REISOLATION AND EMENDATION OF DESCRIPTION OF VIBRIO MARINUS (RUSSELL) FORD'

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1 JOURNAL OF BACTERIOLOGY Vol. 88, No. 4, p October, 1964 Copyright 1964 American Society for Microbiology Printed in U.S.A. REISOLATION AND EMENDATION OF DESCRIPTION OF VIBRIO MARINUS (RUSSELL) FORD' RITA R. COLWELL AND RICHARD Y. MORITA Department of Biology, Georgetown University, Washington, D.C., and Departments of Microbiology and Oceanography, Oregon State University, Corvallis, Oregon ABSTRACT COLWELL, RITA R. (Georgetown University, Washington, DNC.), AND RICHARD Y. MORITA. Reisolation and.inaendation of description of Vibrio marinus (Russell) Ford. J. Bacteriol. 88: Sixteen fresh isolates from the marine environment were subjected to taxonomic analysis. From the taxonomic data, both quantitative and qualitative, these strains appear to form a speciesgroup closely related to the Vibrio marinus isolated by Russell from the gulf of Naples in The V. marinus species description is extended. As a peculiarly marine species, the organisms are distinguished by features of inorganic salt requirement and psychrophilism, as well as by an ability to form acid from glucose anaerobically and a marked curvature of the cells. The role of bacteria in the sea is under increasingly intensive study (Oppenheimer, 1963; Kriss, 1959). In the marine environment, bacteria are particularly important in the mineralization, assimilation, and degradation processes. Other functions ascribed to marine bacteria, aside from their contribution to the total biomass and the various trophic levels of the sea, are the diagenesis of sedimentary material and production of ectocrine compounds. The classification and taxonomy of the microorganisms found in the sea are currently vital areas of study for information purposes. Bacterial species commonly found in the sea have been sketchily studied, and the descriptions are not always helpful in identification of fresh isolates. Furthermore, the testing of bacterial cultures isolated from the marine environment was frequently done by use of media made up with natural seawater, since seawater can be only superficially duplicated with respect to the inorganic ions and trace organic matter. With 1 Published as technical paper no. 1800, Oregon Agricultural Experiment Station. Received for publication 27 March 1964 the information now available concerning the nutrition and growth requirements of marine bacteria (MacLeod and Onofrey, 1956, 1963), descriptions of marine species based on the use of chemically defined media permitting optimal growth can be presented. This is not to say that all previous descriptions of marine strains are not meaningful or useful. The earliest recorded descriptions, such as those prepared by Russell (1891), Fischer (1894), and Benecke (1905), serve as valuable reference for current studies on marine microorganisms, but the use of rigorously defined methods and media in a taxonomic study should improve earlier descriptions by bringing them up to date. Also, it can be noted that the rapid identification techniques resorted to in laboratories aboard research vessels can yield much more significant information, if techniques are based on a larger study of the taxonomy of marine bacteria. MATERIALS AND METHODS Source of strains. Vibrio marinus strain PS 207 was isolated from the skin of a Pacific cod caught off Port Orchard, Wash. Sampling conditions and procedures were described elsewhere (Colwell, 1962). Cultures designated as MP were from seawater off the coast of Oregon. The MP cultures were isolated from a water sample raised from 1,200 m depth in the north Pacific Ocean (Morita and Haight, 1964). The temperature of this water was 3.24 C. The water (ca. 500 ml) obtained was placed immediately under aseptic conditions in a sterile bottle containing about 0.5 g of sterile yeast extract dissolved in 10 ml of distilled water. The water sample was refrigerated (ca. 5 C) until the boat docked, and the sample was then transported back to the laboratory under ice. The time interval between the isolation of the cultures and collection of the water was approximately 5 days. The medium used for the isolation of MP 831

