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 p 2 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 r 4 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 I4 s 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 I 7 141. 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. at\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., noC. 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/irfm) 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?5 l ^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 streaming 1 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 e ther 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 Pj g 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. 6 7 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* Fi g .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. 7 6 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? 8 9 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.* 9 o 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. " 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. 9 6 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 I Y autenschlager oven shown in plate 6 does not require watching and gives a uniform temperature *Fic. 85. Petri dishes wrapped in two layers of Manila paper and ready to be dry sterilized. They are set on edge in the oven. Fig. 85* STERILIZATION. 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 w r all 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,auU j iiM-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. F lg . 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 2O l /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 few r 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 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. I2 3 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 33I C 3 Plant sprayed December 9, 1904; slightly blackened leaf-tooth fixed in 95 per cent alcohol on December 17, 1904. Inked from a photomicrograph. fFic. 116. Cross-section of leaf-tooth of cabbage infected by Bacterium 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 (3 4 -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 y 2 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 II a . 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 I 1 '. 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.tpT ..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 ??.? i 1 ' ' ' 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. v Fic. 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 tcrio 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, I 7 , 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- Fi g . 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. .8 4 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 w r hich 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 arc 1 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 li