Fluorescent method for monitoring cheese starter permeabilization and lysis

A fluorescence method to monitor lysis of cheese starter bacteria using dua l staining with the LIVE/DEAD BacLight bacterial viability kit is described . This kit combines membrane-permeant green fluorescent nucleic acid dye SY TO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iod ide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lact ococcus lactis MG1363 were incubated under different conditions and subsequ ently labeled with SYTO 9 and PI and analyzed by How cytometry and epifluor escence microscopy. Lysis was induced by treatment with cell wall-hydrolyzi ng enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes t he cells. Under nonstabilizing conditions a high concentration of mutanolys in caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydro genase. In the stabilizing buffer, mutanolysin caused membrane damage as we ll but the cells disintegrated at a much lower rate. Stabilizing buffer sup ported permeabilized cells, as indicated by a high number of PI-labeled cel ls. In addition, permeable cells did not release intracellular aminopeptida se N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with thes e stains and confocal scanning laser microscopy the permeabilization of sta rter cells in cheese could be analyzed.