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Applied and Environmental Microbiology, May 2001, p. 2326-2335, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2326-2335.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Flow Cytometric Assessment of Viability of Lactic Acid Bacteria

Christine J. Bunthof, Karen Bloemen, Pieter Breeuwer, Frank M. Rombouts, and Tjakko Abee*

Laboratory of Food Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University and Research Centre, 6700 EV Wageningen, The Netherlands

Received 21 July 2000/Accepted 10 November 2000

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70°C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.

* Corresponding author. Mailing address: Laboratory of Food Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University and Research Centre, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Phone: 31 317 484981. Fax: 31 317 484893. E-mail: Tjakko.Abee{at}micro.fdsci.wag-ur.nl.

Applied and Environmental Microbiology, May 2001, p. 2326-2335, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2326-2335.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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