SOCIETY OF AMERICAN BACTERIOLOGISTS MONOGRAPHS ON SYSTEMATIC BACTERIOLOGY Volume I. General Systematic Bacteriology. R. E. Buchanan, Iowa State College. 597 pages. $6.00, net postpaid. Volume II. A Compilation of Culture Media for the Cultivation of Microorgan- isms. Max Levine and H. W. Schoenlein, Iowa State College, xvi + 969 pages. $15.00 A COMPILATION OF CULTURE MEDIA FOR THE CULTIVATION OF MICROORGANISMS MONOGRAPHS ON SYSTEMATIC BACTERIOLOGY A Compilation of Culture Media FOR THE CULTIVATION OF MICROORGANISMS BY MAX LEVINE, Ph.D. Professor of Sanitary and Technical Bacteriology, Bacteriologist, Iowa Engineering Experiment Station AND H. W. SCHOENLEIN, M.S. Formerly Fellow in the Department of Bacteriology, Iowa State College, Ames, Iowa PREPARED AT THE REQUEST OF THE SOCIETY OF AMERICAN BACTERIOLOGISTS AND FINANCED BY A GRANT FROM THE DIGESTIVE FERMENTS COMPANY, DETROIT, MICHIGAN BALTIMORE THE WILLIAMS & WILKINS COMPANY 1930 Copyright 1930 THE WILLIAMS & WILKINS COMPANY Made in the United States of America Published June, 1930 All rights reserved Composed and Printed at the WAVERLY PRESS, INC. FOR Thk Williams & Wilkins Company Baltimore, Md., U. S. A. CONTENTS I. FOREWORD xi II. PREFACE xiii III. INTRODUCTION xv IV. KEY TO THE MAIN GROUPS OF CULTURE MEDIA 1 V. KEY TO THE SUB-GROUPS OF LIQUID MEDIA. Group I (Med. 1-1394) 1 A. Sub-group lA. Water only as a basal medium 1 B. Sub-group IB. Key to sections of liquid media in which ail constituents are inorganic. (Med. 2-113) 3 1. Nitrogen present as free N2 only. a. Incomplete "basal" solutions requiring addition of other nutri- ents. Section 1 (Med. 2-25) 3 b. Complete nutrient solutions. Section 2 (Med. 26-29) 10 2. Nitrogen supplied as ammonium salts. a. Incomplete or "basal" solutions. Section 3 (Med. 30-56) 11 b. Complete media primarily for organisms oxidizing NH3 to NO2. Section 4 (Med. 57-72) 20 c. Complete media primarily for organisms not oxidizing NH3 to NO2. Section 5 (Med. 73-85) 25 3. Nitrogen supplied as nitrites. Section 6 (Med. 86-95) 28 4. Nitrogen supplied as nitrates. a. Incomplete or "basal" solutions. Section 7 (Med. 96-106) 31 b. Complete nutrient solutions. Section 8 (Med. 107-113) 34 C. Sub-group IC. Key to sections of liquid media in which one or more con- stituents are organic. (Med. 114-1394) 36 1. Chemical composition of all constituents known. a. Nitrogen present only as free N2. Section 1 (Med. 114H90) 37 b. Nitrogen present as ammonium salts. Section 2 (Med. 191-323) 59 c. Nitrogen present as nitrites. Section 3 (Med. 324-327) 91 d. Nitrogen present as nitrates. Section 4 (Med. 328-365) 92 e. Nitrogen present as amino acids. Section 5 (Med. 366-479) 102 f. Organic nitrogen other than amino acids present. Section 6 (Med. 480-515) 135 2. Chemical composition of one or more constituents not known. a. Media containing commercial digests. (1) Additional constituents inorganic. Section 7 (Med. 516-565) 144 -i 3 5 b i) 1 Viii CONTENTS (2) Incomplete or "basal" solutions with additional organic non-nitrogenous materials of known composition. Section 8 (Med. 566-588) 157 (3) Complete media as in (2). Section 9 (Med. 589-647) 167 (4) Additional constituents nitrogenous and of known chemi- cal composition. Section 10 (Med. 648-683) 182 (5) Additional materials of plant origin. Chemical composi- tion not known. Section 11 (Med. 684-712) 191 (6) Additional material of animal origin of unknown chemical composition (other than extracts or infusions). Section 12 (Med. 713-747) 198 (7) Media containing animal extracts or infusions in addition to commercial digests. Section 13 (Med. 748-977) 210 b. Media containing non-commercial digests. Section 14 (Med. 978-1140) 299 c. Media not containing digests. (1) Containing plant derivatives of unknown chemical com- position. Section 15 (Med. 1141-1228) 329 (2) Containing animal derivatives. Section 16 (Med. 1229-1376) 355 (3) Containing extracts of soil, ashes, etc. Section 17 (Med. 1377-1394) 402 VI. KEY TO THE SUB-GROUPS OF REVERSIBLY SOLID AGAR MEDIA. Group II (Med. 1395-2198) 408 A. Sub-group IIA. Key to media containing water and agar only. (Media 1395-1401) 408 B. Sub-group IIB. Key to sections of liquefiable agar media, with all con- stituents (other than agar) inorganic. (Media 1402-1430) 411 1. Nitrogen present only as free N2. Section 1 (Med. 1402-1410) 411 2. Nitrogen supplied as ammonium salts. Section 2 (Med. 1411-1424) 413 3. Nitrogen supplied as nitrites and nitrates. Section 3 (Med. 1425-1430) 417 C. Sub-group lie. Key to sections of liquefiable agar media containing or- ganic materials (in addition to agar). (Med. 1431-2198) 419 1. Composition of all added constituents known. a. Inorganic nitrogen supplied. Section 1 (Med. 1431-1495) 419 b. Organic nitrogen supplied. Section 2 (Med. 1496-1519) 436 2. Composition of some of the added constituents not known. a. Agar media containing commercial digests; other materials of known composition. Section 3 (Med. 1530-1596) 443 b. Agar media containing commercial digests together with ma- terials of plant or soil origin. Section 4 (Med. 1597-1627) 461 CONTENTS ^^ c. Agar media containing commercial digests and materials of ani- mal origin other than extracts or infusions. Section 5 (Med. 1628-1660) ....... 469 d Agar media containing commercial digests and extracts or in- fusions of animal origin. Section 6 (Med. 1661-2028) 481 e. Agar media containing non-commercial digests. Section 7 (Med. 2029-2078) . 650 f Agar media containing unknown constituents of plant origin but no digests. Section 8 (Med. 2079-2141) 674 e Aear media containing unknown constituents of animal origin but no digests. Section 9 (Med. 2142-2192) 690 h Agar media containing unknown constituents of ash or soil origin but no digests. Section 10 (Med. 2193-2198) 708 VII KEY TO THE SUB-GROUPS OF REVERSIBLY SOLID GELATIN MEDIA. Group III (Med. 2199-2371) 711 A Sub-group IIIA. Key to gelatin media containing no other organic materials. (Med. 2199-2204) 711 B. Sub-group IIIB. Key to sections of liquefiable gelatin media containing additional organic materials. (Med. 2205-2371) 713 1 Gelatin media with all other constituents of known composition. Section 1 (Med. 2205-2234) 713 2 Gelatin media containing digests and other materials of unknown composition. Section 2 (Med. 2235-2323) 718 3 Gelatin media containing materials of unknown composition other than digests. Section 3 (Med. 2324-2371) 752 VTII KEY TO REVERSIBLY SOLID MEDIA OTHER THAN AGAR OR GELATIN. Group IV (Med. 2372-2382) 765 IX KEY TO SUB-GROUPS OF IRREVERSIBLE SOLID MEDIA; INITIALLY LIQUID; SOLIDIFYING AGENT ORGANIC. Group V (Med. 2383-2466) 768 A Sub-group VA. Irreversibly solid media, solidified by materials of plant origin. (Med. 2383-2397) 768 B Sub-group. VB. Irreversibly solid media, solidified by blood or its deriva- ■ thres. (Med. 2398-2430) 772 C. Sub-group VC. Irreversibly solid media solidified by materials of animal origin other than blood (eggs, etc.). (Med. 2431-2466) 788 X KEY TO IRREVERSIBLE SOLID MEDIA; SOLIDIFYING AGENT INOR- GANIC. Group VI (Med. 2467-2485) 803 X CONTENTS XI. KEY TO SUB-GROUP OF MEDIA INITIALLY SOLID. Group VII (Med. 2486-2543) 813 A. Sub-group VIIA. Initially solid organic substrates. (Med. 2486-2516) 813 B. Sub-group VIIB. Initially solid inorganic substrates. (Med. 2517-2543) 825 XII. MEDIUM NAME INDEX 833 XIII. CONSTITUENTS INDEX 869 XIV. USE INDEX 891 XV. AUTHOR INDEX 899 XVI. REFERENCES 915 FOREWORD The contributions relating to the various substrates and media used for the cultivation of bacteria, yeasts, and molds, are widely scattered in the hterature. Very frequently they are not adequately indexed. For some years it has been felt that a key to the various media and substrates proposed for the growth of microorganisms would prove helpful. The fact that these formulae were not readily available has led to a great amount of dupHcation m the hterature. A search of the hterature has therefore been undertaken and the formulae tor about 7000 various media that have been proposed brought together. The task of securing this material was performed most acceptably by Mr. H. W. Schoenlein, who was also associated in the development of the keys The task of organizing the compilation, of planning the presentation, and of final editmg was undertaken by Dr. Max Levine. It was found possible greatly to curtail the number of separate formulae by use of the subheading Variants m many The compilation presented in the following pages was made possible by a grant to the Society of American Bacteriologists by the Digestive Ferments Company of Detroit, Michigan. At the request of the Society of American Bacteriologists, the Department of Bacteriology at the Iowa State College accepted the task of compilation. The interest, helpfulness, and support of the Digestive Ferments Company throughout the progress of the work cannot be too greatly appreciated. The system of classification of the media, together with the arrangement of keys, indices, etc., should make possible a ready review of the media which have been proposed for growing different types of organisms and containing different ingredients. The primary subdivisions into Groops on the basis of physical characteristics and nature of the sohdifying agent is the logical one both from the standpoint of history and the standpoint of usefulness. The secondary subdivisions based upon the chemical characteristics of the constitu- ents of the media are in fine with modern trend toward emphasis upon physiol- ogy and metabolism of microorganisms. It is hoped that this compendium of information will prove very generally useful to all bacteriologists and in all bacteriological laboratories. R. E. Buchanan. PREFACE The rapid growth of the science of Bacteriology has led to the development of numerous combinations of materials for the propogation of microorganisms. It was felt that the collection and orderly arrangement of such culture media would be of assistance in the future development of the science of Bacteriology, and the authors were gratified with the opportunity to perform this service. In spite of the efforts to thoroughly cover the literature, this compilation is probably incomplete, particularly with respect to formulae published in bulletins and monographs which were not available or consulted. It is hoped that this compilation will help acquaint the bacteriologists with the numerous types and combinations of materials which have been employed for cultivating micro- organisms, and if it serves to check the needless publication of new formulae, which are really old, the authors will feel well repaid for their time and efforts. To Mr. H. G. Dunham, of the Digestive Ferments Company, we are greatly indebted for helpful criticisms, and suggestions. To Mrs. C. H. Werkman, Miss Ruth Confare and Miss Lois Kratoska, the authors extend cordial thanks for assistance in typewriting and correcting the manuscript. To Dr. R. E. Buchanan the authors wish to particularly express their indebt- edness and gratitude for the fatherly advice, encouragement, and assistance, so graciously rendered at all times. Max Levine, h. w. schoenlein. Iowa State College, Ames, Iowa, July 1, 1929 INTRODUCTION At the request of the Society of American Bacteriologists, the Hterature was surveyed for formulae of culture media reported useful for the growth of bacteria and other microorganisms. The standard periodicals such as the various Cen- tralblatts fiir Bakteriology, Zeitschrift ftir Hygiene, Annals of the Pasteur Institute, Journal of Hygiene, Journal of Pathology and Bacteriology, and all of the American bacteriological journals were perused from their inception to the year 1926. A number of other periodicals, as well as text books, monographs and many Experiment Station bulletins were included in the survey. Formulae of about seven thousand culture media were gleaned from the literature. These were arranged and classified into approximately twenty five hundred quite distinct media. A number and a distinctive name was assigned to each medium which is described under the following headings: — Constituents, Preparation, Sterilization, Use, Variants, so as to facilitate comparison of differ- ent formulae. A dichotomous key, several indices and complete references to all articles from which formulae were taken are also included in this compilation. In the key, and all indices, the numbers refer to medium numbers and not pages. The Medium Name Index includes an alphabetical Ust of all media described herein. In the Constituents Index an attempt was made to list every medium in which a particular substance was employed. The Author Index is self explanatory. The Use Index is necessarily incomplete. It includes only those media for which a specific use was indicated in the original article reviewed. The various media were subdivided into seven "groups" on the basis of their physical states. Thus, all the liquid media are considered in a group by them- selves and the solid media are further subdivided, on the nature of the solidifying agent and physical properties of these agents, as, for example, whether initially hquid, reversibly liquid and solid, or permanently solid. Each group, if sufficiently complicated to warrant further classification, is then subdivided into ''sub-groups" on the basis of the presence or absence of additional organic constituents. The sub-groups are further subdivided into sections on the basis of the nature of the nitrogen, carbon, and inorganic constituents. For each section there is prepared a key, generally dichotomous, by means of which each medium therein may be located. The various keys are distributed throughout the book immediately preceding their respective media. The problem of a suitable means of identification of individual media was a particularly difficult one. The scheme finally adopted was to assign a number in / XVI INTRODUCTION / conjunction with a specific name, the latter including the chief nitrogen and carbon source, where possible, and the name of the author who first listed the medium. Thus "273 Ayers' Glucose Ammonium Phosphate Solution," indi- cates that Ayers first described the medium, the nitrogen source was ammonia, the carbon source glucose, and that the medium is No. 273 in this collection. In addition to the carbon and nitrogen source the nature of the solidifying agent is also employed in naming culture media, as, for example, "1553 Greig Smith's Sucrose Peptone Agar." Where the original article containing a medium was not available the fact is indicated by placing the author from whose publication the medium was ob- tained in parentheses after the name of the medium. Thus "2156 Beck's Glycerol Serum Agar (Klimmer)" indicates that the medium was obtained from Klimmer who referred to Beck as the author. If the same medium is described by different authors, only the earliest avail- able author's name appears in the medium name, but the other authors are given under the heading "references" at the end of the description of the medium. Where several media differ only with respect to the relative quantities of constituents employed, or in the method of preparation, the earliest described medium takes precedence, and all others are described as variants. One of the vexing questions which arose was that of the disposition of media in which it was specified that a number of substances might be substituted for each other. For example, if the availability of various carbon sources is being studied, shall each new combination, as the employment of glucose, sucrose, glycerol, etc., be considered a distinct medium? To do so would have markedly extended the number of media to be considered. The term "basal" was introduced to take care of this situation. This was ap- plied to the formula exclusive of the substituted constituents which then became added nutrients. The term "basal" occurring in the name of a medium there- fore designates that the formula is not in iteslf a complete medium, but that it serves as a base to which other constituents are added. A serious difficulty in naming media arose in connection with the use of beef extracts and meat infusions. This was particularly troublesome when authors did not specify whether extract or infusions were employed. Where the use of extracts was specified in the articles reviewed, these terms appear in the names of the respective media, as e.g., "779 Dunham's Infusion Broth" or "1695 Heinemann's Meat Extract Agar." Where the author did not specify the nature of the material employed (i.e., whether extract or infusion) the term "bouillon" is employed, thus "936 Kendall, Day and Walker's Mannitol Bouillon." COMPOSITION OF CULTURAL MEDIA AND SUBSTRATES USED IN BACTERIOLOGY Any classification of media and substrates used in bacteriology must of necessity be artificial and arbitrary in form. In the classification here adopted an effort has been made to bring closely related media together, although it is realized that in some cases even the primary divisions tend to separate media having much the same essential composition and used for much the same purposes. The 7 primary divisions which have been adopted are as follows: PRIMARY CLASSIFICATION OF MEDIA Ai. Liquid media Group I (Med. 1-1394) A2. Solid media. Bi. Initially liquid. Ci. Reversible. Reversibly liquid and solid. Liquefiable by heat. Di. Solidified by the addition of agar-agar. . Group II (Med. 1395-2198) D2. Solidified by the addition of gelatin. . Group III (Med. 2199-2371) D3. Solidified by the addition of other materials Group IV (Med. 2372-2382) C2. Irreversible. Not reversibly liquid and solid. Di. Solidifying agent organic Group V (Med. 2383-2466) D2. Solidifying agent inorganic Group VI (Med. 2467-2485) B.. Initially solid Group VII (Med. 2486-2543) GROUP I. LIQUID MEDIA The term "Liquid Medium" is here de- fined to include all media containing suffi- SUBGROUP I-A cient water and so lacking in viscosity as to rr^u j. i • xu u x • i • i * , • , The water used in the bacteriological be readily poured from one vessel to another. A liquid medium is considered one which laboratory as a medium or in the prepara- , ,, ,, ,. ,.