Cryopreservation of fluorescent marker-labeled algae (Selenastrum capricornutum) for toxicity testing using flow cytometry

A rapid, two‐stain (fluorescein diacetate and ethidium homodimer‐1) flow cytometric assay to evaluat viability and cytotoxicity of the alga Selenastrum capricornutum in preserved samples is described. For storage, stained cells were fixed in glutaraldehyde, flash frozen in liquid nitrogen, and stored frozen (−20°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 cells, 12% nonstained cells, and 38.7% dead cells. This technique was tested using sodium dodecyl sulfate (SDS) and phenol as toxicants. Threshold 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 showed 96‐h median lethal concentration (LC50) values of 2,340 mg/L and 6,970 mg/L for SDS and phenol, respectively. This two‐stain flow cytometric procedure has proven to be a reliable, sensitive assay for determining viability of S. capricornutum in preserved samples. The sensitivity of this dual‐color assay and the ability to store samples for later analysis are significant improvements over current techniques.