An isothermal absorptiometric assay for viable microbes using the redox color indicator 2,6-dichlorophenolindophenol

Abstract

A simple and rapid isothermal absorptiometric assay for detection of viable microbes using the redox color indicator 2,6-dichlorophenolindophenol (DCPIP) was studied. The absorbance of DCPIP decreased at 600nm because of a redox reaction occurring between DCPIP and the surface membrane of viable microbes and was inversely proportional to the viable cell density. The redox reaction was found not only with bacteria, but also with yeast and a mixture of bacteria and yeast. In this assay, the influence of light scattering and absorption caused by microbial cells and coexisting substances in the sample was excluded by a time difference method. The assay required only 10min for one incubation mixture, and highly repeatable results from three consecutive measurements were obtained by isothermal incubation for specific times at 30°C using a thermostable three-cuvette-stir system. Thus, the cell density of microbial cell suspensions or growth medium was successfully determined, and a practical lower detection limit for food inspection was obtained at 104–106cfu/ml. Single-cell effects on DCPIP reduction were evaluated and compared between species. Consequently, this assay is expected to be a useful tool for the rapid measurement of viable microbes as a preliminary assay for the Hazard Analysis Critical Control Point program.