^ , , , tion of media may be either distilled or a does not set or solidify when cooled to ^^tural water. In theory distilled water is o? wateT''^*"'' ""^ ^°'''*' ^° •'" preferred, although distilled water mi. ,' -n J.- I-1- -1 1- J X 1 unless carefully prepared may contain gas- i he classihcation 01 liquid media adopted „ j. n- ■ -i- i- u -n • j. , . ., ^ , , , ! eous or metallic impurities which will inter- is based primarily upon whether the nitro- j-^j.^ ^^^-^ j^^ ^^^ gen supplied is inorganic or organic. Distilled water' for the bacteriological Subgroups of liquid media laboratory. Ai. Water only Subgroup I-A Surface and ground water for media. A2. Water with other constituents Bi. All constituents of medium inorganic 1, Molisch's Basal Nutrient Solution Subgroup I-B (Med. 2 to 113) B2. One or more constituents organic Constituents: Subgroup I-C (Med. 114 to 1394) 1. Sea water 1000.0 cc. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS Preparation : (1) Dissolve one of the added nutrients in amount indicated in 1. (2) Tube in tall tubes. Fill within a centimeter of the plug. Sterilization: Method not given. Use: To determine the constituents essen- tial for the growth of purpurbacteria, Rhodobacterium capsulatum and Rhodo- hacillus palustris (Molisch). The cul- tures were incubated in diffused light. The author reported generally no growth using sucrose, dextrin, glycerol, ammo- nium tartrate or asparagin alone, but gen- erally growth with the other materials and combinations listed. Added nutrients and variants: The author suggested the following: (a) Add one of the following materials: sucrose 1.0% dextrin 1.0% inulin 1.0% asparagin 1.0% peptone 1.0% glycerol 1.0% peptone 1.0% + sucrose 0.5% peptone 1.0% + glycerol 0.5% peptone 1.0% + dextrin 0.5% peptone 1.0% + asparagin 0.5% peptone 1.0% + inulin 0.5% asparagin 0.5% + dextrin 0.5% asparagin 0.5% + inulin 0.5% ammonium tartrate 1.0% (b) Used Moldau river water added one of the following materials: sucrose 1.0% or 0.5% dextrin 1.0% or 0.5% inulin 1.0% or 0.5% asparagin 1.0% or 0.5% glycerol 1.0% or 0.5% peptone 1.0% peptone 1..% + sucrose 1.0% or 0.5% peptone 1.0% + dextrin 1.0% or 0.5% peptone 1.0%, + inulin 1.0% or 0.5% peptone 1.0% + asparagin 1.0% or 0.5% peptone 1.0% + glycerol 1.0% or 0.5% asparagin 0.5% + dextrin 0.5% asparagin 0.5% + glycerol 0.5 g. dextrin 0.5% + inulin 0.5% ammonium tartrate 1.0% sodium citrate (neutral) 0.05% ammonium citrate 0.05% magnesium citrate 0.05% potassium citrate 0.05% calcium butyrate 0.05% sodium acetate 0.05% iron ammonium citrate 0.05% iron magnesium citrate 0.05% iron ammonium citrate 0.05% + gyp- sum infused hay infused hay + iron ammonium citrate 0.05% iron potassium citrate 0.05% iron ammonium tartrate 0.05% iron ammonium oxalate 0.05% iron glycero phosphate 0.05% iron lactate 0.05% iron magnesium lactate 0.05% manganese peptone 0.025% manganese peptone 0.025% + K2HPO4 0.1% manganese peptone 0.025% + K2HPO4 0.1% + KNO3 0.1% + MgS04 0.1% manganese peptone 0.025% + K2HPO4 0.1% + KNO3 0.1% manganese peptone 0.025% + MgS04 0.1% manganese peptone 0.025% + KNO3 0.1% manganese peptone 0.025% + gypsum 0.1% manganese peptone 0.025% + K4Fe (CN)6 0.1% manganese peptone 0.025% + citrate iron peptone peptone 0.5% peptone + MnCOs in excess peptone + FeCOa in excess iron albuminate 0.05% iron peptonate 0.05% manganese lactate 0.05% manganese phospholactate 0.05% manganese salicylicate 0.05% manganese fluorate 0.05% + peptone 0.5% manganese citrate 0.05% manganese oxalate 0.05% manganese glycerinate 0.05% iron sulphide — peptone 0.05% manganese acetate 0.05% (c) Sea water without any addition. (d) Moldau river water without any addition. Reference: Molisch (1907, p. 68). CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS SUBGROUP I-B Liquid Media in which All Constituents are Inorganic Liquid media in which all constituents are inorganic may be classified most con- veniently on the basis of the type of nitro- gen provided. Key to the sections of Subgroup I-B A I. Nitrogen present as free or elementary nitrogen only. Bi. Incomplete media, so-called "basal" solutions, requiring the addition of other nutrients Section 1 (Med. 2-25) B^. Complete solutions, used as media with- out additions Section 2 (Med. 26-29) A^. Nitrogen supplied as ammonium salts. Bi. Incomplete media, or basal solutions. Section 3 (Med. 30-56) B2. Complete media. Ci. Media primarily for organisms oxidiz- ing ammonia to nitrites for growth energy Section 4 (Med. 57-72) C2. Media not primarily for organisms oxi- dizing ammonia to nitrites for growth energy Section 5 (Med. 73-85) A3. Nitrogen supplied as nitrites. Media primarily for organisms oxidizing nitrites to nitrates for growth energy. Section 6 (Med. 86-95) A4. Nitrogen supplied as nitrates. Bi. Incomplete or basal solutions requiring the addition of other nutrients. Section 7 (Med. 96-106) B2, Complete nutrient solutions. Section 8 (Med. 107-113) SUBGROUP I-B. SECTION 1 Inorganic basal solutions of known chemical composition, nitrogen present only as atmospheric nitrogen; incomplete solutions requiring the addition of other nutrients. Ai. Containing salts of monovalent cations only. Physiological Salt Solution. (Normal saline) 2 Molar Salt Solution 3 A2. Containing salts of monovalent and other cations. Bi. Salts of mono and divalent cations present. Ci. Sodium salts present. Di. Calcium salts present. Ashby's Basal Solution. (Hoffmann and Hammer) 4 von Wahl's Basal Salt Solution 5 Locke's Solution 6 Winogradsky's Basal Inorganic Salt Solution (Heinemann) 7 Charrin and Dissard's Basal Salt Solu- tion 8 Gerlach and Vogel's Basal Solu- tion 9 Rettger, Berman and Sturges' Basal Solution 10 D2. Calcium salts not present. Omeliansky's Basal Salt Solution 11 Percival's Basal Salt Solution 12 Nawiasky's Basal Salt Solution 13 C2. Sodium salts not present. Stoklasa's Basal Solution 14 Heinze's Basal Salt Solution 15 Sohngen's Basal Solution 16 van Delden's Basal Gypsum Solu- tion 17 Czapek's Basal Solution 18 Buchanan's Basal Salt Solution 19 B2. Salts of mono, di, and trivalent cations present. Ci. Sodium Salts present. Fuhrmann's Basal Solution 20 Munter's Basal Salt Solution 21 Meyer's Basal Salt Solution. (Per- otti) 22 C2. Sodium Salts not present. Dox's Inorganic Salt Solution for Fungi (Tanner) 23 Gage's Basal Salt Solution 24 Bijerinck and van Delden's Basal Salt Solution 25 2. Physiological Salt Solution (Normal saline) Constituents : 1. Water 1000.0 cc. 2. NaCl (c.p.) 8.5 g. Preparation: (1) Dissolve 2 in 1. Sterilization: As desired. Uses: Diluent and serological work in the laboratory. Variants: Frost used 6.0 g. NaCl per liter References: Original author not found. This formula was taken from Giltner (1921, p. 399). Frost (1903, p. 18). CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 3. Molar Salt Solution Constituents : 1. Water 1000.0 cc. 2. NaCI (best commercial grade) 60.0 g. Preparation: (1) Dissolve 2 in 1. Sterilization: As desired. Uses: Diluent. References: Giltner (1921, p. 399). 4. Ashby's Basal Solution (Hoffmann and Hammer) Constituents : 1. Distilled Water 1000.0 cc. 2. KH2PO4 0.2 g. 3. MgS04 0.2 g. 4. NaCl 0.2 g. 5. CaS04 0.1 g. 6. CaCOs 5.0 g. Preparation : (1) Dissolve the KH2PO4 in a small amount of water, and add tenth nor- mal NaOH to neutral point to phenol- phthalein. (2) Dissolve 3, 4, 5 and 6 in remainder of 1 (3) Mix (1) and (2). (4) Add suitable carbon source. (5) Distribute in 20.0 cc. lots in 150 cc. Erlenmeyer flasks. Sterilization: Method not specified. Uses: For study of the nitrogen fixing bacteria from soil. Added Nutrients: Hoffmann and Hammer suggest the following list of carbon sources: Mannitol 20 g. Maltose 20 g. Sucrose 20 g. Galactose 20 g. Inulin 20 g. Dextrose 20 g. Levulose 20 g. Dextrin 20 g. RafRnose 20 g. Lactose 20 g. References: Hoffmann and Hammer (1910 p. 128). 5. von Wahl's Basal Salt Solution Constituents : 1. Distilled water 1000.0 cc. 2. Potassium phosphate 1.0 g. 3. CaCh 0.1 g. 4. MgS04 0.3 g. 5. NaCl 0.1 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add one of the added nutrients, or combinations of added nutrients. Sterilization : Not specified. Use: Cultivation of carrot bacillus (Bacil- lus daucarum) and other organisms caus- ing vegetable spoilage. Added nutrients : The author added one of the following materials or combinations of materials: (a) peptone 10.0 g. fsucrose 30.0 g. (b) \ asparagin 10.0 g. [glycerol 10.0 g. . , fsucrose 30.0 g. \asparagin 1-0 g. ,^) [glycerol 30.0 g. \asparagin 1.0 or 10.0 g. , . /lactose 30.0 g. \asparagin 10.0 g. (sucrose 5.0 g. glycerol 10.0 g. ammonium tartrate 10.0 g. (sucrose 5.0 g. glycerol 10.0 g. KNO3 10.0 g. (h)(KNO. 0.5g. ^ ^ [soda 0.5 g. [sucrose 5.0 g. (i) jglucose 5.0 g. [glycerol 5.0 g. -., fglucose 30.0 g. [asparagin 10.0 g. . . /(NH4)2S04 0.0025 g. ^^ \Na2CO3 5.0 g. Reference: von Wahl (1906, p. 496). 6. Locke's Solution Constituents : 1. Water 1000.0 cc. 2. NaCl (0.9 to 1.0%)... 9.0 to 10.0 g. 3. KCl (0.01%) 0.1 g. 4. CaCl2 (0.02%) 0.2 g. Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization: As desired. Uses: Commonly used as a diluent. Tis- sue neither swells nor shrinks in this solu- tion. It is also used as a basis for culture CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS media, various nitrogen or carbon sources being added. Reference: Park, Williams and Krumwiede (1924, p. 122). 7, Winogradsky's Basal Inorganic Salt Solution (Heinemann) Constituents : 1. Distilled Water 1000.0 cc. 2. MgS04 0.5 g. 3. CaCl. 0.01 g. 4. NaCl 2.0 g. Preparation: (1) Dissolve 2, 3 and 4 in 1 Sterilization : Not specified. Uses: Used in soil bacteriological work as a base to which other materials are added. Reference: Heinemann (1922, p. 37). 8. Charrin and Dissard's Basal Salt Solution Constituents: 1. Water 1000.0 cc. 2. KH2PO4 0.1 g. 3. Na2HP04 0.1 g. 4. CaCh 0.05 g. 5. MgS04 0.05 g. 6. KHCO3 0.134 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Adjustment of reaction not given. (3) Distribute in 50.0 cc. lots in flasks and add 1.0 or 0.5 g. of one of the added nutrients to each flask. Sterilization: Autoclave at 120°C. Time not specified. Use: To study pigment production by Bacillus pyocyaneus. The authors re- ported that asparagin gave the best pig- ment; peptone gave a little pigment; no growth using urea. Added nutrients: The authors added 0.5 or 1.0 g. of one of the following organic materials to each 50.0 cc. lot of medium: peptone glycogen asparagin acetic acid urea lactic acid glucose Reference: Charrin and Dissard (1893, p. 182). 9. Gerlach and Vogel's Basal Solution Constituents: 1. Water 1000.0 cc. 2. K2HPO4 0.5g. 3. CaCOs 0.5 g. 4. NaCl 0.5 g. 5. FeS04 some Preparation : (1) Dissolve 2, 3, 4, 5 and one of the car- bon sources listed under added nu- trients in 1. (2) Distribute in flat bottomed flasks. Sterilization : Steam for 30 minutes on each of 3 successive days. Use: Study of nitrogen assimilation by azotobacter and other bacteria from the soil. More nitrogen was assimilated using dextrose as a source of carbon than calcium propionate. Little nitrogen was assimilated if the flasks were sealed after inoculation. Added nutrients : (a) The authors suggested the use of the following carbon sources: Glucose 2.0 g. Calcium propionate 1.0 g. (b) Percival used the same basic solution and added 2.0 g. glucose. Variants: Bonazzi added 10.0 g. of glucose, designated the use of deep well water added 1.0 g. CaCOs, specified the addition of 0.02 to 0.5 g. FeS04-7H20, and also stated that 0.23 g. KNO3 and 1.264 g, Ca(N03)2 might be added if desired. References: Gerlach and Vogel (1902, p. 671), Percival (1920, p. 181), Bonazzi (1921, p. 339). 10. Rettger, Berman and Sturges' Basal Solution Constituents : 1. Water 1000.0 cc. 2. NaCl 5.0 g. 3. Na2S04 2.0 g. 4. CaCl2 1.0 g. 5. KH2PO4 1.0 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add one of the added nutrients listed below. Sterilization: Generally by filtering through a Berkefeld filter, in some cases by heat. Use : To study proteolysis by Proteus vul- garis, B. prodigiosus and B. subtilis. Added nutrients : (a) Various chemically pure proteins added alone or in combination. 6 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS (b) Egg albumin prepared by method of Hopkins and Pinkus (1899); amount not specified. (c) Peptone 10.0 g. Reference: Rettger, Berman and Sturges (1916, pp. 15-33). 11. Omeliansky's Basal Solution Constituents: 1. Distilled water 1000.0 cc. 2. Potassium phosphate 0.5 g. 3. MgS04 0.03 g. 4. NaCl 0.5 g. 5. Na.CO, 1.0 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add one of the added nutrients as indicated under added nutrients. Sterilization: The various media under 'added nutrients' are sterilized differently. The method for each medium will be given with the nutrient. Use: To study nitrate and nitrite produc- tion from organic nitrogen. Author re- ported that in the concentrations used all tests for NH3, nitrites or nitrates were negative. Nessler's reagent was used to determine ammonia amyl iodine reaction for nitrite and diphenylamine for nitrate. Added nutrients: The author added one of the following materials to the basic solu- tion: (a) Asparagin 1.0%. Filter through a Chamberland filter to sterilize. (b) Urea 1.0%. Filter through a Cham- berland filter to sterilize. (c) Bouillon 50.0 cc. Both basic solution and bouillon to be sterile. Method of sterilization not given. (d) Egg white 1.0%. Sterilize in the steamer. (e) Fresh urine 50.0 cc. Filter through a Chamberland filter to sterilize. The urine gave a slight ammonia test with Nessler's reagent. To remove all ammonia add 1.0% soda to the fresh urine, filter and store the filtrate for 2daysat25°C. overHsSOi. Then fil- ter through a Chamberland filter as above to sterilize. Reference: Omeliansky (1899 p. 481). 12. Percival's Basal Solution Constituents : 1. Distilled water 1000.0 cc. 2. K2HPO4 5.0 g. 3. MgS04 2.5 g. 4. NaCl 2.5 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add 10.0 g. of one of the added nu- trients. Sterilization : Not specified. Use: To demonstrate ammonia production from protein. Added nutrients : (a) Percival added 1.0% of fibrin or gelatin. (b) Harvey added 10.0 g. of one of the following materials: peptone, fibrin, gelatin. (c) Omeliansky used 1.0 g. potassium phosphate, 0.5 g. MgSO., 0.1 g. NaCl in the basic solution and added one of the listed proteins. He studied the production of aroma by Bad. ester oar omaticum. Growth with aroma production took place with all materials except casein and keratin. One of the following materials was added: egg albumin (Kahlbaum) 10.0 g. blood albumin (Kahlbaum) . . . 10.0 g. peptone (Okuney) 10.0 g. casein (Hammorsten) (Merck). 10.0 g. keratin (Zyzkia, Merck) 10.0 g. References: Percival (1920, p. 112) Harvey (1921-22, p. 102), Omeliansky (1923, pp. 409, 411). 13. Nawiasky's Basal Salt Solution Constituents : 1. Water 1000.0 cc. 2. NaCl 5.0 g. 3. K2HPO4 2.0 g. 4. MgS04 0.5 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Neutralize and make slightly alkaline (3) Add one of the organic materials listed in added nutrients to (2). Sterilization: Method not given. Use: Cultivation of Proteus vulgaris. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS Added nutrients: asparagin 5.0 g glycocoll 4.0 g alanin 6.25 g isobutylaldehyde 1.6 g leucine 2.0 g aspartic acid 4.5 g Reference: Nawiasky (1908, p. 215). 14. Stoklasa's Basal Solution Constituents : 1. Water (River, Tap) 1000.0 cc. 2. Dipotassium phosphate 0.5 g. 3. CaCOs 1.0 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Add suitable carbon source. (3) Distribute in large Erlenmeyer flasks (40 cm. X 15 cm.) Sterilization: Sterilize in streaming steam or in the autoclave. Uses: To study nitrogen assimilation by Azotobacter and Radiobacter as in- fluenced by various carbohydrates. Added nutrients: The following carbohy- drates were suggested by the author to serve as carbon sources: l-arabinose saccharose 1-xylose rhamnose d-glucose lactose d-galactose maltose d-fructose Reference: Stoklasa (1908 p. 491). 