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Applied and Environmental Microbiology, August 1999, p. 3681-3689, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Rapid Fluorescence Assessment of the Viability of Stressed Lactococcus lactis

Christine J. Bunthof, Sabina van den Braak, Pieter Breeuwer, Frank M. Rombouts, and Tjakko Abee*

Department of Food Technology and Nutritional Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands

Received 17 December 1998/Accepted 11 May 1999

The aim of this study was to establish the use of the fluorescent probes carboxyfluorescein (cF) and propidium iodide (PI) for rapid assessment of viability, using Lactococcus lactis subsp. lactis ML3 exposed to different stress treatments. The cF labeling indicated the reproductive capacity of mixtures of nontreated cells and cells killed at 70°C very well. However, after treatment up to 60°C the fraction of cF-labeled cells remained high, whereas the survival decreased for cells treated at above 50°C and was completely lost for those treated at 60°C. In an extended series of experiments, cell suspensions were exposed to heating, freezing, low pH, or bile salts, after which the colony counts, acidification capacity, glycolytic activity, PI exclusion, cF labeling, and cF efflux were measured and compared. The acidification capacity corresponded with the number of CFU. The glycolytic activity, which is an indicator of vitality, was more sensitive to the stress conditions than the reproduction, acidification, and fluorescence parameters. The cF labeling depended on membrane integrity, as was confirmed by PI exclusion. The fraction of cF-labeled cells was not a general indicator of reproduction or acidification, nor was PI exclusion or cF labeling capacity (the internal cF concentration). When the cells were labeled by cF, a subsequent lactose-energized efflux assay was needed for decisive viability assessment. This novel assay proved to be a good and rapid indicator of the reproduction and acidification capacities of stressed L. lactis and has potential for physiological research and dairy applications related to lactic acid bacteria.

* Corresponding author. Mailing address: Laboratory for Food Microbiology, 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.wau.nl.

Applied and Environmental Microbiology, August 1999, p. 3681-3689, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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