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Selection of Fluorescent Probes for Flow Cytometric Viability Assessment of Listeria monocytogenes Exposed to Membrane-Active and Oxidizing Disinfectants

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Abstract:

The aim of this study was to select fluorescence methods for use as alternatives to plate counting to assess the viability of Listeria monocytogenes cells exposed to benzalkonium chloride (BAC) and hydrogen peroxide, two disinfectants with different mechanisms of action. A further aim of this study was to determine whether growth phase influences fluorescence labeling and whether it is possible to predict whether a probe will be a good viability indicator for cells exposed to a certain disinfectant on the basis of the mechanism of action of the disinfectant and the target of the fluorescent probe. The fluorescence methods used were labeling with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC; dehydrogenase activity), labeling with TOTO1 iodide (TOTO; membrane-impermeant probe), and assessment of pH gradient maintenance in a low-pH buffer after labeling with the pH-sensitive probe 5-(and 6)-carboxyfluorescein succinimidyl ester (CFSE) (the pHin method). Growth phase influenced fluorescent labeling. However, the cutoff value for distinction between viable and nonviable cells was the same for both growth phases. The viability (determined by plate counts) of BAC-exposed cells correlated well with CTC labeling and TOTO exclusion. For both BAC-exposed and hydrogen peroxide-exposed cells, the pHin method gave a good qualitative indication of viability, sublethal damage, and cell death. CTC labeling and TOTO exclusion did not correlate with the viability of hydrogen peroxide-exposed cells. Our results demonstrate that even if the mechanism of action of a disinfectant is known, in some cases it is still difficult to predict whether a certain fluorescent probe is suitable for viability assessment. Thus, the proper selection of fluorescent probes for the assessment of the efficacy of antimicrobial agents is essential.

Keywords:

Document Type: Research Article

Affiliations: 1: Food Hygiene and Microbiology Group & Consumer Technology and Product Use Group, Department of Agrotechnology and Food Sciences, Wageningen University and Research Center, P.O. Box 8129, 6700 EV Wageningen, The Netherlands 2: Food Hygiene and Microbiology Group, Department of Agrotechnology and Food Sciences, Wageningen University and Research Center, P.O. Box 8129, 6700 EV Wageningen, The Netherlands 3: Quality and Safety Assurance, Nestlé Research Center, P.O. Box 44, CH 1000 Lausanne 26, Switzerland 4: Food Hygiene and Microbiology Group, Department of Agrotechnology and Food Sciences, Wageningen University and Research Center, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

Publication date: 2003-08-01

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