxvin. ('82). ScHMiDT-MuEHLHEiM. Untersucluingen iiber fadenziehende Milch. Pfliiger's Archiv., 1882, Bd. xxvii. pp. 490-510, I fig. i '84 1 HUEPPE, FERDINAND. Untersuchungen iiber die Zersetzungen dcr Milch durch Micro- organismen. Mitth. a. d. K. Gesundheits- amte, Bd. n, Berlin, 1884, pp. 309-371. ('89). MENGE. KARI.. Ueber rothe Milch. Centralb. f. Bakt., vi Bd., 1889, pp. 596-602. ('89). BAGINSKY. ADOI.F. Rote Milch. Deutsche Medizinal-Zeitung ,1889, No. 9, pp. 106-107. I'S'i). BAGINSKY, ADOLF. Zum Grotenfelt'schen Bacillus der roten Milch. Deutsche mediz. Wochenschrift, 1889, Bd. xv, p. 212. This organism was isolated from feces. It liquefied gelatin slowly and colored milk a dirty red or red-brown. 1'iji). 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 weitverbrerteten milch- wirthschaftliohen Schadling. Berliner land- wirthschaftliche Jahrbiicher, 1891, Bd. xx, pp. 185-207, i plate. Rev. in Centralb. f. Bakt., ix Bd., 1891, pp. 698-700. ('ijji. XENCKI. L.. ET ZAWADZKI, J. Stir la stenlisi tion du lait. Arch, des Sci. Biol. publiees par L'institut Imperial de Med. Exp. a St. Petersbourg. T. I, 1802, pp. 371-397. Contains a bibliography of .sonumbers. ('93). I>i ii \i \, E. Stir le role protecteur de-; microbes dans la creme et les fromages. Ann. de I'ln^t. I'a-tmr. T. vu. 1893, pp. 305-324. BLEISCH, MAX. Ueber bittciv Milch inn! dk- Sterilisierung der Milch durch Erhitzen unter Luftabschluss. Zeitschr. f. Hyg., 1893, Bd. xni, pp. 81-99. ('94). BORDONI-UFFREDUZZI, Guioo. 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. 463-470. rait 2 Abbildungen. ('94). DUCLAUX, EMILE. Le lait; etudes chimiques et microbiologiques. 2 tirage, augmente de notes sur le role des microbes et sur les phosphates du lait. 376 pp., 121110. Paris, J. B. Bailliere & fils.. 1894. ('94). HENRICI, H. Beitrag zur Bakterienflora des Kases. Arb. a. d. bact. Inst. d. tech. Ffoch- schule 7u Karlsruhe, Bd. I, Heft I, 1894, pp. i-no. ('95). RABINOWITSCH. See xxxiv. C'9S)- JOLLES, MAX, u. WINKI.ER. FERDINAND. Bak- teriologische Studien iiber Margarin uncl Margarinproductes. Zeitschr. f. Hyg., Bd. xx, 1895, pp. 60-108. The bacterial content of margarin products is slight in comparison with that of natural butter. ('95). CONN, H. W. Bacteria in the dairy, vr. 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 aroyia of butter. Cen- tralb. f. Bakt., 2 Abt., Bd. 11, 1806, pp. 409- 415. ('97). CONN. H. W. Butter aroma. Centralb. f. Bakt., 2 Abt.. Bd. in. 1897. pp. 177-179. ('09). MAASZEN, ALBERT. Fruobtatherbildende 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 fluorescens and B. priepolleus. There are numerous references to literature. ('59). MOORE, V. A.. AND WARD, A. R. An inquiry concerning the source of gas and taint-pro- clurini; 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. Curnell Univ. Agr. Exp. Sta. Dairy Div. Bui. 165, 1899, pp. 395-412, 4 figs. Ropiuess attributed to Bacillus lactisviscu-n- i 'Hi. STADLER. See xxxvin. \\"EBEK, A. Die Bakti-rien der srigenannten sterilisirteii 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. Coi). CHODAT, R.. ET HOFMAN-BANG, N. O. Les bacteries lactiques et leur 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. foi). PARK. WM. HALLOCK. The great bacterial contamination of the .milk of cities. Can it be lessened 'by the action of Health authori- ties? The Journ. of Hygiene, vo'l. I, 1901, pp. 391-406. See also N. Y. Univ. Bull, of the Med. Sci., vol. i, 1901, pp. 7I-.86. ('02). FISCHER, BERNHARD. Zur Aetidlogie 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. XUII, I9O2, pp. I-2O. ('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., 1901. pp. 13-80. Middletown, Conn., 1902. Relates to those bacteria occurring naturally in milk and uot to those introduced by (he bacteriologist. I'o2l. GRUBER, TH. Ueber e-inen die Milch rosafar- benden Bacillus. Bacillus lactorubefaciens. Centralb. f. Bakt., Abt. 2, Bd. vui, 1902, pp. 457-462. ('02). GRUBER, TH. Ueber eine in tier ATilch Riiben- geruch und Rubengeschmack erzeugende Bakterie. Molk. Zeitung, Hildesheim, Bd. xvi, 1902, pp. 351-353- ('02). HARDING, H. A., UND ROGERS, L. A. Rost- flecken in Cheddarkase. Centralb. f. Bakt., Abt. 2, Bd. vin, 1902, pp. 442-443- Ascribed to Bacillus rudensis. I'nj). RICHTER, ALBRECHT P. F. Bakterielle-. Yer halten der Milch bei Boraxzusaiz. Arch. f. Hyg., Bd. XLIII, 1902, pp. 151-156. ('02). GRIMM, MAX. Ueber einen neuen anmia- bildenden Bacillus nebst einigen Bemer- kungen iiber Reinkulturen fiir Exportbutter. Centralb. f. Bakt., Abl. 2. Bd vin, 1902, pp. 584-590. ('02). ROSAM, A. Ueber Konservierung de.r Milch mittels Wassers'toffsuperoxyd. Centralb. f. Bakt., Abt. 2, Bd. vui, 1902, pp. 739-744, PP- "69-774 ('03) HARRISON, F. C. AND CUM MING, M. The bac- terial flora of freshly-drawn milk, Part iv. Journal of Applied Microscopy, vol. vi, 1903, No. 2, p. 2,181. Bibliography of 25 titles. '03 i SUITHINBANK, HAROLD, AND NEWMAN, GEORGE. Bacteriology of Milk. With special chapters by 1 >r. Newman on the spread of disease by milk and the control of the milk supply. With chromo-lithographs, Woodbtiry type reproduction, of photographs of cultures, and other illustrations of bacteria and of ;iii|iaratns. 