2 832 COLWELL AND MORITA J. BACTERIOL. cultures consisted of: Neopeptone (Difco), 5 g; ferric phosphate, 0.01 g; yeast extract (Difco), 1 g; agar (Difco), 15 g; aged seawater, 750 ml; and distilled water, 250 ml. The final ph of the medium after autoclaving was 7.2. Petri dishes, containing the medium, were cooled to refrigeration temperature before use. The medium, without agar and dispensed into test tubes (16 by 150 mm) in 5-ml portions, was used to maintain the cultures. The liquid medium was also cooled to refrigeration temperature before use. Portions (ca. 0.1 ml) of the water sample were spread over the surface of cooled agar plates. Colonies which developed on the agar surface at refrigeration temperature (ca. 5 C) were restreaked onto fresh agar plates and incubated at 24 C. All cultures that were capable of growth at 24 C were discarded, and the remaining isolates were then tested for their ability to grow at temperatures in the range of 0 to 24 C. All organisms were maintained in a stock medium which was an artificial seawater broth of the following composition: Proteose Peptone (Difco), 10 g; yeast extract (Difco), 3 g; artificial seawater (Seven Seas Mix, Utility Chemical Co., Paterson, N.J.), 1,000 ml; ph 7.2. Incubation temperature of all inoculated stocks and test media was 18 C. Morphological and cultural characteristics. After 24 hr of incubation at 18 C in artificial seawater broth, the organisms were Gram-stained and stained for demonstration of flagella. Colony appearances on artificial seawater agar [artificial seawater broth to which 2% (Difco) agar had been added] were noted after 3 days at 15 C. Seawater broth cultures were examined at 24 hr under phase contrast for morphology and motility. Environmental and cultural characteristics. Requirement for seawater was established by streaking pure cultures on agar plates containing artificial seawater agar and on agar plates with medium of identical composition, except that distilled water was used as diluent. The requirement for specific ions was determined by the use of several media which contained one or all of the following: NaCl, 2.4%; KCl, 0.07%; MgCl2 6H20, 0.53%; and MgSO4 7H20, 0.70%. All test media subsequently were made up with the four inorganic salts listed above as the "synthetic seawater" diluent. A standard inoculum was 1 drop from a pasteur pipette (ca ml) of a 24-hr artificial seawater broth culture. Temperature range of growth. The ability of the strains to grow in seawater broth at 0, 5, 10, 15, 20, 25, 30, and 37 C was recorded after incubation periods of 1 day to 2 weeks, depending on the incubation temperature employed. Sensitivity to antibacterial agents. The strains were tested for sensitivity to penicillin (10 units), dihydrostreptomycin (10,Ag), chloramphenicol (30,ug), oxytetracycline (30,ug), chlortetracycline (30,ug), erythromycin (15j,g), novobiocin (30,ug), polymyxin B (300 units), kanamycin (30 j,g), tetracycline (30,g), and the vibriostatic compound, 2,4-diamino-6,7-diisopropylpteridine (Shewan, Hodgkiss, and Liston, 1954). Sensi- Discs of the antibiotics (BBL) were used; the pteridine compound was sprinkled, in crystal form, onto moist, freshly streaked plates of the cultures tested. Physiological tests. The following tests were carried out, modified only by the use of synthetic seawater as diluent and incubation of inoculated test media at 18 C: gelatin liquefaction, action on litmus milk, reduction of nitrate to nitrite, ammonia production, indole formation, starch hydrolysis, cellulose decomposition, growth in Koser's citrate medium, growth in Simmons citrate medium, Voges-Proskauer and methyl red tests, and test for catalase (Society of American Bacteriologists, 1957). Other tests which were performed included: hydrogen sulfide formation (Colwell and Quadling, 1962); trimethylamine oxide reduction (Wood and Baird, 1943); production of acid from carbohydrates (Hugh and Leifson, 1953); production of 2-keto gluconic acid (Gaby and Free, 1958); lipolytic activity (Sierra, 1957); digestion of chitin (Benton, 1935; Pohja, 1960); growth and production of pyocyanine or fluorescein, or both, on Sabouraud maltose agar, Sabouraud dextrose agar, Burton's medium (Burton, Campbell, and Eagles, 1948), and King's medium (King, Campbell, and Eagles, 1948); production of acetic acid from ethanol; dihydroxyacetone from glycerol; production of carbonates on calcium lactate medium (Shimwell, Carr, and Rhodes, 1950); presence of oxidase (Kovacs, 1956; Ewing and Johnson, 1960; Gaby and Free, 1958); and casein hydrolysis (Demeter, 1943). The Hugh and Leifson (1953) method for

