Application of the MTT-based colorimetric method for evaluating bacterial growth using different solvent systems

Abstract

Although the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is widely used to measure the growth of animal cells, several interfering factors and obstacles for analyzing bacterial growth and for solubilizing bacterial-formazan have been reported. In this study, growth of Gram-negative Escherichia coli and Gram-positive Leuconostoc mesenteroides were analyzed using the MTT assay, and different solvents, specifically dimethyl sulfoxide, 1% Triton X-100, 1% Bile bovine, 10% sodium dodecyl sulfate, and sodium hydroxide (NaOH) were evaluated for solubilizing bacterial-formazan. MTT formazan that had been reduced by L. mesenteroides had fully developed color within 15 min in all the solvents tested. The solvent with the highest peak absorbance was 1 N NaOH. MTT formazan did not have a fully developed color response in the 0.001–0.1 N NaOH range, and its color intensity was not further enhanced at concentrations higher than 1 N. For all solvents, the color intensity of dissolved formazan was significantly higher in E. coli than in L. mesenteroides. The MTT assay showed sufficient sensitivity to detect bacterial cells during early growth phase that could not be detected by the plate counting method. These results suggest that NaOH (1 N) is a suitable solvent for analyzing bacterial growth in MTT assays.