15. Heinze's Basal Solution Constituents : 1. Distilled water 1000.0 cc. 2. CaCh 0.2 g. 3. MgS04 0.4 g. 4. KH2PO4 2.0 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add 1.0% of one of the listed added nutrients (3) Adjustment of reaction not specified. Sterilization : Not specified. Use : To study oxalic acid formation. Oxa- lic acid was formed in quite small amounts. Heavy formation of HNO3 and ammonia occurred. Added nutrients: The author employed 1.0% of one of the following: peptone hemialbumose gelatin Reference: Heinze (1905, p. 18). 16. Sohngen's Basal Solution Constituents : 1. Water 75.0 cc. 2. K2HPO4 0.05 g. 3. CaCOa Preparation : (1) Dissolve 2 in 1 and add 3. (2) Place in a liter Erlenmeyer flask. (3) Add suitable carbon source. Sterilization : Method not specified. Uses: To study the effect of filter paper (colloid) on bacterial processes, particu- larly fixation of atmospheric nitrogen. Added Nutrients: The author employed the following: (a) 1.0 g. glucose + 0.1 g. CaCOs (b) 1.0 g. glucose + 0.1 g. CaCOs + 10.0 g. filter paper cut in squares of 1 sq. cm. (c) 1.0 g. mannitol + 0.1 g. CaCOa (d) 1.0 g. mannitol + 0.1 g. CaCOj + 10.0 g. filter paper Reference: Sohngen (1913, p. 628). 17. van Delden's Basal Gypsum Solution Constituents : 1. Water 1000.0 cc. 2. Gypsum 0.4 g. 3. K2HP04 0.5 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Additional nutrients are added as in- dicated below. Sterilization: Not specified. Use: To study sulphate reduction by Mi- crospira desulfuricans. Added nutrients and variants : The author used one of the following combinations: (a) Added 0.5 g. glucose and 0.5 g. pep- tone. (b) Added 1.0 g. glucose, 1.0 g. CaCO,, specified the use of tap water and used 1.2 g. gypsum. (c) Specified the use of tap water, added 0.5 g. NH4CI, added 0.5 g. glucose and used 1.2 g. gypsum. (d) Specified the use of tap water, added 0.5 g. sodium acetate, 0.5 g. asparagin and used 1.2 g. gypsum. (e) Specified the use of tap water, added 1.0 g. asparagin, used 1.2 g. gypsum and added 1.25 g. of sodium succinate or potassium succinate or calcium citrate or potassium tartrate or po- tassium malate. 8 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS (f) Specified the use of tap water, added 1.0 g. asparagin and used 1.2 g. gyp- sum. Reference: van Delden (1903-04, p. 83). 18. Czapek's Basal Solution (Waksman) Constituents : 1. Distilled water 1000.0 ce. 2. K2HPO4 1.0 g. 3. KCl 0.5 g. 4. MgS04 0.5 g. 5. FeS04 0.01 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. Sterilization: As desired. Uses: Used by many investigators as a basic mineral or salt solution for com- parison of availability of various nitrogen and carbon nutrients. Reference: Waksman (1918, p. 479). 19. Buchanan's Basal Solution Constituents: 1. Water 1000.0 cc. 2. KH.PO4 2.0 g. 3. MgS04 (0.01%) 0.1 g. Preparation : (1) Dissolve 2, 3 and one of the added nutrients in 1. (2) Cool on ice. (3) Filter to remove insoluble precipi- tates. (4) Tube. Sterilization: Intermittently in flowing steam on each of 3 successive days. Use : To study growth of Bacillus radicicola bacteroids, and to study gum production by Bacillus radicicola. Author reported that generally carbohydrates and gluco- sides favored, while peptones inhibited the growth of B. radicicola; mannitol especially favored growth. Added nutrients: One of the following materials or combinations was added: Arabinose 2.0% Rhamnose (Isodulcitol) 2.0% Glucose 2.0% 2.0% + peptone 1.0% Mannose 2.0% Galactose 2.0% Levulose 2.0% Sucrose 0.1, 10.0, 20.0, 30.0, or 50.0% Sucrose 2.0% + peptone 1.0% Sucrose 2.0% + KNO3 0.05 to 0.5% Sucrose 2.0% + nutrose 1.0% Maltose 2.0% + sodium succinate 1.0% Maltose 2.0% + sodium asparaginate 1.0% Maltose 2.0% + ammonium citrate 1.0% Maltose 2.0% + peptone 1.0% Maltose 2.0% + asparagin 1.0% Raffinose 2.0% Melitose 2.0% Inulin 2.0% Glycerol 1.0, 3.0 or 5.0% Glycerol 1.0, 3.0 or 5.0% + ammonium phosphate 0.5% Inositol 2.0% Mannitol 2.0% Ammonium formate 1.0%, 2.0% or 5.0% Ammonium Tartrate 1.0% Ammonium citrate 1.0% Sodium, potassium or calcium butyrate 0.5%, 1.0% or 2.0% Sodium butyrate (0.5%) 5.0 g. + ammo- nium phosphate 0.5% Calcium butyrate (1.0%) 1.0 g. + am- monium phosphate 0.5% Potassium valerianate 0.5%, 1.0% or 2.0% Sodium succinate 0.5%, 1.0% or 2.0% Sodium succinate 1.0% + ammonium phosphate 0.5% Calcium lactate 0.5%, 1.0% or 2.0% Calcium lactate 1.0% + ammonium phos- phate 0.5% Sodium citrate 1.0% Sodium citrate 1.0% + ammonium phos- phate 0.5% Asparagin 1.0% Asparagin 1.0, 2.0, 3.0 or 5.0% + am- monium phosphate 0.5% Asparagin 1.0% + sodium asparaginate 1.0% Sodium asparaginate 1.0, 3.0 or 5.0% + ammonium phosphate 0.5% Sodium asparaginate 0.5, 1.0,3.0 or 5.0% Nutrose 0.1, 0.5, 1.0, 2.0 or 5.0% Nutrose 1.0% + ammonium tartrate 0.5% Nutrose 1.0% + ammonium phosphate 0.5% Nutrose 1.0% + asparagin 1.0% Peptone (Witte) 1.0% Peptone (Witte) 1.0% + Ammonium phosphate 0.5% Peptone (Witte) 1.0% + sodium aspar- aginate 1.0% Peptone (Witte) 1.0% + asparagin 1.0% CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS Peptone (Witte) 1.0% + KNO3 0.1 or 1.0% Amygdalin 2.0% Amygdalin 2.0% + ammonium phos- phate 0.5% Salicin 2.0% + ammonium phosphate 0.5% Salicin 2.0% Ammonium phosphate References: Buchanan (1909, pp. 382, 391, 62), Tanner (1909, p. 57) 20. Fuhrmann's Basal Solution Constituents : 1. Water 1000.0 cc. 2. KH2PO4 1.0 g. 3. CaCla 0.1 g. 4. MgS04-7H20 0.3 g. 5. NaCl 0.1 g. 6. FesCle 0.01 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Distribute in 100.0 cc. lots. Sterilization: Method not specified. TJses: To study the availability of various nitrogen and carbon sources for Pseudo- monas cerevisiae. Added Nutrients : Make the following addi- tions to each 100.0 cc: (a) Asparagin 1.0 g. (b) Asparagin 1.0 g. + glucose 1.5 g. (c) Asparagin 1.0 g. + sucrose 0.75 g. (d) Potassium nitrate 1.0 g. + glycerol 1.0 g. (e) Potassium nitrate 1.0 g. + sucrose 0.75 g. (f) Potassium nitrate 1.0 g. + glucose 1-5 g. (g) Ammonium chloride 1.0 g. + sucrose 0.75 g. (h) Ammonium chloride 1.0 g. + glucose 1.5 g. Reference: Fuhrmann (1906, p. 319). 21. Miinter's Basal Salt Solution Constituents : 1. Water 1000.0 cc. 2. MgSOi 0.5 g. 3. NaCl 0.5 g. 4. CaCl2 0.1 g. 5. K2HPO4 1.0 g. 6. FeCls trace Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Neutralize with CaCOs and filter. (3) Distribute in 100.0 cc. lots in Erlen- meyer flasks. (4) Add 0.5 g. of one of the added nu- trients to each flask. Sterilization: Not specified. Use: To study the availability of nitrogen for Actinomycetes odorifer, Act. chromo- genes, Act. albus I and II, Act. S. a, b, and c. Good growth was obtained with albumin, hemi albumin, casein, asparagin, and alanine; little growth obtained with tyrosin, while generally no growth with urea, sulphocarbamide, or dicyandiamide. Added nutrients: The author employed 0.5 g. of one of the following nitrogen sources for each 100.0 cc. of medium: albumin urea hemi albumin sulphocarbamide casein alanine asparagin tyrosine dicyandiamide Reference: Munter (1913 p. 373). 22. Meyer's Basal Salt Solution (Perotti) Constituents : 1. Distilled water 1000.0 cc. 2. K2HPO4 10 g. 3. CaCh 0.1 g. 4. MgS04 0.1 g. 5. xNaCl 0.1 g. 6. Iron chloride 0.01 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6 in 1. Sterilization: Not specified. Uses: Used by Perotti as a basis for the preparation of various solutions for the study of the dicyandiamid bacteria of the soil. Variants : (a) Buchanan used a basal solution con- taining the following materials: 1. Water 1000.0 cc. 2. KH0PO4 0.1 g. 3. CaCl. 0.1 g. 4. MgS04-7H20 0.3 g. 5. NaCl 0.1 g. 6. Fe2Cla 0.01 g. Buchanan added one of the following materials, and used the media to deter- mine the constituents essential for the growth of Bacillus thermophilus. Growth was obtained with all materials except as- paragin alone. asparagin 1.0% asparagin 1.0% + glycerol 1.0% 10 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS asparagin 1.0% + filter paper 1.0% urea 1.0% + peptone 1.0% peptone 1.0% + glucose peptone 1.0% + lactose 1.0% peptone 1.0% + sucrose 1.0% (b) Klaeser added 2.0g KNO3 and lO.Og. of glucose. He used the solution to study nitrate reduction. (c) Stapp used l.Og HK2PO4 instead of KH2PO4. He dissolved 3.0g Na2HP04 and 0.5 g uric acid in 45.0 cc. water and added 50.0 cc. of the basal solution. The medium was used for the isolation of uric acid splitting bacteria from the feces and soil. The organisms studied were Bad. co- bay ae, Bad. capri, Bad. guano, Bad. muscili, Bad. hoUandicus. Reference : Buchanan (1906 p. 73). Perotti (1908 p. 220), Klaeser (1914 p. 38a) Stapp (1920 p. 3). 23. Box's Inorganic Salt Solution For Fungi (Tanner) Constituents : 1. Distilled Water 3000.0 cc. 2. MgS04 1.5 g. 3. K2HPO4 3.0 g. 4. KCl 1.5 g. 5. FeS04 0.03 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Sterilization : Not specified. Uses: Cultivation of fungi. Reference: Tanner (1919 p. 65). 24. Gage's Basal Salt Solution Constituents : 1. Water 1000.0 cc. 2. KH2PO4 0.5 g. 3. MgS04 0.2 g. 4. CaCl2 0.02 g. 5. Fe2(S04)3 1 drop of 10% solution. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add 1.5 g. of suitable carbon source. Sterilization: Method not specified. Uses : Study of nitrogen fixation and of the morphology of the nitrogen fixing bacteria. Added nutrients: The author suggests the addition of 1.5 g. of one of the following carbon sources: Mannitol Galacton from slip- Glucose pery elm. Dulcitol Pentosan from dulse Sorbitol Sinistrin Sucrose Lichenin Galactose Maltose Mannan from Salep Reference: Gage (1910 p. 21). 25. Beijerinck and van Delden's Basal Solution Constituents : 1. Water 1000.0 cc. 2. MgS04.7H20 8.0 mg. 3. MnS04.4H20 0.5 mg. 4. FeCl3.3H20 0.5 mg. Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization: Not specified. Uses: Used by Beijerinck and van Del- den as a mineral mixture to be added in small quantities to a considerable variety of liquid media. Reference: Beijerinck and van Delden (1903 p. 41). SUBGROUP I-B. SECTION 2 Inorganic liquid media of known composi- tion, nitrogen as free nitrogen only, com- plete solutions, used as media without addition of other nutrients. "Complete" inorganic non-nitrogenous media have been described for the cultiva- tion of certain iron bacteria and for certain algae. In each case it will be noted that either "well water" or a heavy inoculum of soil or soil extract is used. It is probable therefore that ammoniacal or nitrate nitro- gen is usually present, and may in some cases be essential for the growth of the mi- croorganisms. The important media of this section may be differentiated as follows: Ai. Medium employed primarily for iron bacteria. Ellis' Ferric Hydroxide Solution 26 A2. Media for algae. (Cyanophyceae or blue-green). Beijerinck's Basal Phosphate Solution. 27 Heinze's Basal Solution A 28 Heinze's Basal Solution B 29 26. Ellis' Ferric Hydroxide Solution Constituents: 1. Well water. 2. Ferric hydroxide. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 11 Preparation: (1) Add freshly precipitated ferric hydroxide (amount not specified) to sterile well water in sterile flask. Uses: Inoculate with Spirophyllum fer- rugineum (one of the iron bacteria) and expose to sunlight. Reference: Ellis (1907 p. 511). 27. Beijerinck's Basal Phosphate Solution Constituents : 1. Water 100.0 cc. 2. K2HPO4 0.02 g. Preparation : (1) Dissolve 2 in 1. (2) Place in a 3 liter flask and plug with cotton. Sterilization : Not specified. For purposes used, probably generally unnecessary. Uses: Add an inoculum of 1.0 or 2.0 g. garden earth and incubate at 16°-20°C. (In the light?) Oligonitrophilic organ- isms of the Cyanophyceae (blue-green algae) will develop, the medium becom- ing bluish green. Variants: van Delden specified the use of Grabenwasser." Reference: Beijerinck (1901 p. 562), van Delden (1903-04 p. 85). Lohnis (1913 p. 115). 28. Heinze's Basal Solution A Constituents : 1. Distilled water 1000.0 cc. 2. KHsPOi 2.0 g. 3. CaCla 0.2 g. 4. FeCls in 10% solution... 20 drops. Preparation : (1) Dissolve 2, 3 and 4 in 1. If K3PO4 is used, the solution may be acidified with sulphuric acid and neutralized with soda. (2) Distribute in 100 cc. lots. Sterilization: Not specified. Uses : Inoculate with 1.0 cc. of a suspension of 1.0 g. of soil in 100.0 g. sterile distilled water. Incubate (in the light?). The soil Cyanophyceae (blue green algae) will develop. Nitrogen may possibly be assimilated (fixed) in small amounts. Variants: The author substituted K2HPO4 or K3PO4 for KH2PO4. Reference: Heinze (1906 p. 703). 29. Heinze's Basal Solution B Constituents : 1. Water 1000.0 cc. 2. Potassium phosphate neu- tral 0.2 g. 3. MgS04 0.2 g. 4. K2SO4 0.2 g. 5. CaCOa 0.1 g. 6. Iron chloride trace Preparation: (1) Dissolve 2, 3, 4, 5 and 6 in 1. Sterilization: Not specified. Uses: Inoculate with a suspension of soil. Soil algae, such as Nostoc punctiforme will develop. If soil bacteria are present Nostoc punctiforme will grow and nitro- gen will be assimilated. Reference: Heinze (1906 p. 653). SUBGROUP I-B. SECTION 3 Inorganic liquid solutions of known com- position; nitrogen supplied as ammonium salts; incomplete or basal solutions re- quiring the addition of other nutrients. The several solutions falling in this sec- tion may be differentiated as follows: Ai.* Containing ammonia as ammonium chloride. Boas' Basal Ammonium Chloride Solu- tion 30 Wherry's Basal Ammonium Chloride Solution B 31 Kendall, Walker and Day's Basal Am- monium Chloride Solution 32 Lohnis' Basal Ammonium Chloride Salt Solution 33 A2. Containing ammonia as ammonium sulphate. Bi. Not containing additional salts. Buchanan's Basal Ammonium Sul- phate Solution 34 Cathelineau's Basal Ammonium Sul- phate Solution 35 B2. Containing additional salts. Pere's Basal Ammonium Sulphate Solution 36 Henneberg's Basal Ammonium Sul- phate Solution 37 Omeliansky's Basal Ammonium Sul- phate Solution 38 See A2, A3, A4 and A5 12 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS Kita's Basal Ammonium Sulphate Solution 39 Grimm's Basal Ammonium Sulphate Solution 40 Waksman and Joffe's Basal Sulphur Ammonium Sulphate Solution 41 Bokorny's Basal Ammonium Sulphate Solution, (Vierling) 42 Verkade and Sohngen's Basal Ammo- nium Sulphate Solution 43 Starkey's Basal Ammonium Sulphate Solution 44 A3. Containing ammonia as salts of phos- phoric acid. Ayers and Rupp's Basal Sodium Am- monium Phosphate Solution 45 Committee S. A. B. Basal Ammonium Phosphate Solution 46 Kendall, Day and Walker's Basal Am- monium Phosphate Solution 47 Laurent's Basal Ammoniima Phosphate Solution 48 Fermi's Basal Ammonium Phosphate Solution 49 Palladin's Basal Ammonium Phos- phate Solution 50 Schukow's Basal Ammonium Phos- phate Solution 51 Koser's Basal Ammonium Phosphate Solution 52 A4 Containing ammonia as ammonium nitrate. Munter's Basal Ammonium Nitrate Solution 53 von Bronsart's Basal Ammonium Ni- trate Solution 54 Adolf Mayer's Basal Ammonium Ni- trate Solution (Smith) 55 As. Containing ammonia as Ammonium Carbonate. Prazmowski's Basal Ammonium Car- bonate Solution (Smith) 56 30. Boas' Basal Ammonitun Chloride Solution Constituents : 1. Water 1000.0 cc. 2. NH4CI (0.25%) 2.5 g. 3. KH2PO4 (0.25%) 2.5 g. 4. MgS04 (0.15%) 1.5 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add 5.0% of one of the carbohydrates. (3) Distribute in 40.0 cc. lots in 100.0 cc, Erlenmeyer jena glass flasks. Sterilization: Not specified. Uses : Used to study the formation of starch by molds, Aspergillus orijzae. If starch is formed, a blue color appears when iodine is added to the culture. Added nutrients: The author used 5.0% of one of the following carbon sources: sucrose glucose levulose galactose maltose Reference: Boas (1922 p. 8). 31. Wherry's Basal Ammonium Chloride Solution B Constituents: 1. Redistilled water 1000.0 cc. 2. NajCOa 1.0 g. 3. KCl 1.0 g. 4. CaCl2 1.0 g. 5. MgS04 1.0 g. 6. NH4CI 2.0 g. 7. KH2PO4 2.0 g. Preparation : (1) Dissolve 2, 3, and 4 in 1. This is called solution "B". (2) Dissolve 6 and 7 in (1). (3) Distribute in 50 cc. lots and add one of the test materials to each lot. Sterilization: Not specified. Use: To study acid fastness of tubercle bacilli. Growth started first on methyl alcohol. After 20 days the order of vigor of growth was as follows: Propyl, butyl, ethyl and methyl alcohol. Culture be- came strongly acid fast only in propyl alcohol. Added nutrients: The following carbon sources were used: ethyl alcohol 2.0% propyl alcohol 2.0% amyl alcohol 2.0% butyl alcohol 2.0% methyl alcohol 2.0% leucine 0.1 g. tyrosine 0.1 g. alanine 0. 1 g. glycocoU 0.1 g. aspartic acid 0.1 g. glutamic acid 0.1 g. (Salts to be Kahlbaum C. P.) Reference: Wherry (1913 p. 116). CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 13 32. Kendall, Walker and Day's Basal Am- monium Chloride Solution Constituents: 1. Redistilled water 1000.0 cc. 2. NH4CI 4.0 g. 3. NaCl 5.0 g. 4. Na2HP04 Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add one of the nutrients listed below. (3) Distribute in 100.0 cc. quantities in flasks. Sterilization: Method not given. Use: To study lipase production by tuber- cle bacilli. Different esters were added to clear bacteria free culture broth and incubated for 24 hours. Amount of acid produced measured in terms of N/50 NaOH, determined lipase production. Added nutrients: The authors used ethyl alcohol, glycerol or mannitol as carbon sources. Reference: Kendall, Walker and Day (1914 p. 455). 