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 II. Strained and unstrained milk preserved at 70° and 50°. Rep't of Storrs Vnr. Exp. Sta.. Conn.. 1902-3, pp. 38-51. ('03). CONN, H. W., AND STOCKING, W. A., JR. Series III. 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. 3-15. The author's summary is as follows : 1. Variatious 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 yso-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 hours, 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. T,. So far as the keeping property of inilk 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 vervvandter Praparate. Arb. a. d. Bact. Inst. der techn. Hochschule zu Karlsruhe, m 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 : Micrococciis carniphilus, M. raargiuatus. Streptococcus debilis. Bacterium flaveum, B. iusulosum. Bacillus caruiphilus, B. canaliculatus, B. car- nis. B. inlet-mittens, B. anthraciformis, B. glaciformis. B. inicaus, B. kaleidoscopicus. B. caruiphilus occurs in most meat extracts. Other species found were : Bacte- rium rusticum Kern. Bacillus mesentericus Fliigge, B. vulgatus Fliigge, B. cereus Frauklaiid, B. laevis Frank- land, and B. vegetus Kern. XLV1I1. 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. t.'S8i. ARCANGELI, G. Sulla fermentazione panaria. Atti della Societa toscana di scienze naturali residente in Pisa. Memorie. Pisa, 1888, vol. ix, pp. 190-211. Bibliog. 29 titles. I'l-'o), KRATSCHMER UND NIEMILOWICZ. Ueber eine eigentumliche Brotkrankheit. Wiener klin- ischc Wochenschrift, 1889, Bd. n, 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, hut state that tin <>i - ganism will not grow in acid dough or acid bread, but that it grows luxuriantly in feebly alkaline dough or bread. I:A( TKKIA ix liui-Aii; IRON IIACTERIA; SULPHUR BACTERIA. 261 ('89). PKIKKS. \V. L. Die Orgauismen des Sauer- teige> Mini ilirc Bedeutung fiir die Brot- gahrung. Bot. Zemins, iSSo. lid. xi.vn, col. 405-410. 4-' I -43 1. 437-440. Cpo). UFFELMAXX. J. Verdorbenes Brot. Central!.!, f. Bakt.. vni Bd., 1890, pp. 481-4X5. Coin POPOFF, M. Sur un bacille anaerobic de la fermentation panaire. Ann. de 1'Inst. Pas- teur, 1890, T. iv, pp. 674-676. ('94'). LEHMANN. K. B. Ueber die Sauerteiggaruug mid die Beziehungen des Bacillus levans zum Bacillus coli communis. Centralb. f. Bakt., Bd. xv, 1894, pp. 350-354. ('94). WALDO, F. J.. AND WALSH, DAVID. Does bak- ing sterilize bread? TJie Lancet, London, 1804 (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)- VoGEL, J. Beitrag zur Kenntniss des "faden- ziehenden Brotes." Zeitschr. f. Hyg., Bd. xxvi, 1897, pp. 308-416. ('99). JUCKENACK, ADOLF. Beitrag zur Kenntniss des "fadenziehenden Brotes." Zeitschr. f. Untersuch. d. Nahrungs- und Genussmit- teln, II Jahrg., 1899, pp. 786-788. ('00). THOMANX, 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 ou agar, spreads widely and is wrinkled and gray- white on potato, grows in grape-sugar bouillon without fas-formation, produces a thick pellicle ou peptone ouillon with a clear fluid under it, and grows best at 40° to 42° C. t'oi). DUCLAUX. Pain filant. See Traite, T. iv, pp. 513-515. Toi). BEULSHAUSEN, FRIEDRICH. Zur Kenntnls der Ursache des Klebrigwerdens von Brot. Diss. Rostock (Druck v. C. Hinstorff), 1901, p. 24. ('02). MICHELS, WOLFGANG. Zur Entstehung des fadenziehenden Brotes. Diss. Kiel. Konigs- berg i. Pr. (Durck v. Hartung), 1902, p. 15. ('02). LEHMANN, K. B. Hygienische Untersuchun- gen uber Mebl und Brot. x. Neue Studien iiber die Aciditat des Brotes, ihre Ursachen und i'hre beste Bestimnrungs-methode. Arc!h. Hyg., Miinchen, 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 Prlan/e in ihrer Bezie- hung zum Eisen. Eine physiologische Studie. Jena, 1892, Gustav Fischer, 119 pp., I table. MACAI.LUM, A. B. On the distribution of as- similated iron oimp»nnd~, oilier tlian liaemo- globin and haematins, in animal and vege- table cells. Quarterly journal of microsc. M-iem:e, 1895-96, vol. xxxvm, new series No. 150, pp. 175-274. with 3 plates. The part relating to the bacteria begins on page 254. (95). ('97). MARPMANN, G. Bakteriologische Mitteilungen. I. Ueber einen neuen Nahrboden fiir Bak- terien. n. Ueber ferrophile Bakterien. iir. Ueber den Zusammenhang von pathogenen Bakterien mit Fliegen. Centralb. f. Bakt., xxii 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, 2-3x0.8-2.0^, ends rounded, form plump, with black polar chromatophores and intermediate gray gran- ules. Many cells are entirely black and opa'que. 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. Ou adding ferricyanide of potash after HC1, or with it, ttie bacteria become an intense blue. Ou pep- tone-gelatin the organism was white, but when a trace of iron sulphate was added it became black. MIYOSHI, MANABU. Ueber das massenhafte Vorkommen von Eisenbacterien in den Tihermen von Ikao. Journ. of the Coll. of Science, Imperial Univ., Tokyo, Japan, vol. x, Pt. II, 1897, pp. 139-14^ MIGULA. See in. ('97). LAFAR. See in. ('04). SCHORLER, B. Beitrage zur kemrtnis der Eisen- bakterien. Centralb. f. Bakt., 1904, Bd. xn, pp. 681-695. C86) C87). C89). ('89). I 93) (95) ('95) Cup ('97) 97) (-01) L. Sulphur-Bacteria. CERTES AND GARRIGOU. See xxxiv. WINOGRADSKY, SERGIUS. Ueber Schwefelbak- terien. Bot. Zeittmg, 1887, Bd. XLV, col. 489, 513, 529, 545, 569, 585, 606. 