Membrane potential based characterization by flow cytometry of physiological states in an aerobic anoxygenic phototrophic bacterium

A simplified protocol for determination of bacterial membrane potential (MP) by flow cytometry with 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) as the MP probe was established, and was applied to an aerobic anoxygenic phototrophic bacterium (AAPB strain, Roseobacter clade) with an Escherichia coli strain as a reference. It was shown to be capable of precise indication of the physiological states of the cells as well as cellular responses to environmental factors. Based on the experimental results, a MP curve model was proposed for characterization of species-specific features, which offers parameters including the maximum MP of the curve (MPmax), the time to reach the MPmax (T-max), the slope of the exponential phase of the MP curve (K-MP), and the minimum (threshold) concentration of a particular substrate required for the organism to reach its MPmax (S-max). These parameters are functions of substrates and incubation conditions for a single species, and can be used for comparison between different species under the same conditions. The MPmax, T-max, and K-MP of the AAPB strain were quite different form those of the E. coli strain, and the AAPB strain was shown to be less sensitive to temperature and pH changes than the E. coli strain