3 VOI,. 88, 1964 VIBRIO MARINUS 833 testing for the production of acid from carbohydrates was modified further; in addition to the substitution of synthetic seawater for the salts and distilled water, the agar was omitted and a Durham vial was added to each tube for checking gas production. The synthetic seawater litmus milk was prepared by first sterilizing the Litmus Milk (Difco) and double-strength synthetic seawater and mixing the components aseptically after cooling. Fermentative attack on glucose was checked further by the addition of sodium iodoacetate, sterilized by filtration and at M concentration, to the glucose-synthetic seawater-broth tubes. Hydrolysis of esculin was checked by inoculation of a medium consisting of 0.1% esculin added to synthetic seawater agar, with 0.05% ferric citrate as indicator for the breakdown of esculin. Nutritional requirements of the organisms for growth were tested on a 0.4% vitamin-free Casamino Acids and synthetic seawater medium and on media containing synthetic seawater, 1.5% agar (ph 7.2), and each of the following amino acids at 1.0% concentration: DL-alanine, 3-alanine, L-arginine, L-lysine, L-proline, L-phenvlalanine, and tyrosine. Ammonium phosphate and sodium formate were also tested. To measure quantitatively the homogeneity of the group of strains studied, a numerical analysis with the use of the computer method of Colwell and Liston (1961) was carried out. A set of 123 coded features was obtained for each strain. From the coded data, the triangular matrix of similarity values was calculated by computer (Sneath, 1957). The computer output was further treated according to the methods of Liston, Wiebe, and Colwell (1963) and Quadling and Colwell (1963) for assessment of homogeneity and analysis of variance within the group. Comparison with a number of marine isolates from a previous study (Colwell and Gochnauer, 1963) aided in the initial identification and classification of the strains. An IBM 1620 computer was employed in the calculations; details concerning programs may be obtained from the senior author. RESULTS The 16 strains studied were gram-negative; all, except one strain, MP-21, were actively motile. As a group, the strains were homogeneous, with a high mean similarity index, AS = 81%, determined by numerical analysis of the coded features (Liston et al., 1963). Morphology. The organisms were all slightly curved rods, short to medium length (0.6 to 1.2 Iu), and width of 0.6 to 1.0,I, with rounded ends and occurring singly and in pairs. "Round bodies" or rounded forms were often visible, which corresponded to the observations of Liston (1954) for the gut-group vibrios. Studies which indicate that temperature plays a significant role in the formation of these round body forms will be presented in a separate communication. Cultural characteristics. Colonies on seawater agar were grayish, opaque, and raised (2 to 5 mm diameter), with an entire edge and a smooth surface. No diffusible pigments were observed, either in daylight or under ultraviolet. In seawater broth, the appearance of the growth was uniform with an even, slight turbidity and no pellicle. Growth requirements. The organism designated as PS-207 (Colwell, 1962) was able to grow at temperatures from 20 to 30 C, 30 C being the maximum (Burton and Morita, 1963; Morita and Burton, 1963). Temperature characteristics of isolate MP 1 were investigated in an earlier study (Morita and Haight, 1964). None of the MP cultures grew above 24 C, and some of the isolates had maximal growth temperatures of 20 C or less. The isolates were all seawater-requiring, that is, no growth was obtained on media without seawater. A requirement for Na+ is strongly indicated by the inability of the organisms to grow in media containing various combinations of 0.01 M KCl, M MgCl2 *6H20, M MgSO4-7H20, and 0.1% Trypticase (Tyler, Bielling, and Pratt, 1960). Furthermore, a replacement of NaCl with sucrose did not support growth. The nutritional requirements of the organisms were not complex but were restricted. Four strains were able to grow on a vitamin-free, Casamino Acids medium and on a simple medium of DL-alanine and synthetic seawater. The amino acid, L-proline, was found to be an adequate source of carbon and nitrogen for all strains. However, other amino acids tested did not support growth. No growth occurred when selected carbon sources, including formate, citrate, and lactate, were supplied in the presence of NH4+.

