LIVE/DEAD (R) BacLight (TM): application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water

A rapid epifluorescence staining method using the LIVE/DEAD(R) Bacterial Vi ability Kit (BacLight(TM)) was applied to estimate both viable and total co unts of bacteria in drinking water. BacLight is composed of two nucleic aci d-binding stains: SYTO 9(TM) and propidium iodide. SYTO 9(TM) penetrates al l bacterial membranes and stains the cells green, while propidium iodide on ly penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal incubation conditions were f ound to be 15 to 20 min, at room temperature in the dark. Total (red + gree n) and viable (green) cells can hence be counted simultaneously. Factors af fecting the staining procedure were tested (addition of glutaraldehyde, sta ining time, chlorine impact). In the absence of stress, BacLight viable cou nts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. Ba cLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, g rowth rate or temperature) induced a decrease in viability that was more pr onounced for CTC and plate counts than for BacLight viable counts. (C) 1999 Elsevier Science B.V. All rights reserved.