The extensive microbial contamination of DUW in this study is consistent with that described in previous reports. Comparisons with other common public potable water sources emphasize the relatively high concentrations of microorganisms in DUW, and the low numbers of bacteria in most domestic water samples. Microscopic evidence of amebas and worms in DUW reinforces the need for further qualitative and quantitative studies of these contaminants of dental tubing biofilm. In most cases, flushing for two minutes reduced microbial concentrations in DUW. However, in a few cases, concentrations rose; in others, the reductions were negligible. Moreover, after 30 minutes of stasis, or use of the waterline in a routine dental procedure, microbial counts were frequently restored to preflush levels or higher. The longer-term ineffectiveness 30 minutes after flushing is understandable when the hydrodynamics of laminar flow of water in narrow-bore tubing are considered. Biofilm on the tubing wall remains intact during flushes and quickly generates more bacteria during stasis. Increased bacterial concentrations after flushing probably result from sloughing of biofilm from the tubing wall because of stretching and movement of the line during routine use. These two phenomena undermine the benefits of routine water flushes. It is a common belief that bacterial levels build to extremes during overnight or weekend stasis; we observed more variation in contamination levels during the workday than between overnight stasis and typical workday samples. The presence of hemolytic staphylococci and streptococci in water samples from lines that were supplied only from sterile water reservoirs adds to the growing evidence that part of the microbial flora in DUWLs may be derived from patients' mouths. Additional prophylactic measures to limit bacterial contamination in DUW should be implemented according to standard principles used in other medical fluid delivery devices.