33. Lohnis' Basal Ammonium Chloride Solution Constituents: 1. Water (tap) 1000.0 cc. 2. NH4CI 0.5 g. 3. K2HPO4 0.5 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Add 1.0% of one of the listed nutrients Sterilization: Not specified. Use: Methane production by cellulose de- composers. Added nutrients: The author added 1.0% of one of the following materials: acetate starch butyrate sugar lactate peptone gum wool Reference: Lohnis (1913 p. 93). 34. Buchanan's Basal Ammonium Sulphate Solution Constituents: 1. Water 1000.0 cc. 2. (NH4)2S04 (0.5%) 5.0 g. Preparation : (1) Add 2.0% of one of the carbon sources to water and dissolve. (2) Then add 0.5% ammonium sulphate to (1). Sterilization: Sterilize on each of 3 succes- sive days for 20 minutes in flowing steam. Use : To study gum production of Bacillus radicicola. Added nutrients: The following carbon sources (2.0%) were used by the author: arabinose levulose lactose rhamnose melitose raffinose glucose inositol mannitol mannose sucrose inulin galactose maltose starch Reference: Buchanan (1909 p. 388). 35. Cathelineau's Basal Ammonium Sul- phate Solution Constituents : 1. Water 100.0 cc. 2. (NH4)2S04 1.0 g. Preparation : (1) Dissolve 2 in 1. (2) Add one of the following to (1). (a) Glucose 5.0 g. (b) Sodium succinate 5.0 g. (c) Sodium phosphate 5.0 g. (d) Sodium succinate 5.0 g. Glucose 5.0 g. (e) Glucose 5.0 g. Sodium phosphate 5.0 g. (f) Sodium succinate 5.0 g. Sodium phosphate 5.0 g. Sterilization: Method not given. Use: To study pigment production and fluorescence by Bacillus viridis (Lesage). Pigment production was independent of the amount of phosphates present. Added nutrients: The author added the following carbon sources and salts: (a) Glucose 5.0 g. (b) Sodium succinate 5.0 g. (c) Glucose 5.0 g. Sodium succinate 5.0 g. (d) Glucose 5.0 g. Sodium phosphate 5.0 g. (e) Sodium succinate 5.0 g. Sodium phosphate 5.0 g. Reference: Cathelineau (1896 p. 235). 36. Pere's Basal Ammonium Sulphate Solution Constituents : 1. Water 100.0 cc. 2. Ammonium phosphate 1.0 g. 14 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 3. (NH4)2S04 0.5 g. 4. Potassium phosphate 0.25 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add one of the test materials in amounts given to (1). (3) The reaction must be neutral. If acid after sterilization, add a few- drops of ammonia. Sterilization: Method not given. Use: To study the decomposition of car- bonaceous bodies (polyatomic alcohols, starch, etc.) by Tyrothrix tenuis, Bacilhis mesentericus vulgatus and Bacillus sub- tilis. Added nutrients: The author used the following substances: Mannitol 10.0 g. Glycerol 5.0 g. Glycerose, amount not given Potato starch 2.0 g. Sucrose 5.0 g. Glucose 5.0% = 5.0 g Reference: Per6 (1896 p. 421). 37. Henneberg's Basal Ammonium Sul- phate Solution Constituents: 1. Water 1000.0 cc. 2. KH2PO4 3.0 g. 3. (NH4)2S04 3.0 g. 4. MgS04 2.0 g. 5. Carbon source. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Do not adjust the reaction. (3) Add any desired carbon source. Sterilization: Not specified. Use : To study a suitable carbon source for acetic acid organisms, B. pasteurianum, B. aceti, B. oxydans and B. Kutzingianum. Glucose was found to be a suitable carbon source, but methyl, ethyl or propyl alco- hol, acetic acid or ammonium tartrate were not suitable sources of carbon. Reference: Henneberg (1898 p. 18). 38. Omeliansky's Basal Ammonium Sul- phate Solution Constituents: 1. Distilled water 1000.0 cc. 2. Potassium phosphate 0.1 g. 3. (NH4)2S04 0.1 g. 4. MgS04 0.05 g. 5. NaCl Trace 6. Chalk Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Prepare a 1.0% solution of one of the test materials. (3) Distribute in test tubes. (4) Add chalk to each tube. Sterilization: Not specified. Use: To study the fermentation ability of Bacterium for micicum under either anaero- bic or aerobic conditions. Added nutrients : The author used 10 grams of the following carbon sources: Glucose Dextrin Sucrose Amylum Galactose Inulin Lactose Arabinose Mannitol Gum arable Dulcitol Ethylene glycol Maltose Glycerol Erythritol Reference: Omeliansky (1903-4 p. 258). 39. Kita's Basal Ammonium Sulphate Solution Constituents : 1. Water 1000.0 cc. 2. MgS04 0.25 g. 3. KH2PO4 5.0 g. 4. FeCla solution drops 5. (NH4)2S04 5.0 g. Preparation : (1) Dissolve 2, 3 and 5 in 1. (2) Add several drops of a ferric chloride solution to (1). (3) Add 50.0 g. of any desired carbohy- drate to (2). Sterilization: Not specified. Use: To study utilization of various carbon sources by molds, Aspergillus okazaki, Aspergillus candidus, Aspergillus albus, Aspergillis lamarii, Pseudorhizopus, As- pergillus glaucus. Reference: Kita (1913 p. 434). 40. Grimm's Basal Ammonium Sulphate Solution Constituents : 1. Distilled water 1000.0 cc. 2. K2HPO4 0.5 g. 3. MgS04 0.5 g. 4. (NH4)2S04 5.0 g. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 15 Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Distribute in 30.0 cc. lots in 200.0 cc. Erlenmeyer flasks. (3) After sterilization add one of the test materials to (2). (Amount not speci- fied). Sterilization: Not specified. Use: To study the utilization of various carbon sources by Oospora lactis and Aspergillus repens. Growth with ethyl alcohol, isobutyl alcohol, acetic acid, ethyl ether, propyl, isopropyl and amyl formic acid, methyl, ethyl, propyl, iso- propyl, isobutyl and amyl acetic acid. Other materials give very little or no growth. Added nutrients: The author used the following carbon sources: Pentan Heptan Hexan Octan Methyl alcohol Ethyl alcohol Propyl alcohol Isopropyl alcohol Butyl alcohol Isobutyl alcohol Amyl alcohol Formic acid Acetic acid Propionic acid Butyric acid normal Isobutyric acid Valeric acid Ethyl ether Propyl ether Formaldehyde Acetaldehyde Propionic aldehyde Methyl formic acid Ethyl formic acid Propyl formic acid Isobutyl formic acid Methyl acetic acid Ethyl acetic acid Propyl acetic acid Isobutyl acetic acid Methyl iodine Ethyl iodine Ethyl bromide Propyl bromide Chloroform Reference: Grimm (1914 p. 648). 41. Waksman and Joffe's Basal Sulphur Ammonium Sulphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. (NH4)2S04 2.0 g. 3. K2HP0< 1.0 g. 4. MgS04 0.5 g. 5. KCl 0.5 g. 6. FeS04 0.01 g. 7. Sulfur 10.0 g. 8. Ca3(P04)2, 2.5 or 10.0 g. Preparation : (1) Dissolve 2, 3, 4, 5, 6, 7, 8 and one of the added nutrients in 1. (2) Distribute in 100.0 cc. portions in 250.0 cc. flasks. Sterilization : Sterilize in flowing steam for 30 minutes on 3 consecutive days. Use: Enrichment and isolation of sulphur bacteria, Thiobacillus thiooxidans. If glucose be omitted fungi do not grow. Medium supports the growth of all sul- phur oxidizers. Added nutrients: (a) The authors added 10.0 glucose or any other organic or inorganic stimulator. (b) Used the basic solution without any additions. Reference: Waksman and Joffe (1922 p. 239). 42. Bokomy's (Vierling) Basal Ammonium Sulphate Solution Constituents: 1. Water 1000.0 cc. 2. (NH4)2S04 1-0 g. 3. KH2PO4 0.2 g. 4. MgS04 0.1 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add 2.0 g. of one of the carbon sources Sterilization: Not specified. Use: Author used the solution to study urease production. Added nutrients: The author added the following carbon sources: Glycerol Arabinose Ethyl alcohol Sorbose Lactose Xylose Galactose Mannose Rhamnose Reference: Vierling (1920 p. 31). 16 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 43. Verkade and Sohngen's Basal Ammo- nium Sulphate Solution Constituents : 1. Water 1000.0 cc. 2. K2HPO4 (0.05%) 0.5 g 3. MgS04 (0.05%) 0.5 g 4. FeCU (0.01%) 0.1 g 5. MnS04 (0.01%) 0.1 g 6. (NH4) 2SO4 (0.05%) 0.5 g 7. CaCOa Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Add 0.25% of one of the test materials and an excess of CaCOs. Sterilization: Autoclave at 110°C. for 10 minutes. Use: To study the availability of organic acids as a carbon source for Aspergillus niger and Pcnicillium glaucum. Fumaric, citric aconitic, oleic and erucic acids give good growth. Added nutrients: maleic acid fumaric acid citric acid itaconic acid phenylitaconic acid allocinnamic acid cinnamic acid aconite acid isocrotonic acid crotonic acid BB-dimethylacrylic acid angelic acid tiglic acid oleic acid elaidic acid erucic acid brassidic acid Variants: The authors substituted 0.05% KNO3 for (NH 4)280 4. Reference: Verkade and Sohngen (1920 p. 82). 44. Starkey's Basal Ammonium Sulphate Solution Constituents : 1. Distilled water 1000.0 cc. 2. (NH4)2S04 0.2 to 0.4 g. 3. KH2PO4 3.0 to 4.0 g. 4. CaCls 0.25 g. 5. MgS04 0.5 g. 6. FeSOi 0.01 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Add one of the added nutrients. Sterilization : Heat in flowing steam for 30 minutes on each of three consecutive days. Added nutrients : The author added one of the following: (a) Sulphur 1.0%. The sulphur was weighed out and placed in individual flasks, the basic solution added and sterilized. (b) Sodium thiosulphate 0.5%. The thiosulphate was dissolved in water, sterilized and added to the sterile basic solution in the necessary amounts. Use: Cultivation of Thiobacillus thioxidans and to study oxidation of sulphur. Reference: Starkey (1925 p. 138). 45. Ayers and Rupp's Basal Sodium Ammo- nium Phosphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. Sodium ammonium phosphate. . .1.5 g. Preparation : (1) Dissolve 2 in 1. (2) Add one of the combinations given under added nutrients. (3) Adjust to pH = 7.0 Sterilization: Not specified. Use: To study acid and alkali production. Added nutrients and modifications: The author added one of the following com- binations: (a) Glucose 5.0 g. citric acid (neutralized with NaOH) 2.5 g. KCl 0.2 g. (b) glucose 5.0 g. KCl 0.2 g. (c) citric acid (neutralized with NaOH) 2.5 g. KCl 0.2 g. (d) Used 3.6 g. sodium ammonium phos- phate in the basic solution and added KH2PO4 1.2 g. and glucose 5.0 g. To this solution was added 1.2 g. of one of the following acids neutralized with NaOH: formic acid, acetic acid, lactic acid, succinic acid. (e) Same as (d) without the addition of acids. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 17 Reference: Ayers and Rupp (1918 pp. 199, 209). 46. Committee S. A. B. Basal Ammonium Phosphate Solution Constituents : 1. Water 1000.0 cc. 2. (NH4)H2P04 1.0 g. 3. KCl 0.2 g. 4. Brom Cresol Purple (Satu- rated aqueous solution) 2.0 cc. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Adjust to pH = 7 by addition of NaOH. (3) Add fermentable substances. (4) Tube. Sterilization: Method not specified. Uses : To test the ability of microorganisms to produce acid in a solution having ammonia as the nitrogen source and a suitable fermentable carbon compound. Added nutrients: Suitable amounts of any of the carbohydrates or of the polyatomic alcohols, etc., may be used. Variant. Authors also prepared the me- dium as follows: (1) Dissolve 1.0 g. (NH4)H2P04, 0.2 g. NaCl, 0.2g.MgSO4 and 10.0 g. of one of the added nutrients in 1000.0 cc. water. (2) Adjust to pH = 7.0 by the addition of NaOH. (3) Indicator may be added: Litmus Phenol red, 2.0 cc. saturated aqueous solution per liter Brom cresol purple, 2.0 cc. saturated aqueous solution per liter Brom cresol purple, 1.0 cc. saturated aqueous solution per liter Cresol red, 1.0 cc. saturated aqueous solution per liter Brom cresol green 2.0 cc. of a 2.0% alcoholic solution per liter Brom-chlor phenol blue 5.0 cc. of saturated aqueous solution per liter Reference : Committee Society of American Bacteriologists (1922 p. 523), (1923 p. 11). 47. Kendall, Day and Walker's Basal Ammonium Phosphate Solution Constituents: 1. Redistilled water 1000.0 cc. 2. (xNH4)2HP04 4.0 g. 3. NaCl 5.0 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Add one of the added nutrients listed below. (3) Distribute in 100.0 cc. lots. Sterilization: Not specified. Use: To study metabolism and lipase pro- duction by tubercle bacilli. Authors used alizarin, neutral red and phenol- phthalein to study reaction changes. Ziehl-Nielsen stain for staining. Differ- ent esters were added to clear bacteria free culture broth, incubated for 24 hours and the amount of acid produced, (meas- ured in terms of N/50 NaOH) determined lipase production. Added nutrients: The authors added one of the following nutrients: mannitol 10.0 g. glycerol 30.0 g. sodium acetate Reference: Kendall, Day and Walker (1914 p. 434), Kendall, Walker and Day (1914 p. 455). 48. Laurent's Basal Ammonium Sulphate Solution Constituents: 1. Water 1000.0 cc. 2. Ammonium phosphate (neu- tral) 2.5 g. 3. Potassium phosphate (neu- tral) 2.5 g. 4. MgS04 1.0 g. Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization: As desired; not specified. Uses : Used with the addition of 1.0% of any one of a considerable variety of nitroge- nous and carbon compounds. Added nutrients : (a) The following were employed: saccharose potassium lactate lactose potassium citrate glucose potassium tartrate mannite ammonium bimalate glycerine sodium butyrate peptone sodium hippurate asparagin sodium formate potassium sue- potassium acetate cinate These were used by Laurent in his 18 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS studies of Bacillus fluorescens putidus. (b) Laurent cultivated Sclerotinia Liber- tiana using the basic solution and adding 1.0% peptone, 2.5% lactose, 2.5% glycerol and 2.5% potassium tartrate. Variants : Smith cultivated plant parasites on a medium composed of 2.0 g K2HPO4, 0.1 g ammonium phosphate, 0.19 MgS04, and used 5.09 sodium formate or acetate as an added nutrient. Reference: Laurent (1899 p. 43, 81). Smith (1905 p. 50). 49. Fermi's Basal Ammonium Phosphate Solution Constituents : 1. Water 1000.0 cc. 2. Ammonium Phosphate (0.5 to 1.0%) 5.0 to 10.0 g. 3. KH2PO4 (0.5%) 5.0 g. 4. MgS04 (0.5%) 5.0 g. 5. K3PO4 (0.05%) 0.5 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add one of the added nutrients. Sterilization: Not specified. Use: Cultivation of schizomycetes. Added nutrients: The author added 5.0% glycerol or 5.0% sucrose. Variants: Tanner gave the following as Fermi's Solution: 1. Distilled water 1000.0 cc. 2. MgS04-7H20 0.2 g. 3. K2HPO4 1.0 g. 4. (XH4).HP04 10.0 g. 5. Glycerol 45.0 g. Reference: Fermi (1892 p. 26), Tanner (1919 p. 68). 50. Palladin's Basal Ammonium Phosphate Solution Constituents : 1. Water 1000.0 cc. 2. Ammonium phosphate 4.7 g. 3. Potassium phosphate 3.0 g. 4. MgS04 1.0 g. 5. CaClj 1.0 g. 6. FeCl2 Trace Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Add sufficient carbohydrate to make solution one fourth molar. (3) Distribute in thin layers in Erlen- meyer flasks. Sterilization : Method not specified. Use: Cultivation of Chlorothecium sac- charophilnm. Variants: The author suggests the follow- ing carbon sources: saccharose glucose mannitol raffinose Reference: Palladia (1903 p. 146). 51. Schukow's Basal Ammonium Phosphate Solution Constituents : 1. Water 1000.0 cc. 2. Ammonium phosphate 5.0 g. 3. Potassium phosphate 1-0 g. 4. MgS04 0.5 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add 9.0 to 10.0% of one of the organic acids to (1). (3) Adjustment of reaction not specified. (4) Distribute into fermentation flasks (sealed with H2SO4). (5) After inoculation plug the flask with a cork and seal with paraffin. Sterilization: Sterilize in a steamer (method not given). Use: To study the utilization of acids by yeast. The author found 0.1 g. tartaric, 0.15 g. malic and 0.26 g. citric acid was used in 65 days by the yeast. Acid de- terminations were made by titration with normal NaOH, using litmus as an indi- cator. Added nutrients and variants : (a) The author added 9.0 to 10.0% of one of the following organic acids: tartaric malic citric (b) Henneberg specified the use of 0.3% KH,P04 and used 0.1% MgS04 in- stead of 0.05%. He added 5.0% glu- cose and used it for the cultivation of lactic acid bacteria. Reference: Schukow (1896 p. 607), Henne- berg (1903 p. 7). 52. Koser's Basal Ammonium Phosphate Solution Constituents : 1. Distilled water 1000.0 cc. 2. NaCl 5.0 g. 3. MgS04-7H20 0.2 g. CULTURE MEDIA FOR CULTIVATIOX OF MICRO ORGAXISMS 19 4. (NH4)H2P04 1.0 g. 5. K,HP04 1.0 g. Preparation : (1) Dissolve 2, 3, 4, 5 and one of the test materials in 1. Solution is clear at pH = 6.7 to 6.8. (2) Add one of the test materials. Add the different organic acids to (1) in such amounts so that when brought to pH reading of 6.8 by addition of NaOH the resulting concentration of the Na salt will be approximately 0.2%. (3) Tube in 8 to 10 cc. lots. Sterilization: Autoclave at 15 pounds pres- sure for 15 minutes. Use: To study the utilization of organic salts and acids by the colon-aerogenes group. Glucose and lactose were added as controls to determine if the solution would support the growth of the organism. Added nutrients: The author added ap- proximately 0.2% of the sodium salt of the following acids. In case of citric acid 0.1 to 0.5% was used, glucose 0.2% malic lactose 0.2% lactic sodmm salt of one of glyceric the organic acids citric acetic tartaric propionic mucic n-valeric malonic n-butyric oxalic iso-valeric benzoic n-caproic salicylic succinic o-phthalic Reference: Koser (1923 p. 497). 