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

Content Provider	PubMed Central
Author	Falcioni, Tania Papa, Stefano Gasol, Josep M.
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 μg 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.
Related Links	http://dx.doi.org/10.1128/AEM.01668-07
Starting Page	1767
File Format	PDF
ISSN	10985336
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	6
Volume Number	74
Language	English
Publisher	American Society for Microbiology (ASM)
Publisher Date	2008-03-01
Access Restriction	Open
Rights Holder	American Society for Microbiology (ASM)
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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