Effect of temperature, salinity and nutrient content on the survival responses of Vibrio splendidus biotype I

The aim of this study was to evaluate the survival responses of two strains of Vibrio splendidus, both in natural and in defined media. For this purpose, freshwater and defined media containing different salinities (3-3-0-9%) and nutrient concentrations (17-0(.)005 mg l(-1)) were assayed. The incubation temperatures were established at 4, 10 and 22 degreesC. The acridine orange staining technique was used for total cell enumeration and the number of viable cells was determined using two direct assays, nalidixic acid and tetrazolium salt reduction and plate spreading. Resuscitation assays of viable but non-culturable (VBNC) cells were conducted. According to the counting procedures employed, at least four different subpopulations were found: (i) active (positive response in both nalidixic acid and tetrazolium assays) culturable cells; (ii) active non-culturable cells; (iii) tetrazolium-salt-responsive non-culturable cells and (iv) non-active (responsive to none of the direct viable assays) non-culturable cells. Long-term survival was found at salinities and nutrient concentrations of seawater environments (3(.)3% and 5 mg l(-1) or 1 g 1-1), whereas the strains entered a VBNC state in freshwater and in brackish (0(.)9 or 1(.)6% salinities) or high nutrient content (17 g l(-1)) defined medium. The recovery of VBNC cells was not achieved.