53. Miinter's Basal Ammonium Nitrate Solution Constituents: 1. Water 1000.0 cc. 2. MgS04 0.5 g. 3. NaCl 0.5 g. 4. CaCl. 0.2 g. 5. K2HPO4 1.0 g. 6. NH4NO3 7. CaCOa Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Neutralize by the addition of CaCOs. (3) To each 100.0 cc. of (2) add 0.025 g. of nitrogen in the form of XH4NO3. (4) Add 1.0 g. of one of the test materials to (3). Sterilization: Not specified. Uses: To study the availability of various carbon sources for Actinomyces odorifer, Act. chromogenes, Act. albus and others. Glycerin laevulose, dextrose, galactose, mannite and starch were good carbon sources for all the actinomycetes studied. The remaining carbohydrates serve as suitable carbon sources for some of the actinomyces but not all. Oxalic acid, tartaric acid, and hippuric acid are not suitable carbon sources for the actinomy- cetes studied. The remaining acids are generally fair carbon sources for most of the actinomycetes studied. Added nutrients: The author used the following carbon sources: sucrose glucose mannitol arabinose galactose laevulose lactose glycerol inulin starch Variants: The author omitted the CaCU and CaCOs and used one of the following carbon sources. The acid was neutral- ized and made slightly alkaline by the addition of Na.COs or CaCOs- 0.025 g. nitrogen in the form of NH4NO3 and 0.25 g. of the test material was added to each 50.0 cc. of medium. oxalic acid zitric acid acetic acid hippuric acid succinic acid uric acid malic acid humus acid tartaric acid aspartic acid Reference: Miinter (1913 pp. 368, 371). 54. von Bronsart's Basal Ammonium Nitrate Solution Constituents : 1. Water 1000.00 cc. 2. iMgS04 (0.25%) 2.5 g. 3. KH2PO4 (0.25%) 2.5 g. 4. NH4NO3 (1.0%) 10.0 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Add one of the test materials to (1). Sterilization: Not given. Use: To study utilization of various car- bon sources by Xylaria, Xylaria arhua- cula, Xylaria polymorpha, Xylaria hy- poxylon. Growth best using starch as a carbon source with Xylaria arbuscula, and levulose produces best growth with 20 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS Xylaria polytnorpha, Xylaria hypoxylon growth very poor in any of the solutions. Added nutrients : The author used the fol- lowing carbon sources: Starch levulose inulin glucose dextrin mannitol Reference: von Bronsart (1919 p. 57). 55. Adolf Mayers' Basal Ammonium Nitrate Solution (Smith) Constituents: 1. Distilled water 1000.0 cc. 2. MgS04 10.0 g. 3. NH4NO3 15.0 g. 4. Ca3(P04)2 0.1 g. 5. Potassium phosphate 10.0 g. Preparation : (1) Dissolve 2, 3, 4, 5 and one of the added nutrients in 1. Sterilization: Not specified. Use: Study of phosphorescent and acid forming bacteria. Variants: Add 3.0% NaCl if luminous bac- teria are to be cultivated, and an excess of pure CaCOs if acid forming bacteria are to be grown. Added nutrients : Smith added any desired carbohydrate, alcohol, etc. Reference: Smith (1905 p. 197). 56. Prazmowski's Basal Ammonium Car- bonate Solution (Smith) Constituents : 1. Distilled water 1000.0 cc. 2. K2HPO4 5.0 g 3. MgS04 5.0 g. 4. (NH4)2C03 5.0 g. 5. CaCl, 0.5 g. Preparation : (1) Dissolve 2, 3, 4, 5 and one of the added nutrients in 1. Sterilization: Not specified. Use: Study of phosphorescent and acid forming bacteria. Added nutrients : Smith added any desired carbohydrate, alcohol, etc. Reference: Smith (1905 p. 197). SUBGROUP I-B. SECTION 4 Inorganic liquid media of known composi- tion; nitrogen supplied as ammonium salts; complete nutrient solutions primarily for study of organisms oxidizing ammonia to nitrites. Ai.* Ammonia present as ammonium chlo- ride. Hewlett's Ammonium Chloride Solution (Johnson) 57 Beijerinck and Minkman's Ammonium Chloride Solution 58 Aj. Ammonia present as ammonium car- bonate. Prazmowski's Ammonium Carbonate Solution. (Tanner) 59 A3. Ammonia present as ammonium sul- phate. Bi. Without additional salts. Wimmer's Ammonium Sulphate Solu- tion 60 B2. With other salts. Ci. Magnesium carbonate added. Di. Phosphates not added. Winogradsky's Ammonium Sulphate Solution (Harvey) 61 D2. Phosphates added. El. All additional salts of mono valent cations. Buhlert and Fickenday's Ammonium Sulphate Solution 62 E,i. Additional salts of mono and di valent cations. Fi. Calcium salts present. Winogradsky's Ammonium Sulphate Solution 63 Giltner's Modified Winogradsky's Am- monium Sulphate Solution 64 F2. Calcium salts not present. Winogradsky and Omeliansky's Am- monium Sulphate Solution 65 E3. Containing Additional salts of tri- valent cations. Gibbs' Ammonium Sulphate Solution. (modified by Gowda) 68 C2. Magnesium carbonate not added. Di. Calcium carbonate, chalk or lime added. El. Magnesium sulphate added. Omeliansky's Ammonium Sulphate Solution. (Harvey) 67 E2. Magnesium sulphate not added. Christensen's Ammonium Sulphate Solution 68 D2. Calcium carbonate chalk or lime not added. Gage's Ammonium Sulphate Solu- tion 69 See A2 A3 and A4. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 21 Omeliansky's Ammonium Sulphate Solution. (Lohnis-Arnd) 70 Lolinis' Ammonium Sulphate Solu- tion 71 A4. Ammonia present as a salt of phos- phoric acid. Stutzer's Ammonium Magnesium Phosphate Solution 72 57. Hewlett's (Johnson) Ammonium Chlo- ride Solution. Constituents : 1. Distilled water 1000.0 cc. 2. NH4CI 0.5 g. 3. Potassium phosphate 0.1 g. 4. MgS04 0.02 g. 5. CaCl. 0.01 g. 6. CaCOs 5.0 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add 6 and distribute into flasks. Sterilization: Not specified. Use: For the study of nitrite formation. Reference: Johnson (1912 p. 219). 58. Beijerinck and Minkman Ammonium Chloride Solution Constituents: 1. Water 100.0 cc. 2. K2HPO4 0.02 g. 3. NH4CI 0.02 g. 4. NaHCOs 0.1 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Distribute into Sohngen's culture apparatus. Sterilization: Not specified. Use : Inoculate with soil for study of forma- tion of nitrites. Cultivation of Bacillus sausseri. Reference: Beijerinck and Minkman (1910 p. 60). 59. Prazmowski's Ammonium Carbonate Solution (Tanner) Constituents : 1. Distilled water 1000.0 cc. 2. K2HPO4 5.0 g. 3. MgS04-7H20 5.0 g. 4. (NH4)2C03 5.0 g. 5. CaCla 0.5 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Sterilization : Not specified. Reference: Tanner (1919 p. 63). 60. Wimmer's Ammonium Sulphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. (NH4)2S04 2.0 or 4.0 g. Preparation: (1) Dissolve 2 in 1. Sterilization: Not specified. Use: For study of nitrification (production of nitrites) by soil bacteria. Reference: Wimmer (1904 p. 140). 61. Winogradsky Ammonium Sulphate Solution (Harvey) Constituents : 1. Distilled water 1000.0 cc. 2. (NH4)2S04 1.0 g. 3. K2SO4 1.0 g. 4. MgS04 7.5 g. Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization : Not specified. Use: Study of nitrifying organisms. Reference: Harvey (1921 p. 106). 62. Buhlert and Fickendey Ammonium Sulphate Solution Constituents: 1. Water 1000.0 cc. 2. (NH4)2S04 4.0 g. 3. Potassium phosphate 2.0 g. 4. Basic magnesium carbonate (4MgC03-Mg(OH)2-5H20).. 40.0 g. Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization: Not specified. Use: Study of nitrifying bacteria of the soil. Variants : (a) Fremlin's (described by Gage) solu- tion is simifar; the potassium phos- phate is specified as KH2PO4, and the proportions as follows: 1. Water (nitrite free) 1100.0 g. 2. KH2PO4 1.0 g. 3. (NH4)2S04 1.0 g. 4. MgC03 1.0 g. 2 and 3 dissolved in 1000.0 cc. water and 4 in 100.0 cc. water. These solu- tions are sterilized separately and mixed aseptically. (b) Percival distributed the solution in 25.0 cc. lots, and added 20.0 cc. of a mixture of equal parts soil and sterile tat) water to each lot. 22 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS (c) Abbott gave the following solution: 1. Water 1000.0 cc. 2. (NH4)2S04 1.0 g. 3. Potassium phosphate... 1.0 g. 4. MgCOs (Basic) (1) Dissolve 2 and 3 in 1. (2) Flask in 100.0 cc. quantities. (3) Add 0.5 to 1.0 g. of basic MgCOs sus- pended in a little sterile water to each flask. References: Buhlert and Fickendey (1906 p. 404), Barthel (1910 p. 112), Percival (1920 p. 208), Gage (1910 p. 14), Abbott (1921 p. 603). 63. Winogradsky's Ammonium Sulphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. Potassium phos- phate 1 g. 3. MgS04 0.5 g. 4. CaCla trace 5. MgCOs amount not specified 6. (NH4)2S04 2.0 to 2.5 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Distribute in flasks. (3) After sterilization add MgCOs (amount not specified) and from 2 to 2.5 parts per thousand of (NH4)2S04. Sterilization: Method not specified. Use: To show nitrification by organisms found in the soil. Inoculate with soil. Variants : (a) Burri and Stutzer 1. Water 1000.0 cc. 2. NaCi 0.5 g. 3. KH2PO4 ' 1.0 g. 4. MgS04 0.5 g. 5. CaCU trace 6. (NH4)2S04 2.0 g. 7. MgCOs (b) Heinemann 1. Water 1000.0 cc. 2. Potassium phosphate 1.0 g. 3. MgS04 0.5 g. 4. CaCla 0.01 g. 5. NaCl 2.0 g. 6. MgCOs 7. (NH4)2S04 2.0 g. (1) Dissolve 2, 3, 4 and 5 in 1. (2) Distribute in 20.0 cc. lots. (3) Add several tenths grams of MgCOj to each lot. (4) Sterilize intermittently. (5) Add 1.0 or 2.0 cc. of a sterile 2.0% (NH4)2S04 solution to each lot under aseptic conditions. (c) Harvey modified Burri and Stutzer's solution in that he used 1.0 g. K2HPO4 instead of KH2PO4 and used 2.0 g. NaCl instead of 0.5 g. References: Winogradsky (1891 p. 577), Burri and Stutzer (1896 p. 106), Harvey (1921-22 p. 105) Heinemann (1905 p. 129). 64. Giltner's Modified Winogradsky's Ammonium Sulphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. (NH4)2S04 0.4 g. 3. MgS04 0.05 g. 4. K2HPO4 0.1 g. 5. NaoCOs 0.6 g. 6. CaClo trace Preparation: (1) Dissolve 2, 3, 4, 5 and 6 in 1. Sterilization: Not specified. Use: Study of nitrate formation. Reference: Giltner (1921 p. 369). 65. Winogradsky and Omeliansky's Ammonium Sulphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. (NH4)2S04 2.0 g. 3. Potassium phosphate 1-0 g. 4. MgS04 0.5 g. 5. NaCl 2.0 g. 6. FeS04 0.4 g. 7. MgCOs in excess Preparation : (1) Dissolve 3, 4, 5 and 6 in 1. (2) Distribute in 50.0 cc. lots. (3) Add MgCOs in excess to each flask. (4) After sterilization add 1.0 cc. of a sterile 1.0% solution of pure (NH4)2S04 under aseptic conditions. Sterilization : Method not specified. Use : To study nitrification by soil bacteria. Variants: The appended table gives com- binations used by various authors. The preparation of these variants is practi- cally the same as for the original. Omeli- ansky found that repeated inoculation from this medium into the same medium usually yielded a pure culture of nitrify- CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 23 ing organisms when initially inoculated with soil. Giltner and Ldhnis used Harvey's solution (1.0 g. (NH4)2S04) and suggested the use of either CaCOa or MgCOs. He reported that both nitrite and nitrate bacteria are stimulated if CaCOs is used instead of MgCOs. Tan- ner used KH2PO4 in Harvey's (a) solution instead of K2HPO4. (3) Distribute (1) in 5.0 cc. lots. (4) Add 0.5 cc. of (2) to each 5.0 cc. lot of (3). Sterilization: Not specified. Use: To isolate nitrite formers from the soil and to study nitrification. Tromms- darf's reagents were used to test for the presence of nitrites. Reference: Gowda (1924 p. 252). INVESTIGATOR Distilled water (NH4)2S04 NaCl Potassium phosphate MgS04 FeS04 MgCO, K2HPO4 KH2PO4 KCl OMELTANSKY 1899 HARVEY 1921-22 WIMMER 1904 BOULLANGERAND MASSOL 1903 1000.0 cc. Amount not given 1000.0 cc. 1000.0 cc. 2.0 g. 1.0 or 2.0 g. 2.0 g. 2.0 g. 2.0 g. 2.0 g. 2.0 g. 2.0 g. 1.0 g. l.Og 0.5 g. 0.5 g. 0.5 g. 0.5 g. 0.4 g. 0.4 g. 0.4g. 0.4 g. In excess In excess l.Og. In excess l.Og. 0.0 or 0.28 g. In excess Amount not specified References : Winogradsky and Omeliansky (1899 p. 432), Omeliansky (1899 p. 539), Smith (1905 p. 199), BouUanger and Massol (1903 p. 493), Wimmer (1904 p. 139), Lohnis (1913 p. 97), Percival (1920 p. 144), Harvey (1921-22 p. 105), Giltner (1921 p. 375), Tanner (1919 p. 67), Cun- ningham (1924 p. 150). 66. Gibbs' Ammonium Sulphate Solution (modified by Gowda) Constituents : 1. Conductivity water 1100.0 cc. 2. (NH4).2S04 l.Og. 3. K2HPO4 l.Og. 4. NaCl 2.0 g. 5. MgS04 0.5 g. 6. Fe2(S04)3 trace 7. MgCO, 5.0 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1000.0 cc. conductivity water. (2) Dissolve 7 in 100.0 cc. of conductivity water. 67. Omeliansky Ammonium Sulphate Solu- tion (Harvey) 1000.0 cc 1.0 g- 0.5 g- 1.0 g- 0.06 g- 20.0 g- Constituents: 1. Water 2. K.2HPO4 3. MgS04 4. (NH4)2S04 5. NaCl 6. Precipitated chalk Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add 6 to (1). (3) Distribute in flasks. Sterilization: Not specified. Use: Study of nitrifying bacteria. Variants: Niklewski used the following solution to study nitrification by bacteria from stable manure. It was prepared the same as the above solution. 1. Water 1000.0 cc. 2. (NH4)2S04 (0.05%) 0.5 g. 3. K2HPO4 (0.02%..) 0.2 g. 4. MgS04 (0.01%) 0.1 g. 24 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 5. NaCl (0.01%) 0.1 g. 6. FeClj trace 7. CaCO, Reference: Harvey (1921-22 p. 103), Nik- lewski (1910 p. 410). 68. Christensen's Ammonium Sulphate Solution Constituents : 1. Water 1000.0 cc. 2. (NH4)2S04 1.6 g. 3. K0HPO4 2.0 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Distribute in 50 cc. lots in 500.0 cc. tuberculin flasks. (3) Add 1.0 g. of lime to each flask. Sterilization: 20 minutes at 120°C. in auto- clave. Use : Inoculate with soil to study nitrifying bacteria. Reference : Christensen (1913 p. 418). 69. Gage's Ammonium Sulphate Solution Constituents : 1. Water 1000.0 cc. 2. (NH4)2S04 4.0 g. 3. KH2PO4 2.0 g. 4. MgS04 1.0 g. 5. FeS04 0.8 g. 6. NaCI 4.0 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Distribute in Erlenraeyer flasks. Sterilization: Method not specified. Use: Study of nitrification by soil organ- isms. Reference: Gage (1910 p. 31). 70. Omeliansky 's Ammonium Sulphate Solu- tion (Lohnis-Arnd) Constituents : 1. Distilled water 1000.0 cc. 2. (NH4)2S04 1.0 g. 3. K2HPO4 1.0 g. 4. MgS04 + 7H20 0.5 g. 5. NaCl 2.0 g. 6. FeS04 + 7H20 0.4 g. 7. K2CO, Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) After sterilization distribute under aseptic precautions, in 50.0 cc. lots in 300.0 cc. Erienmeyer flasks, each con- taining 0.5 g. pure K2CO3. Sterilization: (1) is sterilized for one hour in streaming steam. The flasks contain- ing the K2CO3 are sterilized for 20 minutes under a pressure of 2 atmospheres in the autoclave. Use: Inoculate with 5.0 g. of soil to study nitrification. Reference: Arnd (1916 p. 561). 71. Lohnis' Ammonium Sulphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. (NH4)2S04 1.0 g. 3. K2HPO4 1.0 g. 4. MgS04 0.5 g. 5. NaCl 2.0 g. 6. FeS04 0.4 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) During incubation at 20 to 25°C. allow some ordinary gas to enter the incubator. Sterilization: Not specified. Use: To study the assimilation of Ca2 by B. oUgocarbophilus. Reference: Lohnis (1913 p. 106). 72. Stutzer's Ammonium Magnesium Phosphate Solution Constituents: 1. Water 2000.0 cc. 2. K2HPO4 2.0 g. 3. NaCi 0.5 g. 4. Dry ammonium magnesium phosphate 20.0 g. 5. FeS04 0.5 g. 6. MgC03 20.0 g. Preparation : (1) Dissolve 2, 3 and 4 in 1000.0 cc. water. (2) Dissolve 5 and 6 in 1000.0 cc. water. (3) After sterilization mix (1) and (2) in equal amounts and distribute in sterile flasks by means of sterile pipettes. Sterilization: Method not specified. Use: Inoculate with a loop of soil suspen- sion. Transfer from flask to flask, using as an enrichment medium. Variants: Stutzer used the constituents in the following proportions: CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 25 1. Water 1000.0 cc. 2. K2HPO4 10 g. 3. NaCl 0.25 g. 4. FeS04 0.025 g. 5. Ammonium magnesium phosphate (drj-) 20.0 g. 6. MgCOs 20.0 g. The medium was prepared by mixing 5 and 6 in a mortar and adding a teaspoon- f ul or more of the mixture to flat bottomed flasks. Then add 50.0 to 100.0 cc. of a solution of 2, 3 and 4 in 1 to each flask. Sterilize the flasks. Method not given. Reference: Stutzer (1901 p. 173). SUBGROUP I-B. SECTION 5 Inorganic liquid media of known com- position, nitrogen supplied as ammonium salts, complete nutrient solutions, but not used primarily for organisms oxidizing ammonia to nitrites. A few media have been described contain- ing ammonium salts but no organic carbon for the cultivation of microorganisms which do not secure their growth energy by oxida- tion of ammonia. These media are of two general classes, those used with sulphur or its compounds for the cultivation of the bacteria oxidizing sulphur, and those for other types of organisms, including the algae. The various media may be differen- tiated as follows: Ai. Primarily for organisms oxidizing sul- phur or its compounds. Waksman's Ammonia and Sulphur Solution no. 2 73 Waksman's Ammonia and Sulphur Solution 74 Waksman's Sulphide Solution 75 Beijerinck's Thiosulphate Solution.... 