3 figs. WINOGRADSKY, S. Sur la morphologic et la physiologic des stilfobacteries. Beitr. z. Morphol. und Physiol. d. Bakterien., fasc I Leipzig, 1888. Not seen. WINOGRADSKY, S. Recherches stir les sulfo- bacteries. Ann. de 1'Inst. Pasteur, T. in, 1889, pp. 49-60. DE TONI AND TREVISAN. Sulphur bacteria. See Saccardo's Sylloge Fungorurn, vol. 8, p. 1,027. Species granula sulphuris secernentes. . ZELINSKY, N. D. Ueber Schwefelwasserstoff- garung im Schwarzen Meere und den Limans von Odessa. Fortschr. d. russ. chem. u. pliys. (k-sellsch., Bd. xxv, Part v, 1893, pp. 298-303. (In Russian.) . 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. ill, 1895, pp. 381-397. Rev. in Ann. de Micr., T. vn, 1895, pp. 281-282. BEYERIXCK, M. W. Ueber Spirillum desul- furicans als Ursaohe von Sulfatreduction. Centralb. f. Bakt., 2 Abt., Bd. I, 1895, pp. 49-59 and 104-114. Mict'LA. See in. LAFAR. See in. MIYUSHI. MAXAI-.F. Studien iiber Schwefel- rasenbildung und die Schwefelbacterien der Thermen yon Yumoto bei Nikko. Jour. College Sri.. Imp. Univ. Tokyo, vol. x, Pt. ii. pp i (.1 17.1, 1897. I plate." Rev. in The American Naturalist, vol. xxxii, 1898, pp. 456-4?7. CONN. See in. 262 BACTERIA IN RELATION TO PLANT DISEASES. ('02). i 03 > . ('03). ('79). ('95). ('95). ('95). (95). (96). ('96). ('96). ('96) ('96). ('96) ('96). ('97). '97 ' ('97). ('97). (•98). NATHANSOHN, ALEXANDER. Ueber eine iieuc Gnippe von Sclnvefelhacterien nnd ihrc Stoffwechsel. Mitt. a. d. zool. Stat. Neapel., r.ii. \v, 1902. pp. 655-680. HINZE, G. Thiophysa volutans, ein neues Schwefel-Bacterium. Ber. der d. hot. Gesellsch., Bd. xxi. Hft. 6, July, 28, 1903, pp. 309-316. VAN DELDEN. Sec xxiv. LI. Bacteria in Prehistoric Times. VAN TIEGHEM. PH. Sur la fermentation buty- ni|ue (Bacillus amylohacter) 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 micrococcus 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 Pl. RENAULT, B. Les bacteriacees de la houille. C. R. des se. de 1'Acad. des sci., Paris, T. cxxm, 1896, pp. 953-955- RENAULT, B. Les Bacteries devonieiines et le genre Aporoxylon d'Unger. Bull, du Mus. d'Hist. nat., Paris. 1896, T. u. pp. 201-203. RENAULT, B. Notes sur quelques nouvelles lucteries fossiles. Bull, du Mus. d'Hist. nat., Paris, 1896, T. u, pp. 285-288, 4 figs. RENAULT, B. Recherches sur les bacteriacees fossiles. Ann. des sci. nat. bot., vin serie, T. u, 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, 189)1. I'l' 139-142, of the Proces-verbaux des se. Ki '..VAULT, 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 boghead^ a Pilas. Bull. d. Mus. d'Hist. nat., Paris. T. in. 18117, pp. 33-39, 4 figs. RENAULT, B. Les bacteriacees des bogheads. Hull. d. Mus. il'IIUt. nat., Paris, T. in, 1897, pp. 251-258, 6 figs. RENAULT. BEKNAKD. II i.nln-aiK et kirtenacee^ Sue. 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., 1808, pp. 133-147, in the Proces-Verbaux d. se. Coo). 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. (,'So). KAISER, EDUARD. Verfahren zur Herstellimg einer tadellosen Glycerin-Gelatin. Bot. Centralb., Bd. I, 1880, pp. 25-26. i gram best French gelatin, 6 grams aq. dest.; softeu 2 hours. Add 7 grams of c. p. glycerin and i gram of c. p. carbolic acid to each loo grains of the preceding. Warm and stir 15 minute!.. Filter through glass wool previously washed in distilled water. ('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- According to Hillhouse, as reviewed by Dippel in Botau. Centralb.. p. 1 = 9. 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. Hilliiouse says that a drop of glycerin on glass can be covered hv a drop of balsam, and the latter will spread over it and adliere firmly to glass arouml it on all sides, inclosing it completely. ('8/1. SOYKA, J. L'eber ein Verfahren, Dauerprapa- 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 KRAI., F. Vorschlage und Anleitungen zur Anlegung von hacteriolo- gischen Mnseen. Zeitschr. f. Hyg., 1888, Bd. IV, pp. 143-150. ('89). KRAL, FRANZ. Weitere Vorschlage und An- leitungen zur Anlegung von bakteriologis- clien 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 im 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 100 of a I per cent solution of mer- curic chloride. Preparations treated in this way are said to remain unchanged for ye:irv Camphor is said to hinder the growth of molds without interfering seriously with bacteria. ('92). PAWSUN. CIIAKLES F. Eine Methode, Pauer- kulturen von Bakterien hermetisch zu ver- schliessen. Centralb. f. Bakt., xn Bd., 1892, pp. 720-721. STOCK-CULTURES, KTC. ; COLOR-CHARTS; PHOTOGRAPHY, ETC. 263 ('ij.il. H.u'SKu, G. UebcT \\T\vemlung des For- malins zur conservirung von Bacteriencul- tureii. Miinchen. med. Wochenschr, 