Rapid method for total, viable and non-viable acetic acid bacteria determination during acetification process

Abstract

A rapid epifluorescence staining method using the LIVE/DEAD ® BacLight Bacterial Viability Kit ( BacLight™) and direct counts in Neubauer chamber were applied to estimate both viable and total counts of bacteria in different stages of vinegar making. BacLight kit is composed of a mixture of two nucleic acid binding stains: SYTO 9™ and propidium iodide. These stains differ both in their spectral characteristics and in their ability to penetrate viable bacterial cells. SYTO 9 penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal dilution and incubation conditions were found to be 1:5 and 15 min at room temperature in dark respectively. Total (red plus green) and viable (green) cells can be obtained in one staining step and hence counted simultaneously. Results obtained with this technique were compared with those from other measurement techniques (colony counts and flow cytometry).