Resting-cell dehydrogenase assay measuring a novel water-soluble formazan detects catabolic differences among cells

Abstract

A resting-cell assay using the novel tetrazolium compound, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethyl-2-thiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), demonstrates the presence of dehydrogenases in cells. When used with the electron-coupling reagent phenazine methosulfate (PMS), MTS can be reduced by cells into a water-soluble formazan. This assay detects different catabolic activities of cells grown with various carbon sources. In this study, the effect of repression of metabolic pathways was demonstrated in Escherichia coli JL3676, Bacillus subtilis ATCC 6633, and Pseudomonas stutzeri JM300 by assaying for formazan formation. Lactose-grown E. coli substrate as compared to glucose-grown or glucose-plus-lactose-grown cells. In addition, when compared to glucose-grown or glucose-plus-oleate-grown cells, oleate-grown E. coli cells showed at least a twenty-fold increase in activity when oleate was the added substrate. When succinate was the added substrate, succinate-grown B. subtilis cells produced a thirteen-fold increase in activity over glucose-grown or glucose-plus-succinate-grown cells. When glucose was the added substrate, glucose-grown P. stutzeri cells demonstrated at least a twelve-fold increase in activity compared with succinate-grown or succinate-plus-glucose-grown cells. Also, results comparable to the production of [ 14C]CO 2 from [ 14C]oleate by E. coli were obtained demonstrating that the assay is an alternative to the use of radiolabeled substrates.