1893. Bd. xi., 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 under 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: 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 iiecessary to keep them from drying out. ('9.?). HAUSER, G. Weitere Mitteilungen iiber Ver- wendnng des Formalins zur conservirung von Bacterienculttiren. Miinchen. med. Wochenschr., 1893. Bd. XL, pp. 655-656, No. 35. Rev. in Central^, f. Bakt., Bd. xiv, 1893, pp. 468-469. Describes a method of fixing and mounting colonies taken from gelatin plate cultures. ('04). KRUECKM ANN, EMIL. Eine Methode zur Hcr- stellung bakteriologischer Museen und 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. Coi). CONN. H. W. How can bacteria be satisfac- torily preserved for museum specimens? Science, n. s., vol. xm, 1901, p. 326. LIII. Stock-Cultures, How Best Kept; Vitality on Media. ('89). CZAPLEWSKI, E. Zur Anlage b;ikteriologischer Museen. Centralb. f. Bakt., vi Bd., 1889. pp. 409-4II- 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. Cy8). LT'NT. 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 iu ordi- nary culture-media. ('oo). BOU.EY, HENRY L. The duration of bacterial existence and fin ?] 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. Coi). SCHULTZ. See vi. LIV. Color-Charts ; Nomenclature of Colors. ('86). RIIIGWAY, 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 zoologorum. 2d ed. Padua. Typis Semi- narii, 1894, Svo., 22 pp., with two tables con- taining 50 colors. A cheap and useful color scheme for botanists. C9S)- Color chart under Spectrum, in the Standard Dictionary, Funk and Wagnalls, New York, 1895. This may be had separately. C'9S)- 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. (' — ). Chart of Spectrum Scales made from the Bradley colored papers. Milton Bradley Co., Springfield. Mass. A small sheet (about 354X7^ inches) with 90 colors. Also a large folded chart (11x28 inches). Colors are bright, but must be carefully protected from the light. LV. Photography and Photomicrography. ('77). 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. T, 1881, pp. T-.i8. The paper is illustrated by 84 heliotrpes 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. CS/). 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 applinuee a 1'etude des microbes. Ann. de 1'Inst. Pasteur, T. I. 1887. pp. 209-225. C8S) ZETTNOW. E. Das Kunfer-Chrom-Filter. Centralb. f. Bakt. iSSS. Bd. iv. pp. 51-52. This light filter is well adapted to photographing bac- teria, whether they are stained red.blueor 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 I to 2 cm. thick is 175 grams sulphate of copper and 17 grams bichromate of potash dissolved iu 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 s8o to 560: more diluted from 590 to 545. 264 BACTERIA IN RELATION TO PLANT DISEASES. ('88). NEUHAUSS, R. Verschiedenes fiber Micro- photographie. Zeitschr. f. Mikr., Bd. v, pp. 484-486, 1888. ('90). NEUHAUSS, RICHARD. Lehrbuch der Mikro- photographie. pp. xi, 273, with 61 wood cuts, 4 autotypes, 2 collotype plates, and I photogravure. Braunschweig, Harald Bruhn, iSco. A ver>' useful book. ('go). 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; II Theil., 1894. pp. xi, 367; ill Theil., Abt. i, 1807, pp. x, 311, Abt. n, 1899, pp. x, 159. ('99). The photo-miniature. Tennant and Ward, New York. Begun in 1899. A series of small, inexpensive volumes, by various authors, 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. foi). 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, but subjects 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. ('4iV DLIJARDIN, FELIX. Histoire naturelle des zo- ophytes, infusoires comprenant la physiolo- gic et la classification. Paris, 1841. ('65). DAVAINE. See v. <'f>5-'6/). 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 Tr£cul. ('7-'). COHN. See v. ('79). TREVISAN. Introduzzione allo studio die bac- teri. Atti d. Inst. Lombardo, 1879. rRoV WINTER. See in. ('iSiV ZOPF, W. Ueber den genetischen Zusammen- hang von Spaltpilzformen. Monatsbericht d. Konigl. preuss. Akad. d. Wissenschaften, Berlin, iS8i, pp. 277-284. i plate. See also various editions of ZopFs "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. vm 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 I zincography) by R. Friedlauder 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 Banm- garten's Jahresbericht, Bd. vn, p. 344. The bacteria are classified as Gymnobacleria and Trich- obacteria. The latter are subdivided into four groups : Monotricha (one polar flagellmn), Lophotricha (a tuft at one pole). Amphitricha (one fiagellmn 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). MICULA, 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). MIC.ULA. W. Schizomycete^. Engler and Fraud's Die Natiirlichen Pflanzenfamilien, Leipzig, Wilhelm Engelmann, 1895. ('95). FISCHER, ALFRED. Untersuchungen iiber Bak- terien. Jahrb. f. wissensch. Botanik, Bd. xxvii, 1895, Hft. i. pp. 1-163. 