ATP bioluminescence for determining total bacteria in natural water to remove free ATP

ATP bioluminescence is a simple, highly sensitive, and real-time monitoring assay to detect the total number of bacteria in sample. However, other forms of ATP in natural water usually interfere with determination of bacterial ATP. Therefore, how to eliminate other body cells and free ATP is an important preprocessing step in bioluminescence method. This research investigated the optimal concentration and the extraction time of body cell extraction agent (Triton-100). Meanwhile, we also studied the optimal free ATP hydrolase (apyrase) concentration and its effect on bacterial ATP assay, which was also applied to the bacteria counts in the natural waters (body cell-free) system containing Tritonx-100 and apyrase. The result showed that using 0.4% of Tritonx-100 could effectively extract body cells in 2 minutes and 0.2 U apyrase could effectively hydrolyze body cells in water samples. In addition, adding apyrase to samples did not hydrolyze bacteria and had no effect on the bacterial ATP measurement. The body-cell-free system with Tritonx-100 and apyrase could decompose body cells and had no significant impact on the bacterial ATP measurement. Besides, the practical application also proved that this system accurately detected the number of bacteria in environmental samples by ATP bioluminescence method. Therefore, by treating natural water with Tritonx-100 and apyrase, body cells and free ATP can be completely eliminated, leading to actual bacteria counts in natural water.