INFLUENCE OF WATER CHLORINATION ON THE COUNTING OF BACTERIA WITH DAPI(4',6-DIAMIDINO-2-PHENYLINDOLE)

Counting bacteria in drinking water samples by the epifluorescence tec hnique after 4',6-diamidino-2-phenylindole (DAPI) staining is complica ted by the fact that bacterial fluorescence varies with exposure of th e cells to sodium hypochlorite. An Escherichia coli laboratory-grown s uspension treated with sodium hypochlorite (5 or 15 mg of chlorine lit er(-1)) for 90 min was highly fluorescent after DAPI staining probably due to cell membrane permeation and better DAPI diffusion. At chlorin e concentrations greater than 25 mg liter(-1) DAPI-stained bacteria ha d only a low fluorescence. Stronger chlorine doses altered the DNA str ucture preventing the DAPI from complexing with the DNA. When calf thy mus DNA was exposed to sodium hypochlorite (from 15 to 50 mg of chlori ne liter(-1) for 90 min), the DNA completely lost the ability to compl ex with DAPI. Exposure to monochloramine did not have a similar effect . Treatment of drinking water with sodium hypochlorite (about 0.5 mg o f chlorine liter(-1)) caused a significant increase in the percentage of poorly fluorescent bacteria, from 5% in unchlorinated waters (40 sa mples), to 35 to 39% in chlorinated waters (40 samples). The presence of the poorly fluorescent bacteria could explain the underestimation o f the real number of bacteria after DAPI staining. Microscopic countin g of both poorly and highly fluorescent bacteria is essential under th ese conditions to obtain the total number of bacteria. A similar effec t of chlorination on acridine orange-stained bacteria was observed in treated drinking waters. The presence of the poorly fluorescent bacter ia after DAPI staining could be interpreted as a sign of dead cells.