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Applied and Environmental Microbiology, May 2007, p. 3283-3290, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02750-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry{triangledown}

Michael Berney,1 Frederik Hammes,1 Franziska Bosshard,1,2 Hans-Ulrich Weilenmann,1 and Thomas Egli1,2*

Swiss Federal Institute of Aquatic Science and Technology, EAWAG, P.O. Box 611, CH-8600 Dübendorf, Switzerland,1 Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, 8092 Zurich, Switzerland2

Received 24 November 2006/ Accepted 13 March 2007

The commercially available LIVE/DEAD BacLight 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, 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 nonirradiated 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.

* Corresponding author. Mailing address: Swiss Federal Institute of Aquatic Science and Technology, EAWAG, P.O. Box 611, CH-8600 Dübendorf, Switzerland. Phone: 41 44 823 5158. Fax: 41 44 823 5547. E-mail: egli{at}eawag.ch

{triangledown} Published ahead of print on 23 March 2007.

Applied and Environmental Microbiology, May 2007, p. 3283-3290, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02750-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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