Improved method for detection of Vibrio parahaemolyticus in seafood

Abstract

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.

FIG. 1

Appearance of V. parahaemolyticus on CV agar.

FIG. 2

Protocol of the method of enrichment and plating for isolation of V. parahaemolyticus from seafood samples.

FIG. 3

Colonies plated out from enrichment culture of seafoods on CV and TCBS agars. (a, c, and e) CV agar; (b, d, and f) TCBS agar; (a to d) incubation at 37°C for 18 h; (e and f) incubation at room temperature for 24 h followed by incubation at 37°C for 18 h; (a and b) spread plating; (c to f) streak plating. Violet colonies on CV agar and green colonies on TCBS agar were identified as V. parahaemolyticus.