The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities

Abstract

The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) grows in vitro as irregularly rod-shaped hyphal bodies in a complex medium. In order to simplify the medium composition and determine growth-promoting compounds for the cultivation of this fungus, we were looking for a rapid and quantitative method to estimate the number of living cells in small volumes of liquid culture. A colorimetric method for the determination of cell densities using MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] proved to be more accurate and timesaving than conventional hemocytometer counting.

FIG. 1

Comparison of the results from the MTT method with the hemocytometer counts. A dense culture of N. parvispora was diluted to cell densities between 8.45 × 10⁶ and 8.25 × 10⁴ cells/ml of culture volume. The samples were then incubated with MTT, and the absorbance (OD) was measured after 16 h. The results of both methods show a linear relationship (R² = 0.996; n = 8; P < 0.0001). Each data point represents the mean and standard error calculated from five MTT measurements and four hemocytometer counts.

FIG. 2

Comparison between MTT assay results and hemocytometer counts for N. parvispora grown with various medium compositions in order to get different cell densities: Grace’s insect cell culture medium and FBS with increasing hemolymph concentrations (○, 0%, a; 5%, c; 10%, g); Grace’s insect cell culture medium and 10% hemolymph with different concentrations of BSA as a replacement for FBS (●; 0 mg/ml, b; 0.625 mg/ml, d; 1.25 mg/ml, e; 2.5 mg/ml, f; 3.75 mg/ml, i; 5 mg/ml, h; 10 mg/ml, j). The two methods still exhibit a linear relationship (R² = 0.968; n = 10; P < 0.0001). Each data point represents the mean and standard error of four independent replicates.