Application of methylene blue dye reduction test (MBRT) to determine growth and death rates of microorganisms

Abstract

An efficacy of a compound for antimicrobial characteristics is typically evaluated through experiments to obtain the Minimum Inhibitory Concentration (MIC). Similarly, a Minimum Activating Concentration (MAC) can be obtained for a compound necessary for growth. We report a protocol for the determination of MIC and MAC for antibiotic and exhibition assays, respectively using methylene blue reduction test. The assay has been demonstrated on the growth of Escherichia coli on the antibiotic kanamycin, as an inhibitor and on vitamin B12, as an activator. The slope of discoloration of methylene blue was used to determine the growth and death rates of the organism. The use of the rate constants to determine MIC/MAC values established a rational basis for determination of these relevant constants. The methodology presented here is general and can be easily adapted to other systems. The dose-response curves for extent of death/growth can be used to establish the MIC90 or MAC90 for an inhibitor or an activator, respectively. The methodology presented here rationalizes and standardizes the antibiotic and exhibition assays. The main advantage of the reported assay is the rapid and easy determination of the MIC and MAC values.