LIVE/DEAD BacLight : application of a new rapid staining method for direct enumeration of viable and total bacteria...

A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit (BacLight) was applied to estimate both viable and total counts of bacteria in drinking water. BacLight is composed of two nucleic acid-binding stains: SYTO 9 and propidium iodide. 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 incubation conditions were found to be 15 to 20 min, at room temperature in the dark. Total (red + green) and viable (green) cells can hence be counted simultaneously. Factors affecting the staining procedure were tested (addition of glutaraldehyde, staining time, chlorine impact). In the absence of stress, BacLight viable counts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. BacLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, growth rate or temperature) induced a decrease in viability that was more pronounced for CTC and plate counts than for BacLight viable counts.