A medium, solidified with gellan gum, for determining the Na⁺ requirement of Vibrio species

Abstract

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.

The taxonomic significance of the growth response to Na+by strains ofVibrio

“…The characteristics of marine bacteria, growing under these conditions, have been studied by Reichelt and Baumann (1974), , , Berthelet and MacLeod (1989), Manuel et al (1993), and Noble and Gow (1993). In the study presented here, groups of related Vibrio strains were studied for their growth responses to Na+ concentration.…”

“…The isolation of the strains has been described by Martin-Kearley and Gow (1994), who identified these as strains of Vibrio. The composition of the medium used to test for Na+ requirement, and the methods used to maintain the cultures and prepare them for multipoint inoculation have been described (Noble and Gow 1993).…”

“…The test medium, unsupplemented with Na+ and called the basal medium, is known to contain about 1.9 mM background Na+ (Noble and Gow 1993). Plates were incubated at 25"C, and examined for growth every 12 h for the first 72 h and then at 24-h intervals up to 3 12 h. Apositive result was assigned if there was evidence of growth on at least one of the quadrareplicates.…”

“…Aeromonas hydrophila and K harveyi were expected to grow on the basal medium, the former growing within 12 h and the latter growing with a lag period greater that 12 h. Vibrio splendidus was expected to require about 150 mM Na+ for growth. These assumptions were based on results reported by Noble and Gow (1993).…”

“…An examination of the data showed that the solid medium used was adequate to show the Na+ requirement. Noble and Gow (1993) suggested that strains that do not clearly show the presence or absence of the Na+ requirement, using the solid medium, be tested further by using a chemically defined broth medium.…”