Recommended culture methods for monitoring bacterial contamination of H2O, dialysate and bicarbonate concentrate in dialysis centers in the USA involves culturing these fluids for 48 h at 37 °C. A variety of media and commercial culture methods are accepted for monitoring these fluids. Over a 3 month period a comparison was made between an acceptable culture method, tryptic soy agar (TSA) employing the pour plate (PP) technique at 37 °C for 48 h, and PP cultures on standard methods agar (SMA) and R2A agar, incubated at ambient temperature (23°C) for 48, 72, 168 h. Increases in the colony counts over time occurred for all three fluids. However, counts were greater on SMA and R2A than on TSA. The increases over the standard 48-hour TSA cultures ranged as high as 104 times for 23°C cultures at 7 days of incubation. Endotoxin levels even in the most contaminated samples were found to be below the acceptable 5 EU/ml recommended for reprocessor water. Bacterial colonies that appeared at 48, 72 and 168 h were isolated and identified. Pseudomonas, Moraxella, Acinetobacter and CDC group VI C-2 were among some of the common bacteria isolated. This study indicates that the media utilized, the time and temperature of incubation may result in a significant underestimation of the bacterial population of water and dialysis fluids, thus potentially placing the patient at a higher risk.