76 A2. Not primarily for organisms oxidizing sulphur. Bi. Ammonia supplied as ammonium chloride. Kaserer's Hydrogen Ammonium Chlo- ride Solution 77 Beijerinck and van Deiden's Ammo- nium Chloride Solution 78 B2. Ammonia supplied as ammonium nitrate. Richter's Ammonium Nitrate Solu- tion 79 B3. Ammonia supplied as ammonium sul- phate. Lieske's Ammonium Sulphate Solu- tion 80 Gottheil's Ammonium Sulphate Solu- tion. (No. XI) 8. Gosling's Ammonium Sulphate Solu- tion 82 Beijerinck and van Deiden's Ammo- nium Sulphate Solution 83 B4. Ammonia supplied as a salt of phos- phoric acid. Beijerinck and van Deiden's Manga- nese Ammonium Phosphate Solu- tion 84 Beijerinck and van Deiden's Ammo- nium Phosphate Solution 85 73. Waksman's Ammonia and Sulphur Solution No. 2 Constituents : 1. Distilled water 1000.0 cc, 2. (NH4)2S04 0.2 g. 3. iMgS04-7H20 0.5 g. 4. KH2PO4 1.0 g. 5. Ca3(P04)2 (re-precipitated) 2.5 g. 6. Sulfur 10.0 g. 7. H3PO4 Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Weigh sulfur and Ca3(P04)2 sepa- rately into the individual flasks into which the medium is distributed (100.0 cc. portions are usuall}' placed in 250 cc. flasks). (3) Adjust to pH = 3.0 by the addition of M/1 H3PO4. Sterilization : Sterilize in flowing steam for 30 minutes on 3 successive days. Use: Isolation and cultivation of Thio- bacillus thiooxidans. Reference : Waksman (1922 p. 606). 74. Waksman's Ammonia and Sulphur Solution Constituents: 1. Distilled water 1000.0 cc. 2. (NH4)2S04 0.2 g. 3. MgS04-7H20 0.5 g. 4. KH2PO4 3.0 g. 5. CaCla 0.25 g. 6. Sulphur (powdered) 10.0 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Weigh out the sulphur separately in the individual flasks into which the 26 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS (1) is distributed (100 cc. in 250 cc. flasks usually). Sterilization : Sterilize in flowing steam for 30 minutes on 3 consecutive days. Use: Isolation and cultivation of Thio- bacillus thiooxidans. The organisms can grow at a pH = 1.0 with a maximum at pH = 3.0 to 4.0. Variants: The author added a trace of FeS04 and the following combinations of potassium salts of phosphoric acid to the above solution containing no potassium phosphate, (a) KH2PO4 3.0 g. pH = 4.2 'KH2PO4 1.5 g.\ ^TT _ 5 4 (c) K2HPO4 3.0 g. pH = 6.0 . ,. /K2HPO4 3.0 g.l ^„ _.. ^^) icaCOa 10.0 g./ P^ - ^-^ At the various pH values the author found starting with acid medium some Th. thiooxidans give final pH = 1.2. Soil bacteria produce no acid at pH = 4.2 but change pH = 8 to pH = 7.6. Reference: Waksman (1922 pp. 606, 609 to 613). 75. Waksman's Sulphide Solution Constituents : 1. Water 1000.0 cc. 2. K2S 5.0 g. 3. NH4CI 0.1 g. 4. MgClj 0.1 g. 5. Na2HP04 or K2HPO4 0.2 g. 6. NaHCOs 1.0 g. 7. CaC03 10.0 g. or CaClj 0.25 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in part of 1. (2) Dissolve 6 and 7 in rest of 1. (3) Add (1) to (2) by means of sterile pipettes after sterilization. (4) pH = 7.5. Sterilization: Autoclave at 15 pounds pres- sure for 15 minutes. Use: To show oxidation of K2S by Thio- bacillus thiooxidans . Reference: Waksman (1922 p. 609-15). 76. Beijerinck's Thiosulphate Solution Constituents : 1. Water 100.0 cc. 2. Na2S203-5H20 0.5 g. 3. NaHCOa 0.1 g. 4. K2HPO4 0.02 g. 5. NH4CL 0.01 g. 6. MgCl2 0.01 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Adjustment of reaction not specified. (3) Inoculate with a little ditch or canal water or ditch mud. Incubate at 28 to 30 °C. Sterilization: None required. Use: Enrichment and isolation of organ- isms utilizing carbonic acid, Thiobacillus thioparus. Two or three days after inoculation the surface is covered with a layer of free sulphur filled with bacteria. Variants : (a) When Beijerinck used an inoculum from sea water, 3.0% NaCl was added to the solution. (b) Waksman used the above solution with either 0.02 g. Na2HP04 or K2- HPO4 and added one of the following combinations: (1) KH2PO4 3.0 g. pH = 5.4 (2) NaHCOa 1.0 g. pH = 8.8 fNaHCOalLOg. ^3 = 9 4 ^'^' \CaCO3 jlO.Og. P^ ^-^ He found that Thiobacillus thio- oxidans produced a pH = 1.4 in variant (b) (1). (c) Trautwein substituted Na2HP04 for K2HPO4 in the original solution and used the solution to grow Thionic acid bacteria (Omeliansky). Reference: Beijerinck (1903 p. 595), Waks- man (1922 p. 609), Trautwein (1921 p. 518). 77. Kaserer's Hydrogen Ammonium Chlor- ide Solution Constituents: 1. Water 1000.0 cc. 2. K2HPO4 (0.05%) 0.5 g. 3. MgS04 (0.02%) 0.2 g. 4. NH4CI (0.1%) 1.0 g. 5. NaHCOs (0.05%) 0.5 g. 6. Iron chloride trace 7. Hydrogen Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Flask. (3) Following sterilization, if the solu- tion is sterilized, inoculate the solu- tion with soil. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 27 (4) Prepare hydrogen gas from c.p. H2SO4 and zinc. (5) Pass hydrogen and carbonic acid into the flask. Sterilization: Not specified. Use: To show oxidation by soil forms, Bacillus oligocarbophilus and Bacillus methylicus . After 5 days the amount of hydrogen was less in the flask inoculated with soil than in the control flask. Variants : Lantzsch using the same solution found that Actinomyces (Bacillus oligo- carbophilus oxidized hydrogen in a non- sterilized medium, but failed in a steril- ized medium. Reference: Kascrer (1906 p. 686), Lantzsch (1922 p. 316). 78. Beijerinck and van Delden's Ammonium Chloride Solution Constituents : 1. Distilled water 1000.0 cc. 2. NH4CI 0.1 to 1.0 g. 3. Potassium phosphate 0.2 g. 4. Beijerinck and van Delden's basic salt solution (see p. 12) 10 drops. Preparation : (1) Dissolve 2 and 3 in 1. (2) Add 1.0 drop of Beijerinck and van Delden's basic salt solution (see page 12) to each 100.0 cc. of (1). Sterilization: Not specified. Use: Cultivation of Bacillus oligocarbo- philus. Reference: Beijerinck and van Delden (1903 p. 36). 79. Richter's Ammonium Nitrate Solution Constituents: 1. Water 1000.0 cc. 2. NH4NO3 3.0 g. 3. MgS04 1.0 g. 4. KH2PO4, FeS04 and ZnS04 1.0 g. Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization: Not specified. Use: Growth oi Aspergillus niger. Reference: Richter (1910 p. 619). 80. Lieske's Ammonium Sulphate Solution Constituents: 1. Water 1000.0 cc. 2. Magnesium bicarbonate 3. NaHC03 (0.001%) 0.01 g. 4. (NH4)2S04 (0.001%) 0.01 g. 5. Potassium phosphate trace 6. MgS04 Preparation : (1) Prepare Magnesium bicarbonate by adding pure carbonic acid to a sus- pension of Magnesium carbonate. (2) Filter. (3) Dilute the filtrate 1 to 10 with water. (4) Dissolve 3, 4, 5 and 6 in (3). Sterilization: Not specified. Use: Cultivation oi Leptothrix ochracea. Reference: Lieske (1919 p. 422). 81. Gottheil's Ammonium Sulphate Solution (No. XI) Constituents: 1. Distilled water 1000.0 cc. 2. Potassium phosphate 1.0 g. 3. CaCla 0.1 g. 4. MgS04 0.3 g. 5. NaCl 0.1 g. 6. Iron trace 7. (NH4)2S04 0.25 mg. 8. NaoCOa 0.5 g. Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. Sterilization: Not specified. Use : Cultivation of organisms found in the soil on roots and rhizomes. Reference: Gottheil (1901 p. 432). 82. Gosling's Ammonium Sulphate Solution Constituents: 1. Distilled water 500.0 cc 2. NazCOs (dehydrated) 2.3 g 3. CaCOs 0.25 g 4. K.,C03 0.08 g 5. FeS04 0.03 g 6. (NH4)2S04 0.005 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6 in 1. Sterilization: Not specified. Use: To determine sulphate reduction. Reference: Gosling (1904 p. 391). 83. Beijerinck and van Delden's Ammonium Sulphate Solution Constituents: 1. Distilled water 1000.0 cc. 2. (NH4)2S04 1.0 g. 3. Potassium phosphate 0.2 g. 28 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 4. Beijerinck and van Del- den's mineral solution (see p. 12). Preparation : (1) Dissolve 2 and 3 in 1. (2) Add 1.0 drop of 4 to each 100.0 cc. of (1). Sterilization: Not specified. Use: Growth of Bacillus oligocarbophilus. Reference: Beijerinck and van Delden (1903 p. 36). 84. Beijerinck and van Delden's Manganese Ammonium Phosphate Solution Constituents : 1. Distilled water 1000.0 cc. 2. (NH4)2HP04 0.2 g. 3. NaaCOa 1.0 g. 4. Beijerinck and van Del- den's solution (see p. 12). . 20 drops. Preparation : (1) Dissolve 2 and 3 in 1. (2) Add 2.0 drops of 4 to each 100.0 cc. of (1). Sterilization: Not specified. Use: Bacillus oligocarbophilus grows as a snow white scum on the surface of the medium. Reference: Beijerinck and van Delden (1903 p. 41). 85. Beijerinck and van Delden's Ammo- nium Phosphate Solution Constituents : 1. Distilled water 100.0 cc. 2. K2HPO4 0.02 g. 3. (NH4)2HP04 0.02 g. 4. NazCOa 0.1 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) Reaction to be slightly alkaline. Sterilization: Not specified. Use: Bacillus oligocarbophilus grows as a thin white scum on the medium. Reference: Beijerinck and van Delden (1903 p. 41). SUBGROUP I-B. SECTION 6 Inorganic liquid media or basal solutions of known composition; nitrogen supplied as nitrites. The several liquid media which have been published for the growth of organisms oxidizing nitrites may be differentiated as follows: A I. Employed primarily for organisms oxidizing nitrites to nitrates. Bi. Calcium carbonate added. Hewlett's Nitrite Solution (Johnson). 86 Cunningham's Nitrite Solution 87 B2. Calcium carbonate not added. Ci. Magnesium carbonate added. Omeliansky's Nitrite Solution 88 C2. Magnesium carbonate not added. Di. Magnesium sulphate added. Winogradsky and Omeliansky's So- dium Nitrite Solution 89 Winogradsky 's Basal Sodium Nitrite Solution 90 D2. Magnesium sulphate not added. Gartner's Sodium Nitrite Solution ... 91 Wimmer's Sodium Nitrite Solution.. . 92 Bijerinck and van Delden's Basal Nitrite Solution 93 A2. Not employed primarily for organisms oxidizing nitrites to nitrates. Gottheil's Nitrite Solution No. VIII . 94 Bijerinck and van Delden's Potassium Nitrite Solution A 95 86. Hewlett's Nitrite Solution (Johnson) Constituents : 1. Distilled water 1000.0 cc. 2. KNO2 0.3 g. 3. Potassium phosphate 0.1 g. 4. MgS04 0.05 g. 5. CaCOs 5.0 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Sterilization: Not specified. Use: Study of nitrate production. Bacil- lus megatherium produced no nitrates in this solution. Reference: Johnson (1912 p. 219). 87. Cunningham's Nitrite Solution Constituents : 1. Distilled water 1000.0 cc. 2. NaNOo 1.0 g. 3. K2HPO4 0.5 g. 4. MgS04 0.3 g. 5. NaCl 0.5 g. 6. Na.COa 0.2 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Distribute in 10.0 cc. quantities in 50.0 cc. Erlenmeyer flasks. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 29 (3) Add a small quantity of sterile CaCOs to each tube of sterile (2). Sterilization: Sterilize (2) and the CaCOa into autoclave. Use: To study oxidation of nitrites and nitrates. Reference: Cunningham (1924 p. 151). 88. Omelianski's Nitrite Solution Constituents: 1. Distilled water 1000.0 cc. 2. NaNOs (Merck) 1.0 g. 3. NaaCOs (ustum) 1.0 g. 4. Potassium phosphate 0.5 g. 5. NaCl 0.5 g. 6. FeS04 0-4 g. 7. MgS04 0.3 g. 8. MgCOa Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (2) Flask in flat bottomed flasks in 50.0 cc. lots. (3) Add 0.5 g. MgCOa to each flask. Sterilization: Not specified. Use: Isolation of nitrate forming organ- isms. The author found that repeated cultivation on this medium yielded a pure culture. Variants: Boullanger and Massol used the above solution with the addition of slag. Reference: Omeliansky (1899 p. 548), Boul- langer and Massol (1903 p. 494). 89. Winogradsky and Omeliansky's Sodium Nitrite Solution Constituents : 1. Distilled water 1000.0 cc. 2. NaNOa 1-0 g- 3. Potassium phosphate 0.5 g. 4. MgS04 0-3 g. 5. NaCl 0.5 g. 6. Na.COa 1-0 g. 7. FeS04 0.4 g. Preparation : (1) Distill water with permanganate. (2) Dissolve 2, 3, 4 and 5 in (1). (3) Distribute in 50.0 cc. lots. (4) Add 0.0, 0.1, 0.2, 0.4, 0.5 or 0.6 g. of NaaCOa to each flask (1.0% gives best results). (5) Iron may be added. Sterilization: Not specified. Uses: To study nitrate production from nitrites. The author found that the addition of 0.1% Na2C03 gave the best results. The addition of FeS04 had no effect on nitrate production. Added nutrients : The authors added one of the following materials to determine the effect of organic material on nitrification. glycerolO.l, 0.2, 0.4, 0.8, 1.0, 1.4 or 2.0% glucose 0.05, 0.1, 0.2, 0.4, 0.8, 1.0 or 2.0% urea 0.1, 1.0 or 1.6% asparagin 0.1, 0.6, 0.8, 1.0 or 2.0% hay infusion 4.0, 8.0, 16.0 or 32.0% leaf infusion 4.0, 8.0, 16.0 or 32.0% dirt infusion 4.0, 8.0, 16.0 or 32.0% meat infusion 2.0, 4.0, 8.0 or 10.0% urine 2.0, 4.0, 10.0 or 20.0% (NH4)2S04 (1.0% solution) 0.2, 1.0, 1.6, 2.0, 3.0, 4.0 or 6.0% sodium acetate 1.0, 2.0, 3.0, 4.0 or 6.0% sodium butyrate 0.1, 1.0, 2.0, 3.0 or 4.0% Generally organic material slows up oxidation unless it be present in very small quantities. The more organic materials present the slower the oxidation. Variants : (a) Wimmer used the above solution specifyingO.5 g. KH2PO4 and suggest- ing the addition of 0.28 g. KCl. (b) Harvey used this solution employ- ing 0.5 g. K2HPO4 and 0.3 g. anhy- drous Na2C03 instead of 1.0 g. (c) Gibbs (Gowda) used 0.5 g. K2HPO4 and a trace of Fe2(S04)3 instead of 0.4 g. FeS04. Reference: Winogradsky and Omeliansky (1899 p. 333), Wimmer (1904 p. 140), Smith (1905 p. 199), Harvey (1921-22 p. 106), Gowda (1924 p. 258), Percival (1920 p. 149). 90. Winogradsky's Basal Sodium Nitrite Solution Constituents: 1. Distilled water 1000.0 cc. 2. NaN02 (Merck) 1.0 g 3. Potassium phosphate 0.5 g 4. MgS04 0.3 g 5. NaoCOs 0.5 g 6. NaCl 0.5 g Preparation : (1) Distill water two times with "chama- leon." (2) Dis-solve 2, 3, 4 and 5 in 1. The Na2C03 should be water free, heated to a weak glowing. 30 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS (3) Adjustment of reaction not specified. (4) Flask in 50.0 cc. lots. Sterilization: Sterilize in the autoclave. Use: To study nitrification. Added nutrients and variants : The author added glucose, urea, asparagin or peptone to the solution to determine the efTect of organic materials on nitrification. Dis- tribute in 50.0 cc. lots and add: (a) 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2.0, 2.4 or 4.0 g. glucose. (b) 0.05, 0.1, 0.2, 0.4, 0.8, 1.2 or 1.6 g. peptone. (c) 0.1, 0.2, 0.4 or 0.8 g. urea. (d) 0.1, 0.2 or 0.4 g. asparagin. (e) 5.0 cc. of bouillon. The presence of organic materials gen- erally tends to slow up the oxidation. 3. K2CO3 5.0 g. 4. NaNOs 2.0 g. Preparation : (1) Dissolve 2, 3, and 4 in 1. (2) Distribute in 30 cc. lots in flasks. Sterilization: Not specified. Use: To study the oxidation of nitrite by fungus. Nitrite was found to be still present after four weeks. Reference: Gartner (1898 p. 4). 92. Wimmer's Sodium Nitrite Solution Constituents: 1. Distilled water.. 1000.0 cc. 2. Natrium nitro- sum puriss (NaNOo) 1.0, 2.0 or 50.0 g. Preparation: (1) Dissolve 2 in 1. Distilled water NaN02 Potassium phosphate MgS04 NaCl NaoC03 K2HPO4 K2CO3 KCl WINOGRADSKY AND OMELIANSKY 1000.0 cc. 1.0 g. 0.5 g. 0.3 g. 0.5 g. 1.0 g. 1000.0 cc. 2.0 g. 0.3 g. 0.5 g. 1.0 g. 0.5 g. 1000.0 cc. 25.0 cc. 2.0% solution 1.0 g. 0.5 g. l.Og. 0.1 1000.0 cc. l.Og. 0.3 g. 0.5 g. 0.3 g. l.Og. Peptone in small amounts tends to hasten oxidation. Same is true of urea. Asparagin and glucose even in small amounts slow up the oxidation. Variants: The modifications shown in the table have been described. Their prepa- ration is like the original solution. Stutzer and Hartleb bubbled air con- taining CO2 thru their solution. Carbo- nates or CO 2 were essential for nitrate production. References: Winogradsky (1896 p. 423), Stutzer and Hartleb (1897 p. 244), Wino- gradsky and Omeliansky (1899 p. 378), Stutzer (1901 p. 169), Lohnis (1913 p. 97) Giltner (1921 p. 375). 91. Gartner's Sodium Nitrite Solution Constituents : 1. Water 1000.0 cc. 2. Potassium phosphate 0.5 g. Sterilization: Not specified. Use : To study nitrification by soil bacteria. Raference: Wimmer (1904 p. 140). 93. Beijerinck and van Delden's Basal Nitrite Solution Constituents: 1. Water 1000.0 cc. 2. KNO2 0.5 g. 3. K0HPO4 0.5 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Add one of the added nutrients to (1). Sterilization: Not specified. Use: Tostudy denitrification by 5. subtilis, B. mesenlericus and Azo. chrooincoccum. B. subtilis gave no ammonia test. B. mesenlericus gave ammonia using man- nitol as a carbon source. Chroococcum gave ammonia using either mannitol or malate as a carbon source. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 31 Added nutrients: The authors used either 20.0 g. of mannitol or 20.0 g. calcium malate as a carbon source. Reference: Beijerinck and van Delden (1902 p. 41). 94. Gottheil's Nitrite Solution No. VIII Constituents. 1. Distilled water 1000.0 cc. 2. Potassium phosphate 1-0 g. 3. CaClo 0.1 g. 4. MgSOi 0.3 g. 5. NaCl 0.1 g. 6. Iron trace 7. KNO2 0.05 g. 8. Soda 0.05 g. Preparation : Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. Sterilization: Not specified. Use: Cultivation of bacteria found in soil on roots and rhizomes. Reference: Gottheil (1901 p. 432). 95. Beijerinck and van Delden's Potassium Nitrite Solution A Constituents: 1. Water (tap) 100.0 cc. 2. KCl 0.01 g. 3. K2HPO4 0.02 g. 4. Kx\02 0.01 g. Preparation : (1) Dissolve 2, 3 and 4 in 1. (2) The reaction is slightly alkaline. Sterilization: Not given. Use: Bacillus oligocarbophilus grows in a dry thin snow white scum on the surface of the medium. Reference: Beijerinck and van Delden (1903 p. 41). SUBGROUP I-B. SECTION 7 Inorganic basal solutions of known com- positions; nitrogen supplied as nitrates; incomplete solutions requiring the addition of other nutrients. A I. Saltsof monovalent cations only added. Beijerinck's Basal Nitrate Solution. 96 Stoklasa and Vitek's Basal Nitrate Solution 97 A2. Salts of mono and divalent cations added. Czapek's Basal Nitrate Solution (Waksman) 98 Giltay-Aberson's Basal Nitrate Solu- tion (Stoklasa) 99 Henneberg's Basal Nitrate Solution. 