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 double-staining 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.

FIG. 1

Green versus red fluorescence cytograms corresponding to different test experiments. Panels: A1, unstained seawater sample; B1 and B2: 0.2-μm-filtered seawater sample supplemented with PI and SYBR Green II, respectively; C1 and C2, seawater samples supplemented with PI and SYBR Green, respectively. The final probe concentrations were 10 μg of PI cm⁻³ and 1:1,000 (vol/vol) SYBR Green II.

FIG. 2

Single and dual staining of heat-treated seawater samples. Panels: D1 and D2, heat-treated seawater samples supplemented with PI and SYBR Green II, respectively; D3, heat-treated (40 min at 60 to 70°C) seawater sample simultaneously stained with PI and SYBR Green. Killed bacteria essentially appeared as red fluorescent cells. The final probe concentrations were 10 μg of PI cm⁻³ and 1:1,000 (vol/vol) SYBR Green II.

FIG. 3

Cell sorting and esterase activity assay. Green fluorescent bacteria resolved by the NADS protocol (gated cells on the fresh-marine-sample cytogram) were sorted and further stained with CFDA as described in Materials and Methods. The cells exhibited an increase in green fluorescence due to esterase activity and carboxyfluorescein retention (gated cells on the sorted cells-plus-CFDA cytogram).

FIG. 4

Application of the NADS protocol to a Seveso River (Italy) sample before and after ozone treatment. The untreated-sample cytogram corresponds to a freshwater subsample mainly containing green bacteria. The ozone-treated-sample cytogram was obtained after ozone treatment to kill cells, and it shows essentially red bacteria. Quadrants are identified as A to D. The plate count obtained with green bacteria from the untreated sample shows abundant colonies, whereas the plate count made with red cells from the ozone-treated sample yielded no colonies.

FIG. 5

Bacteria distribution in the Adda River (Italy) at four different stations: 1 km upstream from a sewage outflow, at the sewage outflow site, and at 1 and 6 km downstream from the sewage outflow point. Bacteria were resolved according to their fluorescence (green and green-plus-red bacteria and red fluorescent bacteria) generated by the NADS protocol. The values in brackets are the percentages of red fluorescent bacteria relative to the total count.

FIG. 6

Staining classes for bacteria collected from surface water inside Pointe-Rouge harbor (Marseilles, France) and a few kilometers away from the town, at the site facing Maire Island.

FIG. 7

Comparison of single (SYBR Green II; 1:1,000 [vol/vol] final dilution) and double (NADS protocol) staining of bacteria in a seawater sample collected off Marseilles at the site facing Maire Island. The green versus the red fluorescence cytograms corresponding to SYBR Green II staining reveal the presence of bacteria with high (H) and low (L) DNA contents. The cytogram associated with the NADS protocol shows a significant amount of LDNA bacteria appearing as red-negative cells in quadrant A. At the same time, cells that were negative with SYBR Green II were apparent with PI staining in quadrant D. Refer to Table 1 for the counts.