USE OF 5-CYANO-2,3-DITOLYL TETRAZOLIUM CHLORIDE FOR QUANTIFYING PLANKTONIC AND SESSILE RESPIRING BACTERIA IN DRINKING-WATER

Direct microscopic quantification of respiring (i.e., viable) bacteria was performed for drinking water samples and biofilms grown on differ ent opaque substrata. Water samples or biofilms developed in flowing d rinking water were incubated with the vital redox dye 5-cyano-2,3-dito lyl tetrazolium chloride (CTC) and R2A medium. One hour of incubation with 0.5 mM CTC was sufficient to obtain intracellular reduction of CT C to the insoluble fluorescent formazan (CTF) product, which was indic ative of cellular respiratory (i.e., electron transport) activity. Thi s result was obtained with both planktonic and biofilm-associated cell s. Planktonic bacteria were captured on 0.2-mum-pore-size polycarbonat e membrane filters and examined by epifluorescence microscopy. Respiri ng cells containing CTF deposits were readily detected and quantified as red-fluorescing objects on a dark background. The number of CTC-red ucing bacteria was consistently greater than the number of aerobic CFU determined on R2A medium. Approximately 1 to 10% of the total plankto nic population (determined by counterstaining with 4,6-diamidino-2-phe nylindole) were respirometrically active. The proportion of respiring bacteria in biofilms composed of drinking water microflora was greater , ranging from about 5 to 35%, depending on the substratum. Respiring cells were distributed more or less evenly in biofilms, as demonstrate d by counterstaining with 4,6-diamidino-2-phenylindote. The amount of CTF deposited in single cells of Pseudomonas putida that formed monosp ecies biofilms was quantified by digital image analysis and used to in dicate cumulative respiratory activity. These data indicated significa nt cell-to-cell variation in respiratory activity and reduced electron transport following a brief period of nutrient starvation. The result s of this study demonstrate that CTC reduction is a rapid and sensitiv e method for quantification and localization of viable bacteria in dri nking water and other environmental samples. The method is particularl y well suited for exploration of cellular activity in surface biofilms .