5 plates. ('96). MEZ, CARL. Der heutige Stand der bak- teriologischen Systematik. Botanisches Centralb., Bd. LXVIII, 1896, pp. 203-211. ('97). CHESTER, FREDERICK D. A preliminary arrange- ment of the species of the genus Bacterium. Qth 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 in. ('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. xxvni. pp. 481-493. Also a separate, pp. 3-15. Pub. 1903- ('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 ill, 1903, pp. 3-95. 3 tables. Also a separate. CATAI.OCUKS. 265 ('03). GAGE, STEPHEN DE M., AND PHELPS, EARLE B. On the classification and identification of 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 Puhlic 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 F. 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.: CARL 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, So 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 bactcriologie 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, So East l8th St., near Broadway, New York. Koenigliche Porzellan-Manufaotur, 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 iiber chemische Apparate und Gerathschaf- ten, 1900, Berlin, N. W., Luisen-Strasse 58. Preis S mark. pp. 600. DR. PETERS & ROST. (1) Preis-Liste fiber Apparate und Utensilien ftir elektrochemische und elektrolytische Ar- beiten. Liste No. 29. Berlin, 1900, pp. 48. (2) Preis-Liste iiber Apparate und Utensilien fiir Bakteriologie, Hygiene, Mikroscopie. Rea- gentien, Farbstoffe, Nahrboden, Reincul- tnren. mikroscopische Praparate. Liste No. 30. Berlin, 1900, pp. 123. (3) Preis-Liste iiber chemische Apparate und Uten- silien ftir wissenschaftliche und Fabriks- Laboratorien, Chemicalien, Reagentien, Normal-Losungen. Liste No. 28. Berlin, 1902, pp. 534- (4) Preis-Liste iiber physikalische Apparate. Liste No. 37. Berlin, 1902, pp. IV, 383. MAX KAEHLER AND MARTINI, Berlin. Chemical and Bacteriological Apparatus. The above five catalogue? or their equivalent may now be obtained from the following: Vereinigte Fabriken fiir Laboratoriumsbedarf, ges. M. B. H.. Chauseestr. 3. Berlin. N., formerly Max Kaehler & Miartini. Dr. Peters & Rost. New York Office : Laboratory and School Supply Co., 20-24 East 2Oth street, New York. KM n, GREINER, New York. Bacteriological apparatus, glassware, high-grade glass blowing. Very relul'V GREINKR &- FRIEDRICHS, Stiitzerbach, Germany. Glassware. C. P. GoERZ. Price list of doirble-anastigmat lenses and of the Goerz-Anschutz camera. Berlin-Friedenau, and 52 East Union Square, New York. FOLMER 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. 266 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, 3,001-3,10.2, 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 VOIGTLAENDER & SON 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 Thaxter 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. I'.'ith 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 4J4 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 I3th 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. SCHERIKG, 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. Slender, Leipzig, Gerichtsweg 10. Fabrik mid Lager von Glasgegenstaenden zur Anfertigung mikroskopischer Praeparate, u. s. iw. EAGLE OXYGEN Co. (See p. Si.) DEFENDER PHOTO SUPPLY Co., Rochester, N. Y. Argo and other photographic papers. AD DEN DA. Page 46, paragraph b, for '• fat " read casein. The fluid has a soap}- 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 dipheuylamiii 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 INDEX. Abbe camera 130 Abstracts, making of 1 14 Acclimatization to liig-h temperatures 222 Acetic alcohol. as a fixing fluid 202 with mercuric chloride 202 Acetone, for use in fermentation-tubes 52 Acid-fast bacteria iSS 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 planls, 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 Aerobe, 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 flagvlla 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. "J5 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 q; I Agfa-intensifier 142 Air, bacteria infrequent in Arctic 255 bacteria rare in upper strata of 255 of mountains, bacteria rare in 255 over the sen. 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 metbylene blue 188 Amaryllis, inoculated with B. hyacinth! 66 Amido-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/O BACTERIA IN RELATION TO PLANT DISEASES. Page. Anilin-dyes, differential diagnosis with 230 watery solutions of 187 Analin-dyes (basic), affinity of bacteria for 27 use of, in vegetable tissues 29 Anilin fuchsin 187 Anilin gentian violet 187 Anilin methyl violet 187 Anilin stains, alcoholic solutions of 187 Anilin-water 187 Animal-fluids for culture-media, beef-broth 45 blood-serum 48 egg-albumen 48 egg-yolk 49 litmus-milk 48, 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 So, branching in 216 generic name for I71 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 140 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 219 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 frezing 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 106, 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 262 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, 176 prevention of 93 Bacterium, Conn'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, it, 12 useful for comparison 29 Bacterium hyacinth!, effect of desiccation on /I 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, flagellum of 237 green fluorescence of 65 Page- Bacterium pericarditidis — continued. viscidity in fluid culture-media 42 vitality of 72 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 Stewarti, 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 Barf oed'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, Berkcfeld's, for filtration of culture-fluids.. 