4 834 COLWELL AND MORITA J. BACTERTOL. TABLE 1. Selected characteristics descriptive of the species Vibrio marinus Characteristic* Resultt Salt requirement (seawater), ca. 0.5 M (100) Temperature relations (growth), ca. Oto 20 C (100) Oxidase-positive (100) Catalase-positive (100) Glucose fermented, without production of gas (100) Acid produced from maltose (87.5) Starch hydrolyzed... 5 (31.5) Chitin digested (100) Gluconate oxidized to ketogluconate 0 (0.0) Cellulose decomposed... 0 (0.0) Growth on calcium lactate medium. 0 (0.0) Growth on Simmon's citrate medium (0.0) Visible, diffusible pigment produced... 0 (0.0) Litmus milk acid... 1 (6.3) Gelatin liquefied (87.5) Casein hydrolyzed (100) Growth on L-proline-synthetic seawater medium (100) Indole produced... 0 (0.0) Methyl red-positive... 1 (6.3) Voges-Proskauer-positive... 1 (6.3) * Total of 16 strains were examined. Test media were prepared with synthetic sea salts. See Materials and Methods. t Number of strains; figures in parentheses are percentages of the total number of strains positive. Sensitivity to antibacterials. All 16 strains were sensitive to chloramphenicol, novobiocin, and oxytetracycline; 15 of the strains were sensitive to erythromycin and tetracycline; 14 to chlortetracycline; and 13 to polymyxin B and kanamycin. None was sensitive to penicillin or streptomycin. All except five of the strains exhibited a very marked sensitivity to the vibriostatic compound, but the results were variable, depending on the age of the culture, the medium, and the incubation time and temperature. Catalase and oxidase formation. All isolates were catalase-positive and oxidase-positive. Gelatin liquefaction. All but two strains, MP-21 and PS 207, liquefied gelatin; the reaction was usually observed to occur within an incubation period of 1 week. Liquefaction was stratiform. Action on litmus milk. None of the strains grew in litmus milk without added inorganic salts. In the seawater-litmus milk, only one strain, PS 207, produced any reaction, a slight acidity and a slight reduction of the litmus. Reduction of nitrate to nitritc. All strains but one reduced nitrate to nitrite. In addition, one strain, PS 207, reduced nitrite to ammonia or nitrogen gas. Hydrogen sulfide formation. One strain, PS 207, produced hydrogen sulfide when grown on a lead acetate-seawater medium. Further examination showed that this organism produced hydrogen sulfide from cysteine but not from sodium thiosulfate. None of the other strains tested was able to produce hydrogen sulfide. Indole formation. None of the strains gave an indole-positive reaction. Carbohydrate utilization. All strains produced acid from glucose; 14 strains produced acid from maltose, 7 from galactose, and none from fucose. None oxidized gluconate to the ketogluconate. Five strains hydrolyzed starch. All strains tested were fermentative in attack on glucose; that is, they produced acid but no gas both aerobically and anaerobically (Hugh and Leifson, 1953). No acid was produced from glucose in the presence of iodoacetate. Only two strains grew in the glucose-iodoacetate medium, and the growth observed was very slight. None of the strains produced dihydroxyacetone from glycerol. Voges-Proskauer and methyl red tests. Only one strain was able to produce positive Voges- Proskauer and methyl red reactions, and only when tested in a medium made up with seawater or a seawater substitute. Digestion of chitin. All strains grew on the chitin medium and produced measurable zones of clearing, indicating hydrolysis of the chitin. Degradation of cellulose. None of the cultures was able to attack cellulose. Lipolytic activity. Lipolysis, as measured by reaction on media containing the fatty acid esters, was positive. No pigment production was observed on any of the media used. Esculin. Only one strain, PS 207, was able to hydrolyze esculin. Casein. Of the 16 strains, 13 were able to hydrolyze casein. In Table 1, the data for the V. marinus strains are summarized.

