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RAPID TETRAZOLIUM DYE REDUCTION ASSAY TO ASSESS THE BACTERICIDAL ACTIVITY OF OYSTER (CRASSOSTREA VIRGINICA) HEMOCYTES AGAINST VIBRIO PARAHAEMOLYTICUS
Citation:
Volety, A. K., L M. Oliver, F J. Genthner, AND W S. Fisher. RAPID TETRAZOLIUM DYE REDUCTION ASSAY TO ASSESS THE BACTERICIDAL ACTIVITY OF OYSTER (CRASSOSTREA VIRGINICA) HEMOCYTES AGAINST VIBRIO PARAHAEMOLYTICUS. AQUACULTURE 172(1/2):205-222, (1999).
Description:
An assay was developed to assess the ability of oyster, Crassostrea virginica, hemocytes to kill the human pathogenic bacterium, Vibrio parahaemolyticus (ATCC 17802). Bacterial killing was estimated colorimetrically by the enzymatic reduction of a tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet razolium (MTS), and phenylmethasulfazone (PMS). The assay proposed here provides an indicator of immunocompetence of oysters against V. parahaemolyticus. The assay involved: (1) exposure of plasma-free oyster hemocytes to a streptomycin (SM)-resistant mutant strain of V. parahaemolyticus in a 96-well plate for 3 h at 17|C in SM-augmented sea water; (2) growout of surviving bacteria in nutrient broth for 2 h at 37|C; (3) addition of MTS and PMS; and (4) measurement of MTS/PMS reduction product (formazan) at 490 nm using a microplate reader. Advantages of this assay include the absence of radio-isotopes used in some killing assays and requirement of low volumes of plasma and numbers of hemocytes. In addition, we demonstrated greater precision than traditional, plate counting methods for bacterial estimation. This technique has the potential to evaluate oyster capacity to eliminate microbial agents and to assess effects of environmental changes and pollutant stress on defense capabilities of oysters.