RESTING-CELL DEHYDROGENASE ASSAY MEASURING A NOVEL WATER-SOLUBLE FORMAZAN DETECTS CATABOLIC DIFFERENCES AMONG CELLS

A resting-cell assay using the novel tetrazolium compound, imethyl-2-t hiazolyl)-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 c ells into a water-soluble formazan. This assay detects different catab olic activities of cells grown with various carbon sources. In this st udy, the effect of repression of metabolic pathways was demonstrated i n Escherichia coli JL3676, Bacillus subtilis ATCC 6633, and Pseudomona s stutzeri JM300 by assaying for formazan formation. Lactose-grown E. coli cells exhibited approximately a one-hundred-fold increase in dehy drogenase activity when lactose was the added 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-g rown E. coli cells showed at least a twenty-fold increase in activity when oleate was the added substrate. When succinate was the added subs trate, succinate-grown B. subtilis cells produced a thirteen-fold incr ease in activity over glucose-grown or glucose-plus-succinate-grown ce lls. When glucose was the added substrate, glucose-grown P. stutzeri c ells demonstrated at least a twelve-fold increase in activity compared with succinate-grown or succinate-plus-glucose-grown cells. Also, res ults comparable to the production of [C-14]CO2 from [C-14]oleate by E. coli were obtained demonstrating that the assay is an alternative to the use of radiolabeled substrates.