A double layer agar plate method results in an improvement for enumerating Vibrio vulnificus and Vibrio parahaemolyticus exposed to nutrient deficiency and refrigeration temperature.

This study sought to І)investigate the effect of varying concentrations of salt on the survival of Gram-negative bacteria in artificial seawater (ASW; pH 6) stored at 4 °C, ІІ)examine the culturable populations of Vibrio vulnificus persisting in ASW with or without 1000 U/mg catalase or 0.1% sodium pyruvate (pH 6) under aerobic and oxygen-restricted environments at 4 °C, and ІІІ)determine whether double layer agar plate (DLAP) methods can improve the culturability of V. vulnificus and Vibrio parahaemolyticus in ASW (pH 6) stored at 4 °C. Once incubated in ASW containing 10% salt at 4 °C, Escherichia coli O157:H7, Salmonella Typhimurium, and Shigella flexneri survived at levels over at least 3.42-4.65 log CFU/mL on day 28, but V. vulnificus underwent the inability to produce colonies within 7 days, while maintaining its membrane integrity. A positive correlation between sodium pyruvate and maintenance of platable colony-forming capability was observed in V. vulnificus incubated aerobically. Using tryptic soy agar containing 3% salt (TSAS) overlayed by thiosulfate-citrate-bile salts-sucrose agar (TCBS), a higher platable colony count of Vibrio spp. was recovered from ASW after 25 or 30 days of storage at 4 °C, followed by TSAS, TSAS overlayed by marine agar (MA), MA, and TCBS.