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Appl. Environ. Microbiol. doi:10.1128/AEM.01668-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

EVALUATING THE FLOW CYTOMETRIC NUCLEIC ACID DOUBLE STAINING PROTOCOL (NADS) IN REALISTIC PLANKTONIC BACTERIAL DEATH SITUATIONS

Center of Cytometry and Cytomorphology, University "Carlo Bo", Urbino, Italy, Institut de Ciències del Mar, CSIC, Barcelona, Spain

^* To whom correspondence should be addressed. Email: pepgasol{at}icm.csic.es.

	Abstract

Since hetrotrophic 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 effect of environmental variables on that state are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the NADS protocol (Grégori et al. 2001, AEM 67:4462-4670), 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: actively respiring cells by 5 cyano-2,3 ditolyl tetrazolium chloride (CTC), and incorporation of tritiated leucine. We show that concentrations in the range 5-20 µg ml^-1 of Propidium iodide (PI) simultaneous to 10x of SYBRGreen I are best to detect two separated populations of "Live" (green cells) and "Dead" (red cells) organisms. During exposure to heat and UVC we observed that the amount of Live cells declined concurrently to that of actively respiring cells (CTC+) and to 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 NW Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.