43 Chamberland's, for filtration of culture- fluids 43 Chamherland's, sterilization of, necessity for frequent 44 Bowhill's flagella stain 192 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 apbides 215 Bromide-prints 151 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, 211, 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 I'ln-bon dioxide, assimilation of, by bacteria 64 behavior in 59, 223 cultures in, factors to be considered 59 jii'Mi-ration 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 5^ Carbon monoxide, effect on bacteria 58 Carnoy'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 17^ 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, Migula's system 159 Fischer's system 157 [NDEX. 273 Page. location — continued. water-bacteria 203 \Yinslow & Rogers 265 with reference to nitrogen-nutrition 175 Cleaning, glassware 100, 226, 227 Cleaning mixture, chromic acid 200 Clostridium 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 appearaii. e 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 "f 101 waste, for laboratory use 107 Counting-plates, ruled 36, 227 Cover-glasses, cleaning of -'-7 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 -.;5 Page. Cross-inoculations 186 Cross-level 149 Crystals. formation of, in milk-cultures 46 in old cultures 66 prevent making lliin sections 122 Cucumber, natural infection in 92,178,215 Cultural characters, value of 178 variations in, din- In environment 182, 183 Culture-fluids, o ild 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-j elly 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 Icrmo, fla- gella of 20 Dangeard, green bacteria 237 Dannappel, spores sensitive to beat 22,246 274 BACTERIA IN RELATION TO PLANT DISEASES. Page. Dark-room, arrangement of ISO 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 /o, 248 Developer, hydrochinon 146,201 lantern-slide 201 ortol 14° pyrogallol 200, 201 Development of negatives 140 Dewar glasses, for liquid air So 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 126, 129 Distribution, geographical 7 Divided-plate method for study of antago- nism 73 1 )i vision of cells, arrangement after 22, 218 Dowries & 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 195 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 119 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 injured 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 10 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 witli 229 when not usable 208, 234 1NDF.X. 2/5 Page. 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 61 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, 6 1 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 Ficker, glass, influence of substances dis- solved out of 223 Filaments, bacteria in form of 22 Filters, Berkefeld and Chambcrland, for cold steril- ization of fluids 43 Zettnow's copper-chrom 20 r 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. Flagella-staining, cultures best suited for 190 literature on 219 technique 189 Flasks, for storing distilled water 129 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 Formulas 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 r \CTKRIA IN RELATION TO PLANT DISEASES. Page. C.-iuze, surgeon's, uses in laboratory 102 Gelatin, antiseptic salts in 30 brands recommended for bacteriological work 31 composition of 3° impurities in 30 melting point of 30 nitrates in 224 per cent recommended for media 30 plate-cultures, stabs, streaks 20 standard nutrient, preparation of 196 sterilization of. p$ variation of same organism nn 180 with cane-sugar 51 with high melting point 225 with malic acid 51 with soluble starch, etc S1 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-watcr 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 TOO 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 < iriess-Ilosvay's reagent 229 C.rimbert, on value of syuthrtir media 51 Growth of bacteria, manner and rapidity of 27 on media exhausted for other 55 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 Ice, 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, 2ir 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 1 86 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 -3, 177 Iodine-starch test for nitrites 63 Iron-haematoxylin iSS, iS 26° 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 J73 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 '9° M-"->re & Kellerman, action of copper on P.. typhosus in water 97 Morphological characters, change in, due to changed environment 222 value of I7<5 Morphology r9 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 I72 Myconostoc 161 Myxobacter 165 Myxobacteriaceae, characters of 164 Myxococcus !O5 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-indium, for making trans- fers 43 Negatives, development of 140 records on M2 NYssler's test, how used 61 Neisser's spore-stain 194 Neumann, variability of pigment- formation. . . 