Evaluation of a double layer agar plate for direct enumeration of Vibrio parahaemolyticus

A double layer agar plate (DLAP) was developed according to the thin agar layer (TAL) method as a 1-step procedure for direct enumeration of injured Vibrio parahaemolyticus cells based on the formation of unique purple colonies by V parahaemolyticus. The DLAP was prepared by overlaying an equal volume (10 mL) of a nonselective medium (tryptic soy agar supplemented with 1.5% NaQ onto a selective medium (Bio-Chrome Vibrio medium). The DLAP was capable of detecting V parahaemolyticus in mixed cultures containing nonVibrio bacteria. Production of purple colonies by V parahaemolyticus on DLAP was not affected by the growth of other bacteria, even when V parahaemolyticus was only a small fraction (5%) of the entire bacterial population. Direct plating on DLAP was found as effective as the most probable number (MPN) method for recovering beatand cold-injured V parahaemolyticus cells, which could not be detected by direct plating on Bio-Chrome Vibrio medium or thiosulfate-citrate-bile salts-sucrose agar. The DLAP offers an alternative to the MPN method for detecting injured V parahaemolyticus cells and can be used as a simple 1-step procedure for quick screening of V parahaemolyticus in foods.