A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis

Abstract

A novel cellular cytotoxicity assay using two fluorescent dyes was developed as an alternative method to the standard direct count of viable protozoa under light microscopy. The compound calcein AM is a non-fluorescent substance that diffuses passively across intact cell membranes and is converted by intracellular esterases to the green fluorescent calcein, which is retained in viable cells. The addition of EthD-1 that binds to DNA stained nuclei of dead cells red. The experiments were carried out in order to assess viability in the freshwater ciliate Tetrahymena pyriformis after exposure to eight surfactants, two of each representing one of four ionic class (non-ionic, anionic, cationic and amphoteric), and two heavy metals, copper and zinc, at several concentrations. In earlier time exposure, less than one hour of contact with surfactants at sublethal concentrations, the fluorescent method is more sensitive and provides more accurate results than direct counting under light microscopy. In contrast, with increasing time exposure, the results obtained by the two methods were similar. Calcein was shown to be a poor viability marker in the presence of zinc and copper since the fluorescence intensity was affected by the metal presence. However, the fluorescent method offers new opportunities to use advanced techniques, such as flow cytometry, to assess cytotoxicity in protozoa.