Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

Abstract

Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 g ml ؊1 of propidium iodide, simultaneous to a 10؋ concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.Heterotrophic prokaryotes (Bacteria and Archaea) play a relevant role in planktonic marine microbial food webs (e.g., reference 5). As a relevant difference from other planktonic organisms, prokaryotes are known to sometimes be inactive or dead, but in the way that they are commonly enumerated (e.g., with DAPI [4Ј,6Ј-diamidino-2-phenylindole]), these cells are accounted for in budgets for prokaryote biomass since they are not distinguished from live and active cells.From an ecological point of view, at least three cell categories within microbial communities can be distinguished: the viable and active cells, which play a functional role and participate in biomass production at the time of sampling; the live but inactive cells (often called dormant cells), which do not participate in production at the time of sampling but have the potential for doing so; and the dead and therefore inactive cells, which do not have a role in the cycling of chemical elements, even though they might be retaining nutrients. The discrimination of these cellular categories is not without dispute: for example, the "active" term is often confounded with the "live" one and the "inactive" with the "dead" one (26). Moreover, different intrinsic levels of metabol...

High-resolution imaging of pelagic bacteria by Atomic Force Microscopy and implications for carbon cycling

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

“…Immediately after collection, samples (0.4 ml) were incubated in the dark with 4 μl of SYBR Green (10x final conc.) and 4 μl of propidium iodide (10 μg ml − 1 final) for 15 min (Falcioni et al, 2008). NADS+ and NADS − cells were enumerated by flow cytometry and differentiated in a scatter plot of FL1 (green)-FL3 (red emission after blue-light excitation).…”

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