100 A3. Salts of mono, di and tri valent cations added. Bi. Calcium salts added. Stoklasa and Vitek's Basic Nitrate Salt Solution 101 Harvey's Basal Nitrate Solution 102 Bo. Calcium salts not added. Beijerinck and van Delden's Nitrate Solutions, B, E, F, H, N 103 Kita's Basal Nitrate Solution 104 Stoklasa's Basal Nitrate Solution . . . 105 Lantzsch's Basal Nitrate Solution. . . 106 96. Beijerinck's Basal Nitrate Solution Constituents: 1. Water 1000.0 cc. 2. KNO3 1.0 g. 3. K2HPO4 0.2 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Add one of the materials listed under added nutrients. Sterilization: Not specified. Use: To study denitrification by Azoto- bacter chroococcum. Added nutrients and variants : (a) The author added 20.0 to 100.0 g. mannitol. (b) Beijerinck and van Delden used 0.2 g. KNO3 in the basic solution without additional nutrients for the cultiva- tion of Bacillus oligocarbophilus . (c) Beijerinck and van Delden used 0.2 g . KNO3 and 0.2 g. Na2HP04 in the basic solution and added 0.2 g. KCl for the cultivation of Bacillus oligocarbo- philus. (d) Beijerinck and van Delden used 0.1 g. KNO3 and 0.1 g. K2HPO4 in the basic solution and added 0.1 g. Na2C08 for the cultivation of Bacillus oligo- carbophilus. (e) Beijerinck and Minkman used 1.0% KNO3 and 0.05% K.2HPO4 in the basic solution and added 2.0% man- nitol, glycerol, sodium acetate or sodium propionate to study deni- trification. (f ) Beijerinck and van Delden used 0.5 g. KNO3 and 0.5 g. K2HPO4 in the basic solution and added 2.0 g. of calcium 32 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS malate or mannitol to study deni- trification. References: Beijerinck (1901 p. 575), Beij- erinck and van Delden (1902 p. 41), (1903 p. 41), Beijerinck and Minkman (1910 pp. 35, 36). 97. Stoklasa and Vitek's Basal Nitrate Solution Constituents: 1. Water 1000.0 cc. 2. NaNOs 8.5 g. Preparation : (1) Dissolve 2 in 1. (2) Add a suitable carbon source. Sterilization: Not specified. Use: To study nitrate reduction by Bad. Hartlebi and other bacteria. Added nutrients : The author suggested the addition of the following carbon sources: xylose 15.0 g. glucose 18.0 g. sucrose 34.2 g. Reference: Stoklasa and Vitek (1905 p. 205). 98. Czapek's Basal Nitrate Solution (Waksman) Constituents: 1. Water 1000.0 cc. 2. K2HPO4 1.0 g. 3. MgS04 0.5 g. 4. KCl 0.5 g. 5. FeS04 0.01 g. 6. NaNOa 2.0 g. Preparation : (1) Prepare a solution of 1, 2, 3, 4, 5 and 6 using chemically pure materials. (2) Add 20.0 g. of one of the test ma- terials. Sterilization: Autoclave at 15 pounds pres- sure for 15 minutes. Use: To study the metabolism of actino- mycetes. The author found that the Actinomycetes utilized the materials in order listed. Starch, glucose, lactose, maltose, glycerin, sucrose, cellulose and then organic acids. Added nutrients: The author used 20.0 g. of one of the following carbon sources: arabinose starch glycerol sodium salts of : lactose acetic acid sucrose malic acid maltose sodium salts of: mannitol tartaric acid inulin oxalic acid glucose lactic acid cellulose in form of Whitman filter paper 1.0 cc. of 0.5% cellulose suspension. Reference: Waksman (1919 p. 317). 99. Giltay-Aberson's Basal Nitrate Solution (Stoklasa) Constituents: 1. Water 1000.0 cc. 2. NaNOs 2.0 g. 3. MgS04 2.0 g. 4. K2HPO4 2.0 g. 5. NaCl 0.2 g. 6. Iron chloride less than 0.2 g. Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Adjust to slightly alkaline reaction by adding Na2C03. (3) Add one of the combinations given in added nutrients. Sterilization: Not specified. Use: To study denitrification and influence of carbohydrates on denitrification by soil forms. Added nutrients: The author added one of the following combinations: (a) Citric acid 5.0 g. and glucose 2.0 g. (b) Glucose 2.0 g. (c) Glucose 2.0 g. with stable manure. (d) Glucose 2.0 g. and citric acid 5.0 g. with stable manure. (e) Glucose 2.0 g. with Chilisaltpeter. (f) Glucose 2.0 g. and citric acid 5.0 g. with Chilisaltpeter. (g) Chilisaltpeter, peat, or soil rich in humus. Reference: Stoklasa (1907 p. 28). 100. Henneberg's Basal Nitrate Solution Constituents: 1. Water 1000.0 cc. 2. MgS04 1.0 g. or 2.0 g. 3. KNO3 2.0 g. or 3.0 g. Preparation: (1) Dissolve 2 and 3 in 1. (2) Add one of the test materials to (1). Sterilization: Not specified. Use: To determine the fermentation of acetic acid bacteria, B. oxydans, B. acetosum, B. aceti, B. Kutzingianum, B. Pasteurianum, B. acetigenum. B. oxy- CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 33 dans ferments arabinose, levulose, dex- trose, galactose, maltose, dextrin, ethyl and propyl alcohol, erythritol. B. aceto- sum ferments arabinose, dextrose, galac- tose, ethyl and propyl alcohol. B. aceti, B. Kiitzingianum, B. Pasteurianum and B. acetigenum ferment dextrose and ethyl and methyl alcohol. Added nutrients: The author added the following carbon sources: arabinose inulin levulose methyl alcohol glucose ethyl alcohol galactose propyl alcohol sarobose amyl alcohol sucrose glycerol maltose erythritol lactose mannitol dextrin dulcitol starch melampyrit glycogen quercitol Reference: Henneberg (1898 p. 19). 101. Stoklasa and Vitek's Basal Nitrate Salt Solution Constituents: 1. Water 1000.0 cc 2. NaNOs 2.0 g. 3. K2HPO4 1.25 g. 4. K2SO4 0.2 g. 5. CaCl2 0.05 g. 6. MgCU 0.05 g. 7. NaiCOs 0.1 g. 8. FeP04 0.05 g. Preparation : (1) Prepare solutions as indicated in the table by dissolving the salts in water. (2) Add 2.0 or 2.5 g. of test material. Sterilization: Not specified. Use: To study reduction of nitrates by denitrifying organisms. Added nutrients: The author added a variety of carbohydrates (2.0 o;- 2.5 g.) or 2.0 g. of organic acids. Neutralize the acids with NaoCOs. butj-ric acid glucose succinic acid levulose lactic acid galactose citric acid arabinose malic acid xjdose tartaric acid saccharose asparagin lactose Variants: The authors substituted 0.25 g. Na2HP04 for 1.25 g. K2HPO4. Reference: Stoklasa and Vitek (1905 p. 104) (1901 p. 262). 102. Harvey's Basal Nitrate Solution Constituents: 1. Distilled water 1000.0 cc. 2. NaNOa 1.0 g. 3. NaCl 0.02 g. 4. MgS04 0.02 g. 5. K2HPO4 0.5 g. 6. FeCla trace Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) Add one of the added nutrients. Sterilization: Not specified. Use: Study of nitrate reduction. Added nutrients: The author added 10.0 g. glucose or 10.0 g. glycerol. Variants : The author used the following solution: 1. Distilled water 3000.0 cc. 2. KNO3 7.0 g. 3. K0HPO4 1.5 g. 4. MgS04 1.5 g. 5. NaCl 1.5 g. 6. CaS04 5.0 g. 7. FeCl 3 solution few drops Reference: Harvey (1921-22 p. 104). 103. Beijerinck and van Delden's Nitrate Solution B, E, F, H, N Constituents : Solution B: 1. Distilled water 1000.0 cc. 2. KCl 0.2 g. 3. KNO., 1.0 g. 4. K2HPO4 0.2 g. Solution E: 1. Distilled water 1000.0 cc, 2. KCl 0.1 g. 3. KNO3 1.0 g. 4. K2HPO4 0.2 g. Solution F: 1. Distilled water 1000.0 cc. 2. KNO3 0.1 g. 3. Na2HP04 0.2 g. Solution H: 1. Distilled water 1000.0 cc. 2. KCl 0.2 g. 3. KNO3 0.2 g. 4. Na2HP04 0.2 g. Solution N: 1. Distilled water 1000.0 cc. 2. KNO3 0.1 to 1.0 g. 3. K2HPO4 0.1 g. 34 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS Preparation : (1) Dissolve the salts in water as indi- cated in the table. (2) Add one drop of Beijerinck and van Delden's mineral solution (see Med. 25) to each 100.0 cc. of (1). Sterilization: Not specified. Use: Bacillus oligocarbophilus grows as a dry thin snow white scum on the surface of the medium. Reference: Beijerinck and van Delden (1903 p. 41). lOi. Kita's Basal Nitrate Solution Constituents : 1. Water 1000.0 cc. 2. MgS04 0.25 g. 3. KH2PO4 5.0 g. 4. FeCls solution few drops 5. KNO3 50 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add 5.0% of any carbohydrate. (3) Adjustment of reaction not specified. Sterilization: Not specified. Use: Cultivation of molds, Aspergillus albus, Aspergillus Okazaki, Aspergillus candidus, Aspergillus tamarii, Pseudo- rhizopus, Aspergillus glaucus. Added nutrients: The author did not specify the carbohydrates used. Reference: Kita (1913 p. 434). 105. Stoklasa's Basal Nitrate Solution Constituents : 1. Distilled water 1000.0 cc. 2. K2SO4 10 g. 3. MgCl2 0.5 g. 4. Iron sulphate 0.1 g. 5. NaNOa 6. Monodiferric phosphate Fe203(P205)3 or monodialu- minum phosphate AI2O3P2O5 1.0 g. Preparation : (1) Dissolve 2, 3, 4 and one of 6 in 1. (2) Add 2.0 g. of nitrogen in the form of NaN03 and 20.0 g. of test material to (1). (3) Distribute in Fernbach flasks. Sterilization: Sterilize in the autoclave. Use: To study the cycle of the phosphate ion in the soil. Bact. fiuorescens lique- faciens and Bact. Hartlebi were the organisms used. Added nutrients: The author added 20.0 g. of any carbohydrate. Reference: Stoklasa (1911 p. 479). 106. Lantzsch's Basal Nitrate Solution Constituents : 1. Water (tap) 100.0 cc. 2. K2HPO4 (0.01%) = 0.01 g. 3. NaN03 (0.01%) = 0.01 g. 4. MgS04 trace 5. FeCls trace Preparation : (1) Dissolve 2, 3, 4 and 5 in 100.0 g. tap water or in 29 parts tap water + 80 parts distilled water. (2) Place in an atmosphere of one of the test materials. Sterilization: Not specified. Use : To study utilization of gaseous carbon compounds. Added nutrients: The author prepared the various atmospheres of test materials as follows: (a) Decompose 0.6 g. lead formate with H2SO4, in a 2500.0 cc. flask. (b) 2.0 cc. of a 4.0% formalin solution in a 2000.0 cc. flask. (c) 0.125 g. CH3COOH in 2500 cc. flask. (d) 0.1 g. acetone in a 2000.0 cc. flask. (e) 0.2 cc. butyric acid in 5 liter flask. (f) 0.12 cc. of benzol or xylol in 2000.0 cc. flask. Reference: Lantzsch (1922 p. 310). SUBGROUP I-B. SECTION 8 Inorganic liquid media of known compo- sition; nitrogen supplied as nitrates; com- plete nutrient solutions. Ai. Containing oxidizable sulphur. Bi. Sulphur added as elementary sulphur. Beijerinck's Sulphur Nitrate Solution 107 B2. Sulphur added as thiosulphate. Lieske's Thiosulphate Nitrate Solu- tion ^(Gehring) 108 Trautwein's Thiosulphate Nitrate Solution 109 Nathansohn's Thiosulphate Nitrate Solution (Trautwein) 110 A2. Not containing oxidizable sulphur. Beijerinck's Ammonium Nitrate Solution Ill Nabokich and Leedeff's Hydrogen Nitrate Solution 112 Bokorny's Calcium Nitrate Solution. 113 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 35 107. Beijerinck's Sulphur Nitrate Solution Constituents : 1. Ditch water or dis- tilled water 100.0 cc. 2. Sulphur (powder) 10.0 g. 3. KNO3 0.05 g. 4. NasCOa 0.02 g. 5. CaCOs 2.0 g. 6. K2HPO4 0.02 g. 7. MgCl. 0.0 or 0.01 g. Preparation : (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (2) Completely fill a well stoppered flask with (1). Sterilization: Not specified. Use: To study denitrification with sulphur o.xidation by Thiobacillus denitrificans . The author found that the nitrate was reduced, and the sulphur o.xidized to sul- phate, with the liberation of nitrogen gas. Reference: Beijerinck (1903-4 p. 597). 108. Lieske's Thiosulphate Nitrate Solution (Gehring) Constituents : 1. Distilled water 100.0 cc. 2. NaaSaOs 0.5 g. 3. KxNOs 0.5 g. 4. NaHCOs 0.1 g. 5. K2HPO4 0.02 g. 6. MgCl2 0.01 g. 7. Fe.Cle trace 8. CaCl2 trace Prepj ration : (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. (2) Distribute in test tubes 45 cm. high. Sterilization: Not specified. Use: Isolation and cultivation of denitri- fying thiosulphate bacteria, Thiobacillus denitrificans. Reference: Gehring (1914 p. 405), Traut- wein (1921 p. 518). 109. Trautwein's Thiosulphate Nitrate Solution Constituents: 1. Distilled water 100.0 cc. 2. Na2S203 0.5 g. 3. KNO3 0.5 g. 4. NaaCOa 0.1 g. 5. K2HPO4 0.02 g. 6. MgClj 0.01 g. 7. KCl trace 8. Iron chloride trace Preparation : (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. (2) Distribute in tall glass cylinders. (3) Inoculate with waste water filtrate. Sterilization: Not specified. Use: Enrichment and isolation of thionic bacteria (Omeliansky). A slight opal- escence is formed after 6 days. After a milky turbid culture results, a snow white membrane is formed on the surface. Variants : The author suggested the follow- ing solution: 1. Distilled water 1000.0 cc. 2. MgCl2 0.01 g. 3. KNO3 0.1 g. 4. Na2HP04 0.02 g. 5. Na2S203 0.2 g. 6. NaHCOa 0.1 g. Reference: Trautwein (1920 p. 515) (1921 p. 515). 110. Nathansohn's Tliiosulphate Nitrate Solution (Trautwein) Constituents : 1. Distilled water 100.0 cc. 2. NaCl 3.0 g. 3. MgCl. 0.25 g. 4. KNO3 0.1 g. 5. Na2HP04 0.05 g. 6. NaoSoOs 0.2 or 0.1 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6 in 1. Sterilization: Not specified. Use: Cultivation of thionic bacteria (Ome- liansky). Reference: Trautwein (1921 p. 518). 111. Beijerinck's Ammonium Nitrate Solution Constituents : 1. Water (tap) 100.0 cc. 2. NH4NO3 0.2 g. 3. Potassium bi-phosphate 0.05 g. Preparation: (1) Dissolve 2 and 3 in 1. Sterilization: Not specified. Use: Cultivation of gonidia of algae. Variants: Beijerinck used 0.02 g. NH4NO3 and 0.02 g. K2HPO4 in 100.0 cc. of tap water to cultivate Chlorophijcae, Chloro- coccum and Cyanophycee. References: Beijerinck (1893 p. 371), Beij- erinck (1901 p. 563). 36 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 112. Nabokoch and Lebedeff's Hydrogen Nitrate Solution Constituents : 1. Water 1000.0 cc. 2. Na,HP04 0.5 g. 3. KNO, 2.0 g. 4. MgSO, 0.2 g. 5. NaHCOa 1.0 g. 6. FbsCU some 7. Hydrogen Preparation : (1) Dissolve 2, 3, 4, 5 and 6 in 1. (2) The reaction is slightly alkaline (about 0.050 g. H2SO4 to 100.0 cc. of solution). (3) Distribute in 100 or 150.0 cc. lots in vacuum flasks with side tubes. (4) Inoculate. (5) Remove all the air by means of an oil pump. (6) Replace the air by a mi.xture of oxygen and hydrogen ("Knallgase") contain- ing carbonic acid. Sterilization: Method not given. Use: To study the oxidation of hydrogen by microorganisms. Reference: Nabokich and LebedefT (1907 p. 352), Lohnis (1913 p. 106). 113. Bokomy's Calcium Nitrate Solution Constituents : 1. Water 100.0 cc. 2. Ca(N03)2 0.1 g. 3. MgS04 (crystalline) 0.02 g. 4. KH2PO4 0.02 g. 5. Iron chloride trace 6. KCl 0.0 or 0.05 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6 in 1. Sterilization : Not specified. Use: To study the metals required for the growth of Spirogyra majuscula. It was found that potassium was necessary. Reference: Bokorny (1912 p. 125). SUBGROUP I-C Liquid Media with One or More Constituents Organic The liquid media in which one or more constituents are organic may be divided conveniently into sections based upon the character of the nitrogen and carbon sources supplied. The sections recognized may be differentiated as follows: Key to the sections of Subgroup I-C Ai. Chemical composition of all constit- uents known. Bi. Nitrogen inorganic, carbon organic. Ci. Nitrogen present as free or elementary nitrogen only. . Section 1 (Med. 114-190) C2. Nitrogen present as ammonium salts. Section 2 (Med. 191-323) Cj. Nitrogen present as nitritesj. Section 3 (Med. 324-327) C4. Nitrogen present as nitrates. Section 4 (Med. 328-365) B2. Nitrogen organic. Ci. Nitrogen present as amino acids. Section 5 (Med. 368-479) C2. Organic nitrogen other than amino acids present . . . Section 6 (Med. 480-515) A2. Chemical composition of one or more constituents not definitely known. Bi.* Containing digests. Ci.* Containing commercial peptones or digests. Di. Additional constituents, if any, in- organic Section 7 (Med. 516-555) D2. Containing additional organic constit- uents. El. Chemical composition of all additional materials known. Fi. No additional nitrogen compounds added. Gi. Basal solutions. Employed with the addition of various nutrients. Section 8 (Med. 556-588) B2. Complete nutrient solutions. Section 9 (Med. 589-647) F2. At least one additional nitrogenous constituent. Section 10 (xMed. 648-683) E2. Composition of one or more additional materials not definitely known. Fi. Additional organic material exclusively of plant origin Section 11 (Med. 684-712) F2. Containing additional materials of animal origin; plant derivatives may also be present. G:. Not containing extracts or infusion. Section 12 (Med. 713-747) G2. Containing extracts or infusion. Section 13 (Med. 748-977) See page 37 for B2 and C2. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 37 Cj. Containing non-commercial digests. Section 14 (Med. 978-1140) Bj. Not containing digests. Ci. Containing plant constituents of un- known composition. Section 15 (Med. 1141-1228) C2. Containing animal constituents of un- known composition. Section 16 (Med. 1229-1376) C3. Containing extracts of soil, ashes, etc. Section 17 (Med. 1377-1394) SUBGROUP I-C. SECTION 1 Liquid media or basal solutions with con- stituents of known chemical composition containing free or elementary nitrogen only, carbon organic. Ai. Inorganic salts not added. Bi. Carboh3'drates added. Pringshein's Glucose Cellulose Solu- tion 114 Waksman and Joffe's Basal Glucose Solution 115 Buchanan's Sucrose Solution 116 B2. Alcohols added. Waksman and Joffe's Basal Glycerol Solution 117 Buchanan's Glycerol Solution I 118 A2. Inorganic salts added. Bi. Potassium salts, only, added. Ci. Containing carbohydrates. Beijerinck and van Delden's Phos- phate Glucose solution 119 Beijerinck's Phosphate Sucrose Solu- tion 120 C2. Containing alcohols. Beijerinck's Phosphate Mannitol Solution 121 C3. Containing salts of organic acids, van Delden's Basal Lactate Solution. 122 Beijerinck and van Delden's Ace- tate Solution 123 B2. Other salts added. Ci.