Method Validation for Bacterial and Viral Analysis of Geoducks

Thesis (Master's)--University of Washington, 2016-06

Background The shellfish industry in Western Washington is an integral part of the State economy; with products being distributed in both domestic and international markets. Oysters and geoducks are two widely harvested shellfish in Western Washington, and ensuring clean growing areas with low levels of bacterial and viral contamination is essential for protecting consumers from disease. Microbiological methods for assessing viral and bacterial contamination in oysters have been established; however, similar testing procedures for geoducks have not been validated. Our study adapted viral and bacterial testing methods for oysters to better suit geoduck meat. Methods To validate our viral and bacterial testing methods, four trials were performed with MS2 coliphage and five trials were performed with E. coli CN-13 seeded into geoduck meat harvested in Western Washington. Each MS2 trial contained four seeding levels for which we calculated plaque forming units (PFU)/100g and percent recovery. MS2 recovery was analyzed using the double agar layer method. The E. coli CN-13 trials contained three seeding levels with the most probable number (MPN) calculated for each. MPN was assessed using the APHA’s 5-tube fermentation method. Fermentation on seeded geoduck meat was first performed in Lauryl tryptose broth (LST) and then passaged into EC broth and EC-MUG medium. In addition, six unseeded field samples were also processed using these methods to evaluate the ability of the method to detect naturally occurring levels of E. coli and male-specific coliphage. Results The average percent recoveries for MS2 trials ranged from 106.7% to 146.6%, with higher recoveries occurring at more concentrated seeding levels. Recoveries of over 100% are likely due to MS2 aggregation. The average recovery ratios for E. coli CN-13 trials were greater than 1, with higher recoveries also occurring at more concentrated seeding levels. Recovery ratios greater than 1 are likely due to bacterial aggregation and potential bacterial replication in geoduck meat. All geoduck meat used in the E. coli trials was shown to have prior levels of fecal coliforms but tested negative for E. coli. In unseeded geoduck, male-specific coliphage levels, though typically low, ranged from 9.55 to 139.39 PFU/100g and E. coli levels ranged from .20 to 79.0 MPN/g. Conclusion Our findings suggest that, with modifications, the current oyster viral and bacterial testing methods can be applied successfully to geoduck meat. The percent recoveries and MPN ratios indicate that both methods provide good assessments of bacterial and viral contamination in geoducks.