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Applied and Environmental Microbiology, September 2001, p. 4264-4271, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.4264-4271.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

Christine J. Bunthof,1 Saskia van Schalkwijk,2 Wilco Meijer,2 Tjakko Abee,1 and Jeroen Hugenholtz2,*

Laboratory of Food Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6700 EV Wageningen,1 and Department of Flavor and Natural Ingredients, NIZO Food Research, 6710 BA Ede,2 The Netherlands

Received 5 February 2001/Accepted 1 June 2001

A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.

* Corresponding author. Mailing address: NIZO Food Research, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31-318-659540. Fax: 31-318-650400. E-mail: hugenhol{at}nizo.nl.

Applied and Environmental Microbiology, September 2001, p. 4264-4271, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.4264-4271.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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