Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining

Abstract

The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid doublestaining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions

“…Membrane-intact and -damaged prokaryotic cells (named 'live' and 'dead' for simplicity) were enumerated in non-fixed samples following the NADS protocol (Gregori et al, 2001;Falcioni et al, 2008). NADS+, green cells (assumed to be live, with intact membranes), and NADS − , red cells (assumed to be inactive, with compromised cell membranes), were identified by simultaneous double staining with a membrane-permeable (SYBR Green; Molecular Probes) and impermeable (propidium iodide) probe.…”

The percentage of living bacterial cells related to organic carbon release from senescent oceanic phytoplankton

“…To do so, we used the nucleic acid double-staining (NADS) (Grégori et al, 2001) flow cytometric protocol. This technique consists of the use of two nucleic acid fluorescent dyes, SYBR Green I (SG1; Molecular Probes) and propidium iodide (PI; Sigma Chemical Co.).…”

Grazing rates and functional diversity of uncultured heterotrophic flagellates

“…Pictures of DAPI-stained bacteria were taken with a digital camera (Spot RT Slider; Diagnostic Instruments Inc., Sterling Heights, MI, USA) and processed with the Image Pro Plus software analyzer (Media Cybernetics Inc., Bethesda, MD, USA) to calculate the biovolume of 100-500 cells after the measured area and perimeter (Massana et al, 1997). Bacterial viability was assessed with the nucleic acid double-staining (NADS) protocol (Grégori et al, 2001) that uses SYBR Green to stain all cells and propidium iodide to stain cells with compromised membranes. Both populations were counted by flow cytometry and cells with intact membranes were considered 'alive' (Falcioni et al, 2008).…”