5 VOL. 88, 1964 VIBRIO MARINUS 835 DISCUSSION Bacteria isolated from marine sources are particularly difficult to identify, since very few full descriptions of marine species are available in the literature. ZoBell and Upham (1944) published a list of 60 "new species," but these descriptions are based predominantly on morphology, appearance of growth on gelatin, agar, or potato, and in broth; several other features such as attack on litmus milk, nitrate reduction, indole production, and acid production from eight simple carbohydrates were included. The media used by ZoBell and Upham (1944), with the exception of freshwater broth, litmus milk, and potato slants, were made up with filtered seawater collected at the end of Scripps Pier. Reproducing these particular test conditions, therefore, for identifying freshly isolated marine bacteria is very difficult. Other workers, more recently, have concerned themselves with the problem of differentiation of bacterial isolates from the marine environment (Kriss, 1959; Shewan, 1963). However, "aged sea water" was also used in the test media on which the determinative schema are based. Even though it can be conceded that there are taxa of marine bacteria that can be isolated and identified only by using natural seawater, it is nevertheless useful to present descriptions of those groups indigenous to the sea which can be isolated, examined, and identified under standardized conditions. Such information, collected in marine laboratories located in various geographical areas, will then be compatible and easilv duplicated for purposes of identification and classification. The particular group of bacteria described in this paper demonstrate a detectable requirement for NaCl, and grow only at low temperatures (O to 30 C). It should be recognized that the ambient temperature of the oceans is considered to range from -1.5 to 27.4 C, but well over 90% of the ocean volume is at a temperature below 5 C. Since representatives of the bacterial group described in this work have been isolated at different times of the year, in various areas of Puget Sound and off the Pacific Coast (Colwell, 1962), it appears that they may constitute a truly marine group and not adventitious organisms collected from the marine environment. Russell (1891) described a bacterial species which he had isolated from samples taken in the Bay of Naples. The species, Spirillum marinum, as described by him included the following salient features: "small bent or slightly curved bacillus, usually in pairs. Several elements may be joined together, producing a characteristic spirillum. Motility.-rapid progressive and rotatory movements... Grows only at low temperatures. No growth at 37 C... liquefaction with turbidity of the liquid gelatin." The name of this organism was later listed in Bergey's Manual (Bergey et al., 1939) as V. marinus, according to emendation by Ford (1927). The characterization of the organism emphasized the marked halophilic [sic] tendencies and low temperature of growth. The group of bacteria described here appears to resemble this organism sufficiently, so that we have applied the epithet, V. marinus (Russell) Ford. A direct comparison of the original isolates and our isolate was not possible, since Russell's isolate was not available in any culture collection. The description of V. marinus (Russell) Ford is emended and extended as follows. Gram-negative asporogenous rods. Cells generally curved, with round bodies occasionally observed. Soma size, 0.6 to 1.2 y. Occurring as singles and pairs. Polarly flagellated. Growth on synthetic seawater agar (NaCl, 2.4%; KCl, 1.07%; MgC92*6H20, 0.53%; MgSO4 7H20, 0.70%; Proteose Peptone (Difco), 10 g; yeast extract (Difco), 3 g; agar, 15 g; distilled water, 1,000 ml; ph = 7.2; incubated at 18 C for 3 days: grayish, opaque, raised, 2 to 5 mm diameter, with entire edge. No visible diffusible or fluorescent pigment produced. Even, slightly turbid growth in seawater broth with slight or no pellicle formation. Catalase-positive. Oxidase-positive. Sensitive to vibriostat 0/129. Not sensitive to penicillin (10 units). Litmus milk-negative or very slight acidity produced. Gelatin usually liquefied. No growth on skim-milk agar but casein hydrolysis produced on sodium caseinate-synthetic seawater agar. Indole-negative. Voges-Proskauer and methyl red tests usually negative. Reduces nitrate to nitrite and may reduce nitrite. Ferments glucose without production of gas and is inhibited by sodium iodoacetate in the fermentation reaction. Gluconate not oxidized to the ketogluconate. Starch and esculin not usually hydrolyzed. Chitin digested. Cellulose and fucose not utilized. No growth on formate,

