Evaluation of Alternative Colony Hybridization Methods for Pathogenic Vibrios

Abstract

Vibrios, such as Vibrio parahaemolyticus, are naturally occurring halophilic bacteria that are a major cause of foodborne illness. Because of their autochthonous nature, managing vibrio levels in marine and estuarine environments is impossible. Instead, it is crucial to reliably enumerate their abundance to minimize human exposure. One method of achieving this is the direct plating/colony hybridization (DP/CH) method, which has been used to efficiently quantify pathogenic vibrios in oysters and other seafood products. Although successful, the method relies on proprietary resources. We examined alternative approaches, assessed the influence of the reagent suppliers’ source on enumeration accuracy, and made experimental adjustments that maximized efficiency, sensitivity, and specificity. We report here that in-house conjugation via Cell Mosaic is a viable alternative to the previously available sole-source distributor of the alkaline phosphatase-conjugated probes used to enumerate vibrios in oysters. We also report that milk was a viable alternative as a blocking reagent, pH must be eight, an orbital shaker was a viable alternative to a water bath, and narrow polypropylene containers were a viable alternative to Whirl-Pak bags. These modifications will be crucial to scientists enumerating vibrios and other pathogens in food products.