238 Neutral red 230 Nicolle, modification of Gram's method 222 Night-blue, capsule-stain • 221 stain for fiagella 220 Nitrate bacteria, fluid culture-medium for isolation of 199 nutrient agar for isolation of 199 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 N i I rogen-bacteria 175 Nitrogen-free media Sr» J9S Nitrogen, fixation of, by bacteria 64 growth in, apparatus for testing. . 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 '4° i . I excellent definition 265 Objectives, apochromatie. Spencer lO mm 14° Zeiss .....130 Occlusion of vascular system due to bacteria . . 8, 12 Oculars r3O for photomicrography 139 Ohlmuller, germicidal action of ozone ^51 Oleomargarine, bacteria in 259 Olive-knot organism, artificial inoculations JO crystals produced by 00 optimum temperature for 85 Omelianski, blocks lor nitrifying organisms 2OO isolation of hydrogen-cellulose ferment. 100,209 sodium li'imute medium 5° 1>< >ie readily stained by anilin dyes 22 Page. Optimum reaction of medium for bacterial growth 69 Optimum temperatures . 75. §5 Origin of bacteria 177 Ortol developer 140 Ostwald-Pfeff er water-bath 78 Oven, paraffin . H9 Oversteaming, effect of 9§ Oxidases 67 Oxidation, pigments due to 66 Oxygen, compressed, where obtained 81 little, in deep layers of agar. . . 230 pigment-formation, dependent on 238, 239 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 T"2 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 I23 Paraffin-oven T !9 Parasite, relation of, to tissues of host.. 8, 10 reaction of host to means of gaining access to tissues of host. 8,92 Parasites, animal, harbored by plants 89 Paratrophic bacteria • Parenchyma, intercellular spaces of, occupied by bacteria Park, effect of freezing on B. typhosus 247 Pasteur, culture-fluid J97 definition of aerobe and anaerobe 230 influence on bacteriology '52 Pathogenesis, rules of proof. . 9 1'athogenicity, developed by association of plant parasites to animals. . regeneration of Pane, Pneumococcus, origin of capsule Payen, agar-agar, composition of 223 Pear-blight organism, action of light on 71 blackening of foliage attacked by 65 differentiated from B. coli by KNO3. ... 5', "3 hosts of 87 natural infection 92 result of inoculations with 202 Pedesis Peirce, root-tubercles of bur clover 64 Pelargonium, natural infection in Pencils for writing on glass Pepsin in 00 INDEX. 28l gg I5O Page. Peptone, o immen-ial .............................. 45 reaction of cln.Irra organism with .......... 229 tests fur purity of ........................ 229 Peptone-bacteria ........................... i-- Peroxidcs, organic, germicidal notion of ...... 25;, Petri, nitrates in commercial gelatin ......... 224 l'> in-dislies ........................... . 226 for excluding light ........................ 228 for quantitative work and photographing. . . 36 of good quality, where obtained ............ 82 sterilization of ............................ 100 Fctri und Maassen, hydrogen sulphide from bacteria .............................. 243 Phenol .................................... -^ tests for ................................. 63 Phenolphthalein, tor use in titration neutral point of Photographic enlargements .................. 147 Photographic plates, for special work ........ 136 Photographic prints, drawings on .............................. jjl making of Photographing, of bacteria in tissues ...................... 1-56 of poured-plate colonies ................... 134 of test-tube cultures ...................... 135 Photography, focusing planars .......................... !32 lenses for ............................. l3Oi 133 Photomicrography ...................... 263, 264 Photomicrographic apparatus ......... 23, 129, 136 care of ................................... !3p Photomicrographs, making of ................................ I3g 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 ............................ IOo Pitfield's flagella stain ....................... 191 Planococcus ............................... 160 Plant acids, sensitiveness of bacteria to ...... 69 Plant juices used as culture-media ........... 41 Page. Plasmolysis, concentration necessarv for. . 