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Applied and Environmental Microbiology, November 2002, p. 5209-5216, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5209-5216.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

Kaouther Ben Amor,1,2 Pieter Breeuwer,1 Patrick Verbaarschot,1 Frank M. Rombouts,1 Antoon D. L. Akkermans,2 Willem M. De Vos,2,3 and Tjakko Abee1*

Laboratory of Food Microbiology, Wageningen University, EV 6700 Wageningen,1 Laboratory of Microbiology, Wageningen University, 6703 CT Wageningen,2 Wageningen Center for Food Sciences, 6700 AN Wageningen, The Netherlands3

Received 8 February 2002/ Accepted 5 August 2002

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

* Corresponding author. Mailing address: Laboratory of Food Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University and Research Center, P.O. Box 8129, EV 6700 Wageningen, The Netherlands. Phone: 31 317 48 49 81. Fax: 31 317 48 49 78. E-mail: Tjakko.Abee{at}micro.fdsci.wau.nl.

Applied and Environmental Microbiology, November 2002, p. 5209-5216, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5209-5216.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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