* Magnesium salts added. Di. Phosphates not added. Gage's Glucose Salt Solution 124 Stoklasa's Phosphorous Free Glucose Salt Solution 125 Dj. Phosphates added. El.* Calcium salts not added. Fi. Carbon present as carbohydrates. Gi. Monosaccharides added. *See next page for C2, E2. Gerlach and Vogel's Basal Glucose Salt Solution 123 Henneberg's Basal Glucose Di-so- dium Phosphate Solution 127 Beijerinck's Glucose Salt Solution. . . 128 Kisch's Basal Glucose Salt Solution. 129 Beijerinck's Phosphate Glucose Salt Solution (Harvey No. 1) 130 Laborde's Basal Invert Sugar Tar- tarate Solution 131 Calmette, Massol and Breton's Basal Glucose Salt Solution 132 Lohnis' Basal Glucose Salt Solution . . 133 G2. Disaccharides added. Will's Basal Sucrose Salt Solution. . . 134 Henneberg's Basal Sucrose Salt Solu- tion 135 Czapek's Sucrose Salt Solution (Waksman) 136 von Bronsart's Basal Sucrose Salt Solution 137 Bokorny's Basal Sucrose Salt Solu- tion 138 Smith's Sucrose Salt Solution 139 Munter's Basal Galactose Salt Solu- tion 140 Buchanan's Basal Maltose Salt Solu- tion 141 Qs. Polysaccharides Added. Omeliansky's Basal Cellulose Salt Solution 142 F2.* Carbon present as alcohols. Gi. Glycerol added. Percival's Glycerol Phosphate Solu- tion 143 Buchanan's Glycerol Salt Solution II. 144 Gerlach and Vogel's Basal Glycerol Salt Solution 145 Kuhne's Basal Lactate Solution. (Proskauer and Beck) 146 Calimard and Lacomme's Basal Glyc- erol Salt Solution (KoUe-Wasser- man) 147 G2. Mannitol added. Bijerinck's Modified Mannitol Salt Solution. (Omeliansky and Sswe- rowa) 148 Murray's Mannitol Salt Solution... 149 Percival's Mannitol Phosphate Solu- tion 150 Capaldi and Proskauer's Basal Man- nitol Salt Solution 151 * See ne.xt page for F3. 38 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS F3. Carbon present as organic acids or their salts. Percival's Succinate Solution 152 E2. Calcium salts added. Fi. Carbon present as carbohydrates. Gi. Glucose added. Hi. Glucose only organic carbon source. Winogradsky's Salt Glucose Solution. 153 Stutzer, Burri and Maul's Glucose Salt Solution 154 Stoklasa's Basal Glucose Salt Solu- tion 155 Charpentier's Salt Glucose Solution. 156 Krzemieniewska's Glucose Salt Solu- tion (Vogel) 157 Kriiger and Schneidewing's Basal Glucose Salt Solution (Heinze) 158 Charpentier's Basal Glucose Salt Solution 159 Stoklasa's Basal Glucose Salt Solu- tion 160 Henneberg's Basal Glucose Salt Solu- tion 161 H2. Glucose with other organic carbon. Gottheil's Carbohydrate Glycerol Solution 162 Harvey's Citric Acid Glucose Solu- tion 163 Munter's Basal Mannitol Salt Solu- tion 164 G2. Sucrose added. Chraszcz's Basal Sucrose Salt Solu- tion 165 Maze's Sucrose Salt Solution 166 Linde's Basal Sucrose Salt Solution. . 167 Gs. Polysaccharides added. Lohnis and Lochhead's Basal Cellu- lose Salt Solution 168 van Iterson's Basal Cellulose Salt Solution 169 F2.* Carbon present as alcohols. Gi. Mannitol added. Jones' Modified Ashby's Mannitol Solution 170 Fred and Loomis' Mannitol Salt Solu- tion 171 Krzemieniewska's Mannitol Salt Solution (Vogel) 172 Harvey's Mannitol Salt Solution. . . . 173 Sackett's Modified Lipman's Man- nitol Salt Solution 174 See next column for F3. Winogradsky's Mannitol Salt Solu- tion (Lantzsch) 175 G2. Glycerol added. Koser and Rettger's Basal Glycerol Salt Solution 176 F3. Carbon present as organic acids or their salts. Puriewitsch's Tartaric Acid Salt Solution 177 C2. Magnesium salts not added. Di. Phosphates not added. Omeliansky's Cellulose Solution 178 Killer's Mannitol Solution 179 Ringer's Salt Solution 180 D2. Phosphates added. Ei. Calcium salts not added. Bijerinck's Glucose Salt Solution. . . . 181 Stoklasa's Glucose Salt Solution. . . . 182 Gag's Maltose Salt Solution 183 Sohngen's Colloid Mannitol Solution. 184 E2. Calcium salts added. Fi. Carbon present as carbohydrates. Bijerinck's Phosphate Glucose Solu- tion 185 Buhlert and Fickendey's Phosphate Glucose Solution 186 F2. Carbon present as alcohols or acids. Wojtkiewicz's Mannitol Salt Solu- tion 187 Kranisky's Mannitol Salt Solution . . . 188 Peklo's Modified Bijerinck's Man- nitol Salt Solution 189 Butcher's Basal Chinate Salt Solu- tion 190 114. Pringsheim's Glucose Cellulose Solution Constituents: 1. Water 1000.0 cc. 2. Cellulose 25.0 g. 3. Glucose 0.2 or 0.4 g. Preparation: (1) Add 2 and 3 to 1. Sterilization: Not specified. Use: To study nitrogen assimilation meth- ane fermenting bacteria. Variants: The author used the following combinations to study nitrogen assimila- tion by Clostridium and azotobacter: (a) 10.0 g. cellulose +0.4 g. de.xtrose in 500.0 cc. solution (b) 5.0 g. cellulose +0.4 g. dextrose in 250.0 cc. solution (c) 10.0 g. cellulose +0.2 g. dextrose in 1000.0 cc. solution CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 39 (d) 5.0 g. cellulose +0.2 g. dextrose in 1000.0 cc. solution (e) 2.5 g. cellulose +0.2 g. dextrose in 500.0 cc. solution (f) 5.0 g. cellulose +0.2 g. dextrose in 500.0 cc. solution (g) 2.5 g. cellulose +0.2 g. dextrose in 500.0 cc. solution (h) 5.0 g. cellulose + 0.2 g. mannite in 1000.0 cc. solution Reference: Pringsheim (1909 p. 303), (1910 p. 224). 115. Waksman and Joffe's Basal Glucose Solution Constituents: 1. Water 1000.0 cc. 2. Glucose 30.0 g. Preparation : (1) Dissolve 2 in 1. (2) Add 2.0 g. of one of the added nu- trients. (3) Tube in 10 to 12 cc. lots. Sterilization: Sterilize at 15 pounds for 15 minutes. Use: To study reaction changes in Actino- mycetes metabolism. Added nutrients: The authors added 2.0 g. of one of the following nitrogen sources: (NH4)2S04 (pH of solution = 5.8) (XHOsCOa (pH of solution = 6.8) Urea (pH of solution = 7.4) Reference: Waksman and Joffe (1920 p. 39). 116. Buchanan's Sucrose Solution Constituents : 1. Water (tap) 1000.0 cc. 2. Sucrose (2.0%) 20.0 g. Preparation : (1) Prepare a 2.0% saccharose solution in tap water. (2) Distribute in 500.0 cc. lots in liter Sterilization: Method not given. Use: Development of gum produced by Bacillus radicicola. The author found that 1.0% N/1 malic acid or citric acid was sufficient to completely inhibit growth. 1.0% N/1 NaOH stimulated growth and gum production, while 2.0% prevented growth. To isolate the gum add 100.0 cc. of 95% alcohol, to the 2 week old culture, filtering the white pre- cipitate, wash in alcohol and dissolve in distilled water. Repeat the precipita- tion 5 times. Finally dry the precipi- tate. Saccharose may be used in higher concentrations. Reference: Buchanan (1909 p. 371). 117. Waksman and Joflfe's Basal Glycerol Solution Constituents : 1. Water to make 1000.0 cc. 2. Glycerol 30.0 g. Preparation : (1) Dissolve 2 in 1. (2) Add one of the test materials (added nutrients) to (1). When using casein and egg albumin, dissolve casein and egg albumin in N/10 NaOH. In using fibrin, add fibrin to each tube. (3) Solutions vary in pH = 7.2 to 7.7, urea pH = 8.0. (4) Tube in 10-12 cc. lots. Sterilization: Autoclave for 15 minutes at 15 pounds pressure. Use: To study change in reaction in Actinomycetes metabolism. Added nutrients: The authors added 5.0 g. of one of the following nitrogenous com- pounds: fibrin leucin casein glycocoll egg albumin urea peptone acetamide 5.0 g. asparagin Variants: The authors used 3.0 g. of glyc- erol and added 2.0 g. of NaNOa, NaNOs, or (NH4)2S04 as inorganic nitrogen sources. Reference: Waksman and JofTe (1920 pp. 36-37). 118. Buchanan's Glycerol Solution I Constituents: 1. Water (tap) 1000.0 cc. 2. Glycerol 5.0 cc. Preparation: (1) Dissolve 2 in 1. Sterilization: Not specified. Use: Cultivation of Monascus purpurens from silage. Variants: The author used a 10.0, 20.0 or 40.0% glycerol solution instead of 5.0%. Growth in the 5.0 and 10.0% solution, but little or no growth in the 20.0 or 40.0% solutions. Reference: Buchanan (1910 p. 102). 40 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 119. Beijerinck and van Delden's Phosphate Glucose Solution Constituents : 1. Water 1000.0 cc. 2. Glucose 20.0 g. 3. K2HPO4 0.5 g. Preparation: (1) Dissolve 2 and 3 in 1. Sterilization: Not given. Use: To study nitrogen assimilation by chroococcum. Variants: Stoklasa prepared a solution of 25.0 g. of glucose and 1.5 g. K2HPO4 in 1000.0 cc. of Moldau river water. He studied the relation between the assim- ilation of phosphoric anhydride and nitrogen by azotobacter. It was deter- mined that 1.0 g. of phosphoric acid anhydride and 2 to 2.3 g. elementary N were assimilated. Reference: Beijerinck and van Delden (1902 p. 8), Stoklasa (1911 p. 461). 120. Beijerinck's Phosphate Sucrose Solution Constituents: 1. Water 1000.0 cc. 2. Sucrose 20.0 g. 3. K2HPO4 0.2 g. Preparation: (1) Dissolve 2 and 3 in 1. Sterilization: Not specified. Use: Growth of Bacillus radicicola. Reference: Beijerinck (1901 p. 575). 121. Beijerinck's Phosphate Mannitol Solution Constituents : 1. Water 1000.0 cc. 2. Mannitol 20.0 g. 3. K2HPO4 0.2 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Reaction is slightly alkaline. (3) Inoculate with 0.1 to 0.2 g. earth. Sterilization: Not specified. Use: Used for the enrichment of Azotobac- ter chroococcum and for nitrogen assim- ilation studies. Variants : (a) Beijerinck specified the use of ditch water. (b) Jacobitz specified the use of distilled water and studied nitrogen fixation hy Bacillus ellenhachensis a (Caron). (c) Christensen specified distilled water and suggested that CaCOs might be added if desired. (d) Percival specified the use of tap water. (e) Beijerinck and van Delden used 0.5 g. K2HPO4 instead of 0.2 g. K0HPO4. (f ) Stoklasa specified Moldau river water and used 0.5 g. potassium phosphate instead of 0.2 g. K2HPO4. Reference: Beijerinck (1901 pp. 568, 578), Jacobitz (1903 p. 101), Christensen (1907 p. 110), Percival (1920 p. 179), Beijerinck and Van Delden (1902 p. 8), Stoklasa (1908 p. 489). 122. van Delden's Basal Lactate Solution Constituents : 1. Water (ditch) 1000.0 cc. 2. K2HPO4 0.2 g. 3. Sodium lactate 1.0 g. Preparation : (1) Dissolve 2 and 3 in 1. (2) Additional nutrients may be added as indicated below. Sterilization : Not specified. Use : Cultivation and enrichment of Micro- spira desulfuricans . Used also to study sulphate reduction. Variants : (a) The author used 0.5 g. sodium lactate. (b) The author used 0.5 g. K2HPO4 and 1.0 g. of calcium lactate. Added nutrients: (a) The author did not specify the use of ditch water, and added (NH4)2S04 andO.Og. MgS04-7H20. (b) The author did not specify the use of ditch water and added 0.2 g. glycocoU andO.Og. MgS04-7H20. (c) The author used 0.5 g. sodium lactate with 0.5 g. K2HPO4 and added 1.0 g. asparagin and 0.0, 0.3, 0.6, 0.9, or 1.2 g. gypsum. (d) The author specified the use of tap water, used 0.5 g. sodium lactate with 0.5 K2HPO4, added NH4CI and 1.2 g. gypsum. (e) The author specified the use of tap water, used 0.5 g. of sodium lactate with 0.5 g. K2HPO4 and added 0.5 g. asparagin. (f) The author specified the use of tap water, used 0.5 g. of sodium lactate with 0.5 g. K2HPO4 added 1.0 g. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 41 asparagin and 1.0 g. gypsum or 4.0 g. MgS04-7H20. (g) The author specified the use of tap water, used 0.5 g. of sodium lactate with 0.5 g. K2HPO4 added 2.5 g. asparagin and 4.0 g. MgS04-7H20. (h) The author specified the use of tap water, used 10.0 g. sodium lactate, added 1.0 g. of asparagin and 2.0 or 5.0 g. of MgS04-7H20. Reference: van Delden (1903-04 p. 85). 123. Beijerinck and van Delden's Acetate Solution Constituents: 1. Water 1000.0 cc. 2. K2HPO4 0.5 g. 3. Sodium acetate 5.0 g. Preparation: (1) Dissolve 2 and 3 in 1. Sterilization: Not specified. Use: Cultivation of chroococcum and granulobacter. The medium was in- oculated with 20.0 g. of fresh earth. Reference: Beijerinck and van Delden (1902 p. 29). 124. Gage's Glucose Solution Constituents: 1. Water 1000.0 cc. 2. Glucose 5.0 g. 3. MgS04 0.25 g. 4. CaCl2 0.01 g. 5. Iron sulphate solution 1 drop Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Sterilization: Not specified. Use : To study the fixation of nitrogen by Pseudomonas radicicola and nitroso bac- teria. It was found that nitrites and possibly nitrates were produced by Bacil- lus radicicola in this medium. Reference: Gage (1910 p. 34). 125. Stoklasa's Phosphorus Free Glucose Salt Solution Constituents : 1. Distilled water 1000.0 cc. 2. d-glucose 25.0 g. 3. K2SO4 1.0 g. 4. MgCl2 0.5 g. 5. Iron sulphate 0.1 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. Sterilization: Not specified. Use : To study the role of phosphorus in the soil. It was found that growth of nitro- gen fi.xing organisms was very poor in this solution. Phosphorus is required for luxuriant and rapid development. Variants : (a) The author suggested the addition of 1.0 g. of nitrogen in the form of (NH4)2S04or NaNO.,. (b) The author suggested the addition of 0.1 g. of aluminum sulphate and 1.0 g. of one of the following phosphorus compounds: dicalcium phosphate CaHP04-2H20. Monodiferric phosphate Fe203(P205)3. Monodialuminum phosphate AI2O3- (P205)2-8H20. Tricalcium phosphate Ca3(P04)2-2H20. Ditriferric phosphate (Fe03)4(P205)5- 3H2O. Triferric phosphate Fe203P205 + 4H2O. Trialuminum phosphate AloOj-PjOs + 4H2O. (c) The author used variant (b) and sub- stituted arabniose for d-glucose. (d) The author added aluminum sulphate and the equivalent of 0.2 g. of phos- phoric anhydride in the form of monomagnesium phosphate, mono- dialuminum phosphate, monodiferric phosphate or trimagnesium phos- phate to the original solution. References: Stoklasa (1911 pp. 492, 441, 490). 126. Gerlach and Vogel's Basal Glucose Salt Solution Constituents: 1. Distilled water 1000.0 cc. 2. Glucose 5.0 g. 3. Potassium phosphate 0.5 g. 4. MgS04 0.3 g. 5. NaCl 0.5 g. 6. Na.COs 0.5 g. 7. FeS04 0.2 g. Preparation : (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (2) Add one of the nitrogen sources listed in added nutrients. (3) Adjustment of reaction not given. Sterilization : Method not specified. When utilizing urea as nitrogen source sterilize the urea at 100°C. dry heat. Use: Cultivation of albumin formers from soil and stable manure. These organ- isms grow very well on agar, gelatin and 42 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS bouillon which contain 0.3% NaNOs. The nitrate in this medium is changed to nitrite after a few days and then quanti- tively to insoluble albuminous materials. Ammonia salts and urea also are suitable nitrogen sources for albumin production by these organisms. Albumin produc- tion using urea however goes on very slowly. Added nutrients : The author employed the following nitrogen sources: NaNOa (c.p. Merck) 3.0 g (NH4)2S04 3.0 g NH4NO3 2.0 g NH4CO3 2.0 g urea 20.0 g Reference : Gerlach and Vogel (1901 p. 612) 127. Henneberg's Basal Glucose Disodium Phosphate Solution Constituents : 1. Water 1000.0 cc. 2. Na2HP04 3.0 g. 3. MgS04 2.0 g. 4. NaCl 2.0 g. 5. Glucose 20.0 g. Preparation : (1) Dissolve 2, 3, 4 and 5 in 1. (2) Add one of the added nutrients (amounts not specified). (3) Adjustment of reaction not given. Sterilization: Method not given. Use: To study the food requirements for acetic acid organisms, B. Pasteurianum, B. oxydans, B. aceti and B. Kiltzingi- anum. The author found that KNO 3 was the poorest source of nitrogen and (NH4)2S04 was the best. Added nutrients : The author employed one of the following salts or carbon sources: KNO3 asparagin (NH4)2S04 peptone ammonium tartrate Reference: Henneberg (1898 p. 18). 128. Beijerinck's Glucose Salt Solution Constituents : 1. Water 1000.0 cc. 2. KH2PO4 0.5 g. 3. MgS04 0.5 g. 4. Glucose 10.0 g. Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization: Not specified. Use: Growth oi Streptothrix chromogena. Variants : (a) Peklo used the solution to grow plant actinomyces. He specified that the solution might be neutralized by the addition of 0.5 g. NaoCOs. (b) Malenkovic used 100.0 g. glucose, 1.0 g. K2HPO4 and added 2.0 g. of KNO3or2.0g.NH4H2PO4. He culti- vated coniophora cerebello (Corti- cium putaneum) and reported NH4H2PO4 a better nitrogen source. Reference: Beijerinck (1900 p. 7), Peklo (1910 p. 514). Malenkovic (1906 p. 412). 129. Kisch's Basal Glucose Salt Solution Constituents: 1. Water 1000.0 cc. 2. K2HPO4 1.0 g. 3. MgS04 0.5 g. 4. NaCl 0.02 g. 5. FeS04 trace 6. Calcium phosphate trace 7. Glucose 10.0 g. Preparation : (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (2) Add Na2C03 until the reaction is slightly alkaline to litmus. (3) Add one of the nitrogen sources listed under added nutrients to (2). Sterilization: Not specified. Use: To study the nitrogen requirements of members of the colon-typhoid group. Added nutrients: The author added one of the following nitrogen sources. (a) KNO3 0.29 g. Bad. paratyphi B. shows growth. Bad. enter itidis Gartner little growth. (b) KNO2 2.43 g. No growth. (c) Urea 8.6 g. Bad. coli and Bad. enteritidis Gart- ner show some growth. (d) Urea 8.6 g. KNO2 2.4 g. B. coli shows growth. (e) (NH4)2S04 1.9 g. B. paratyphi shows good growth. B. coZt shows good growth. B. enteri- tidis Gartner, and dysentery bacillus show good growth. (f) NH4H2PO4 1.42 g. Bad. paratyphi shows good growth. CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 43 Bad. colt shows good growth. B. enteritidis Gartner and dysentery bacillus show good growth.