Studies of the marine bacterium Alteromonas haloplanktis 214 (formerly referred to as marine pseudomonad B-16) showed that as the Na+ concentration in the growth medium decreased from 230 to 34 mM, the lowest concentration permitting growth, the length of the lag period preceding exponential growth increased. Once growth had begun, except for a slight reduction in rate of growth at 34 mM Na+, the generation time and extent of growth remained essentially constant over the range of Na+ concentrations tested. Plate counts showed that during the lag period the numbers of viable cells introduced as inoculum into a complex medium containing 33 mM Na+ decreased exponentially before increasing. Repeated subculture of the cells at 33 mM Na+ failed to eliminate the lag period or reduce the loss of viability of the cells. The viability loss and the lag period could be eliminated either by raising the NaCl concentration to 130 mM or by adding sufficient sucrose to make the osmotic pressure of the medium equal to that obtained by adding 130 mM NaCl. In a chemically defined medium, sucrose added to maintain tonicity reduced but did not eliminate the lag periods obtained at suboptimal Na+ concentrations. Increasing the number of cells plated on trypticase agar medium reduced the Na+ concentration required to permit growth. Evidence was obtained of a requirement of A. haloplanktis for Ca2+ for growth. Ca2+ spared to a small extent the requirement for Na+ for growth. Some 10(10) cells of a histidine-requiring, streptomycin-resistant mutant of A. haloplanktis 214, still viable after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, were screened for capacity to grow in the absence of Na+. Since no non-Na+-requiring mutants were isolated, the requirement of this organism for Na+ would appear to be extremely stable.

Purification of,-hemolysin was achieved by ammonium sulfate precipitation, Sephadex G-100 gel filtration, carboxymethyl cellulose column chromatography, and density gradient electrophoresis. Active fractions eluted from carboxymethyl cellulose contained at least one nonhemolytic protein, and omission of this step was not detrimental to the purification process. Density gradient electrophoresis yielded approximately 1.6 mg of highly active purified ,B-hemolysin per liter of culture supernatant liquid. Purified f-hemolysin gave a single line on gel double diffusion and immunoelectrophoresis. A single symmetrical peak formed in the analytical ultracentrifuge, and the sedimentation coefficient was calculated to be 1.7S. The purified ,B-hemolysin was stable at 4 C and could be lyophilized. Magnesium cations were required for full expression of,-hemolytic activity. f3-Hemolysin was lethal for rabbits when injected intravenously in amounts between 40 and 160 ,ug. Crude ,B-hemolysin was more stable than purified f-hemolysin when heated at 60 C for 30 min. Purified f3-hemolysin lost almost all of its activity on subsequent heating at 100 C for 10 min.

Until now there has not been a satisfactory solid medium for determining the growth responses, to Na+, of marine and other bacteria that have specific growth requirements for Na+. A solid medium would be useful to investigators who would like to take advantage of the efficiency of multipoint inoculation when testing for a Na+ requirement. By using 1% gellan gum (Gel-GroTM) as the solidifying agent a medium was formulated that had a contaminating level of Na+ of slightly less than 2 mM in the basal medium. Two species of Aeromonas, which do not require Na+ for growth, and 31 species of Vibrio, which require Na+, were tested for their growth responses to Na+ on this medium. The Aeromonas strains grew well, within 24 h, at all of the Na+ concentrations tested. Approximately 75% of the Vibrio strains did not grow on the basal medium even after a prolonged incubation period. The remaining species were able to grow on the basal medium, but not without a lag period. These lag periods were as short as 36 h for two of the species and in some instances as long as 312 h. These lag periods were of sufficient duration to determine that Na+ stimulated the growth of the Vibrio strains that were able to grow on the basal medium. Approximately 75% of the strains, representing most species of Vibrio, were able to grow if as little as 25 mM Na+ was present in the medium.Key words: low-sodium medium, Na+ requirement, gellan gum, agar substitute, marine bacteria.

Eighty regional strains of Vibrio isolated from the seasonally cold waters of coastal Newfoundland, and a number of Vibrio reference cultures, were studied. The regional strains had been isolated from the brown macroalga Alaria esculenta and the giant scallop Placopecten magellanicus and were known to grow at 4 degrees C. The strains were grouped according to their arginine-dihydrolase reactions and examined by numerical analysis. According to phenotypic properties the arginine-dihydrolase positive strains closely resembled Vibrio splendidus biovar I. Most clusters of the arginine-dihydrolase negative strains appeared to be unique but the closest phenotypic resemblance among some strains was with Vibrio ordalii. Some strains were examined using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique for fingerprinting and it was shown that the regional strains were significantly different from either V. splendidus biovar I or V. ordalii. Generally, the strains from seaweed clustered separately from those that were from scallops. Strains in some clusters, especially those from the seaweed, were able to utilize most of the compounds that were tested as sole sources of carbon and energy.

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