2=54 Plastids of Hallicr " $ 1 'late-cultures, advantages of. for isolating bacteria 11 arrangement of rods in colonies on 27 characters to be noted 29 discarded, care of 107 how to avoid contaminations in 103 Kncli's, for obtaining pure cultures 226 labeling of nr 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-indium, 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, 14^ 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 I '\ uktanin 74 Pyrogallol, with caustic potash as absorbent of '•ien 57 Pyrogallol developer 200, 201 282 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, thertnophilic bacteria 248 Radium rays 245 Raulin's culture-fluid 197 Ravenel, bacteria exposed to liquid air 246 Razors, sharpening of 121 Torrey, Rogers, Lentz 123 Reaction, for maximum growth in liquid media. .. 69,203 of host to parasite 8 Record-books, for culture-media 109 for field-notes no Records, methods of keeping 109 Reducing negatives I42 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 16 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 114. .'40,241 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. IO 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 endosporcs 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 25 1 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 Schutz, 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 118 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 form-; 237 Slide-boxes 117 Slides, rare of discarded 107 numbering of 121 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 Page. Sodium chloride, effect on luminous bacteria 60 restraining influence of 70, 252 Sodium fluoride, germicida! action of 253 Sodium hydrate, method of increasing tolera- tion of 70 Sodium nitrite, used for indol test 62 Si ifU'ning 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 161 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. . oiitimird. resistance to high temperatures and steam heat 21,84,246 resistant, present in meat-extracts 260 resistant, present in mill; 46 stains for 194 Spraying, inoculation by 108 Stadlcr, 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 bactei la, 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 149 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 101 culture-media 85 dry, of pipettes, scalpels, etc 41 glassware, instruments 100, 107 infectious material 106 milk 46 oven for IQO silicate-j elly 39 soils 85 solid vegetable media 40 starch-jelly 50 surface of plants before making cultures from 13 surface of plants to be inoculated 41, ioS syringes 102 with metallic copper 97, 253 Still, for water, in small quantities 128 for water, on a large scale 124 284 BACTERIA TN RELATION TO PLANT DTSEAS1.S. Page. Stock-cultures, how best kept 72, 123 Stoddart, media for differentiation of motile bacteria 26 Stolz, peculiar growths in Pnemnococcus, etc. 217 Stomata, artificial infections by way of 108, 126 natural infection through 90.92 Stored media, .effect of loss of water on 99 Streak-cultures, characters to be noted 30 Streblothrichia 162 Streptococcus 160 Streptococcus mesentcrioides, isolation of by heat 106 Streptothrix 162 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 13 Synthetic culture-media, kinds of 49, 197 value of 51 Syringes, hypodermic 102 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 1 1 1 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-re >< un 75 Thermi >stats 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 IO3 Trenkmann, flagella-stain 219 Trevisan, genus Pacinia 772 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 L'ltramicroscopical organisms 18,211,213 Unguentum resinae 228 Urocephalum 159, 264 Uschinsky's solution, cultures in dilute, for llagella staining 20 formula 197 in silicate-idly 39 modified 197 INDI.X. 285 Page U-tubes, for testing growth in iiitrn-.-ii 5^ Vacuo, concentration of fluids in 68 growth in 54 Yallin. cleaning filters 227 Van Hrmengem, llagella-stain 191 germicidal effect of ozone 25 1 van't Hoff, modification of plate-method 228 van Tieghem, thermophilic bacteria 247 Varietal resistance to disease 93, 186 Vascular system occupied by bacteria Velox-prints IS1 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, Miiller's genns 172 Viscidity 42 of bacterial growths 39 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, pigtncnted-media for differ- entiation 229 V-shaped forms 23, 217 Wager exposure scale 143 Waldo & Wulsh. 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 XVater, apparatus for distilling 124 bacteria in, literature on 258 Water-bacteria, blue and violet -'37 Hesse & Niedner's nutrient agar for 196 Page. bacteria -continued. ho\v best kept 261, media for 258 Water, examination of. English metli.nl; for. 22O Water-bath. Ostwald-Pfeffer 78 Writer-pore?, infection through 92.102.124 Wax-mixture, Darwin's 200 Wehmer, fermentation of sauerkraut 2=;8 Wfiyert, 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. 109 nitrifying bacteria 240 retting of flax 257 silicate-jelly 36. 240 red sulphur-bacteria 263 Winogradsky & Onu'Iianski. fluid-media for isolating nitrate and nitrite bacteria... 199 Winogradsky-Sleskin silicate-jelly 198 \Vins1ow & Rogers, Coccaceae revised by.... 26; Wood-vinegar, an energetic disinfectant 252 Wounds, disinfection of 107 natural infection through nj 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 I'Mfl • •• ; • ••• •'••" -