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Appl. Environ. Microbiol. doi:10.1128/AEM.02750-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Assessing and interpreting bacterial viability using LIVE/DEAD® BacLight^TM kit in combination with flow cytometry

Swiss Federal Institute of Aquatic Science and Technology, Eawag, P.O. Box 611, CH-8600 Dübendorf, Switzerland; Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, 8092 Zurich, Switzerland

^* To whom correspondence should be addressed. Email: egli{at}eawag.ch.

	Abstract

The commercially available LIVE/DEAD® BacLight^TM kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret Live/Dead-staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium (S. typhimurium), Shigella flexneri and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the Gram-negative bacterium E. coli as well as in the two enteric pathogens the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated non-irradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in Gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD® stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

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