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PERKINSUS-"CIDAL" ACTIVITY OF OYSTER HEMOCYTES USING A TETRAZOLIUM DYE REDUCTION ASSAY: OPTIMIZATION AND APPLICATIONS
Citation:
Volety, A. K., L M. Oliver, AND W S. Fisher. PERKINSUS-"CIDAL" ACTIVITY OF OYSTER HEMOCYTES USING A TETRAZOLIUM DYE REDUCTION ASSAY: OPTIMIZATION AND APPLICATIONS. Presented at 15th Biennial International Estuarine Research Federation Conference, New Orleans, LA, Sept. 25-30, 1999.
Description:
A bactericidal assay developed to assess the ability of oyster (Crassostrea virginica) hemocytes to kill the human pathogen Vibrio parahaemolyticus was optimized to estimate killing of the oyster parasite Perkinsus marinus. Assay variables, temperature, hemocyte:parasite ratio, incubation period, growout time and medium volume, were optimized. The assay involves exposure of oyster hemocytes in vitro to cultured P. marinus cells in 100 |L sea water in microtiter plate wells for 3 hours, growout of surviving P. marinus parasites for 24 hours, addition of the colorimetric reagent MTS/PMS, and measurement of the reaction product of MTSM/PMS reduction at 490 nm on a microplate reader. Since MTS/PMS reduction is proportional to the number of surviving parasite cells, a killing index can be calculated by comparing absorbances of hemocyte-free parasite preparations to those exposed to hemocytes. Potential applications for this assay include quantitative evaluation of oyster defense capacity against a natural pathogen, P. marinus, and assessment of environmental and pollutant stresses on oyster cellular defenses. We exposed oyster hemocytes in vitro to sub-lethal concentrations of tributyltin (TBT) and determined that in vitro exposure of hemocytes to various concentrations of TBT reduced hemocyte ability to kill P. marinus.