DEVELOPMENT OF A ROBUST FLOW CYTOMETRIC ASSAY FOR DETERMINING NUMBERSOF VIABLE BACTERIA

Several fluorescent probes were evaluated as indicators of bacterial v iability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide , and changes in membrane potential, monitored by using fluorescent ca tionic and anionic potential-sensitive probes. Of the fluorescent reag ents examined, the fluorescent anionic membrane potential probe bis-(1 ,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(4)(3)] proved the b est candidate for use as a general robust viability marker and is a pr omising choice for use in high-throughput assays. With this probe, liv e and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable count s determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sens itive analytical technique that can rapidly monitor physiological chan ges of individual microorganisms as a result of external perturbations . The membrane potential probe DiBAC(4)(3) provided a robust flow cyto metric indicator for bacterial cell viability.