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Applied and Environmental Microbiology, October 2001, p. 4662-4670, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4662-4670.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Gérald Grégori,1,* Sandra Citterio,2 Alessandra Ghiani,2 Massimo Labra,2 Sergio Sgorbati,2 Spencer Brown,3 and Michel Denis1

Laboratoire d'Océanographie et de Biogéochimie, Université de la Méditerranée, CNRS UMR 6535, 13288 Marseille,1 and Institut des Sciences du Végétal, CNRS UPR 2355, 91198 Gif-sur-Yvette,3 France, and Dipartimento di Scienze dell'Ambiente e del Territorio, Università di Milano-Bicocca, 20126 Milan, Italy2

Received 20 March 2001/Accepted 29 June 2001

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.

* Corresponding author. Mailing address: Laboratoire d'Océanographie et de Biogéochimie, Université de la Méditerranée, CNRS UMR 6535, 163 avenue de Luminy, Case 901, 13288 Marseille Cedex 9, France. Phone: 33-4-91-82-91-14. Fax: 33-4-91-82-65-48. E-mail: gregori{at}com.univ-mrs.fr.

Applied and Environmental Microbiology, October 2001, p. 4662-4670, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4662-4670.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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