6 836 COLWELL AND MORITA J. BACTERIOL. citrate, or lactate medium. Good growth on proline-synthetic seawater agar. Working neotype cultures (MP-1 and PS 207) were deposited with the American Type Culture Collection, and were given the accession numbers and 15382, respectively. ACKNOWLEDGMENTS This work was supported by grants GB-1623 and G from the National Science Foundation. LITERATURE CITED BENECKE, W Ueber Bacillus chitinovorus, einem Chitinzersetzenden Spaltpilz. Botan. Z. Abt. 1 63: BENTON, A. G Chitinovorous bacteria. J. Bacteriol. 29: BERGEY, D. H., R. S. BREED, E. G. D. MURRAY, AND A. P. HITCHENS Bergey's manual of determinative bacteriology, 5th ed. The Williams & Wilkins Co., Baltimore. BURTON, M. O., J. J. R. CAMPBELL, AND B. A. EAGLES The mineral requirements for pyocyanine production. Can. J. Res. 26C: BURTON, S. D., AND R. Y. MORITA Denaturation and renaturation of malic dehydrogenase in a cell-free extract from a marine psychrophile. J. Bacteriol. 86: COLWELL, R. R The bacterial flora of Puget Sound fish. J. Appl. Bacteriol. 25: COLWELL, R. R., AND M. B. GOCHNAUER The taxonomy of marine bacteria. Bacteriol. Proc., p. 40. COLWELL, R. R., AND J. LISTON Taxonomic relationships among the pseudomonads. J. Bacteriol. 82:1-14. COLWELL, R. R., AND C. QUADLING Miniature tube tests in diagnostic bacteriology. Can. J. Microbiol. 8: DEMETER, J. K Bakteriologische Untersuchungs Methoden von Milch, Milcherzeugnissen, Molkereihilfsstoffen und Wersandsmaterial, p. 48. Berlin. EWING, W. H., AND J. G. JOHNSON The differentiation of Aeromonas and C27 cultures from enterobacteriaceae. Intern. Bull. Bacteriol. Nomen. Taxon. 10: FISCHER, B Die Bakterien des Meeres. Ergeb. Plankton-Expedition der Humboldtstiftung 4:1-83. FORD, W. W Text-book of bacteriology, p W. B. Saunders Co., Philadelphia. GABY, W. L., AND E. FREE Differential diagnosis of Pseudomonas-like microorganisms in the clinical laboratory. J. Bacteriol. 76: HUGH, R., AND E. LEIFSON The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J. Bacteriol. 66: KING, J. V., J. J. R. CAMPBELL, AND B. A. EAGLES The mineral requirements for fluorescin production. Can. J. Res. 26C: KoVACs, N Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178:703. KERISS, A. E Marine microbiology (deep sea). [In Russian.] Acad. Sci. U.S.S.R., Moscow. LISTON, J A group of luminous and nonluminous bacteria from the intestine of flatfish. J. Gen. Microbiol. 12:i. LISTON, J., W. WIEBE, AND R. R. COLWELL Quantitative approach to the study of bacterial species. J. Bacteriol. 85: MACLEOD, R. A., AND E. ONOFREY Nutrition and metabolism of marine bacteria. II. Observations on the relation of sea water to the growth of marine bacteria. J. Bacteriol. 71: MACLEOD, R. A., AND E. ONOFREY Studies on the stability of the Na+ requirements of marine bacteria, p In C. H. Oppenheimer [ed.], Symposium on marine microbiology. Charles C Thomas, Publisher, Springfield, Ill. MORITA, R. Y., AND S. D. BURTON Influence of moderate temperature on growth and malic dehydrogenase activity of a marine psychrophile. J. Bacteriol. 86: MORITA, R. Y., AND R. D. HAIGHT. 19e4. Temperature effects on the growth of an obligate psychrophilic marine bacterium. Limnol. Oceanog. 9: OPPENHEIMER, C. H Symposium on marine microbiology. Charles C Thomas, Publisher, Springfield, Ill. POHJA, M. S Micrococci in fermented meat products. Acta Agral. Fennica 96:1-80. QUADLING, C., AND R. R. COLWELL The use of numerical methods in characterizing unknown isolates. Develop. Ind. Microbiol. 5: RUSSELL, H. L Untersuchungen uber im Golf von Neapel lebende Bakterien. Z. Hyg. Infektionskrankh. 11: SHEWAN, J. M The differentiation of certain genera of gram-negative bacteria frequently encountered in marine environments, p In C. H. Oppenheimer [ed.], Symposium on rmarine microbiology. Charles C Thomas, Publisher, Springfield, Ill. SHEWAN, J. M., W. HODGKISS, AND J. LISTON A method for the rapid differentiation of certain

7 VOL. 88, 1964 VIBRIO MARINUS 837 non-pathogenic, asporogenous bacilli. Nature SOCIETY OF AMERICAN BACTERIOLOGISTS :208. Manual of microbiological methods. McGraw- SHIMWELL, J. L., J. G. CARR, AND M. E. RHODES. Hill Book Co., Inc., New York Differentiation of Acetomonas and TYLER, M. E., M. C. BIELLING, AND D. B. PRATT. Pseudomonas. J. Gen. Microbiol. 23: Mineral requirements and other charac- SIERRA, G A simple method for the detection ters of selected marine bacteria. J. Gen. Microof lipolytic activity of microorganisms and biol. 23: some observations on the influence of the WOOD, A. J., AND E. A. BAIRD Reduction of contact between cells and fatty substances. trimethylamine oxide by bacteria. J. Fisheries Antonie van Leeuwenhoek Ned. Tijdschr. Res. Board Can. 6: Hyg. Microbiol. Serol. 23: ZoBELL, C. E., AND H. C. UPHAM A list of SNEATH, P. H. A The application of com- marine bacteria including descriptions of sixty puters to taxonomy. J. Gen. Microbiol. 17: new species. Bull. Scripps Inst. Oceanog :