Miniaturization and application of the MTT assay to evaluate metabolic activity of protozoa in the presence of toxicants

Abstract

This paper describes a critical evaluation of a miniaturised colorimetric assay, using MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction, applied to protozoan viability testing. The toxic substances used were copper, zinc, Triton X-100 (a membrane surfactant) and cycloheximide (an inhibitor of the protein synthesis). The viability assay of the ciliate protozoan Tetrahymena pyriformis was optimised in terms of MTT concentration and incubation time. Since protozoa are non adherent cells the MTT assay was modified in order to maintain the medium in the well. MTT proved to be effective in the measurement of Tetrahymena pyriformis viability. Four hours of MTT incubation followed by 30 minutes of incubation with DMSO were found to be the best incubation times for optical density reading. Furthermore, 10 mg/ml of MTT solution was the concentration that gave higher values of optical densities with minor medium interference.