Resolution of viable and membrane-compromised free bacteria in aquatic environments by flow cytometry

In aquatic environments, free heterotrophic bacteria play an extremely important role because of their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic-acid double-staining protocol (NADS) for flow cytometry that can distinguish the fractions of viable, damaged, or membrane-compromised cells within the free-bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains, membrane-permeant SYBR Green and membrane-impermeant PI. The efficiency of the double staining is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids. Full quenching of SYBR Green fluorescence by PI will identify cells with a compromised membrane, partial quenching will indicate cells with a slightly damaged membrane, and lack of quenching will characterize cells with an intact membrane. Samples do not require any pretreatment and this protocol can be performed almost anywhere.