USE OF A TETRAZOLIUM-BASED CELL-PROLIFERATION ASSAY TO MEASURE EFFECTS OF IN-VITRO CONDITIONS ON PERKINSUS-MARINUS (APICOMPLEXA) PROLIFERATION

Because the in vitro cell cycle of the apicomplexan oyster pathogen Pe rkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for d etermining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell prol iferation assay yields a soluble formazan chromophore upon intracellul ar reduction by P. marinus, at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected cultur e system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populati ons propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F- 12. Temperatu res of 10-40 degrees C permitted growth, which was maximized at 35 deg rees C. pH 6.0-8.5 permitted growth, which was maximized at 7.0-7.5. O smolalities of 340-1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1-10% (v/v) did not enhance log phas e growth, but enhanced stationary phase metabolic activity in proporti on to concentration. Our isolate (ATCC 50439) has a 13 h log phase dou bling time when propagated under optimized conditions: 28 degrees C, 8 00 mOsm, pH 7.0, 1:1 DME/Ham's F-12 medium, 5% (v/v) FBS. It is tolera nt of antibacterial agents at concentrations commonly used in vertebra te tissue culture, but is inhibited by several antimycotics at similar concentrations.