CRYOPRESERVATION OF FLUORESCENT MARKER-LABELED ALGAE (SELENASTRUM-CAPRICORNUTUM) FOR TOXICITY TESTING USING FLOW-CYTOMETRY

A rapid, two-stain (fluorescein diacetate and ethidium homodimer-l) Ro w cytometric assay to evaluate viability and cytotoxicity of the alga Selenastrum capricornutum in preserved samples is described. For stora ge, stained cells were fixed in glutaraldehyde, flash frozen in liquid nitrogen, and stored frozen (-20 degrees C) for assessment at a later date. Weekly analysis of frozen samples showed that fluorescence was stable for 7 weeks. A mixture of 50% healthy and 50% heat-killed cells of S. capricornutum showed 36.1% healthy cells, 13.2% compromised cel ls, 12% nonstained cells, and 38.7% dead cells. This technique was tes ted using sodium dodecyl sulfate (SDS) and phenol as toxicants. Thresh old concentrations for toxicant impact were in the range of 1 to 10 mg /L for SDS and 10 to 100 mg/L for phenol. Estimates of mortality showe d 96-h median lethal concentration (LC50) values of 2,340 mg/L and 6,9 70 mg/L for SDS and phenol, respectively. This two-stain flow cytometr ic procedure has proven to be a reliable, sensitive assay for determin ing viability of S. capricornutum in preserved samples. The sensitivit y of this dual-color assay and the ability to store samples for later analysis are significant improvements over current techniques.