DVC Methods for Assessing Living Bacterial Numbers in Natural Waters : Measurement of metabolic activity in bacterial cells by substrate-enhanced INT-reduction and digitised image analysis

Two DC methods AO and DAPI, were optimised and applied to quantify total populations of bacterial cells in natural waters, with expected differing bacterial numbers, nutritional contents, temperature and salinity. Various culture-based methods were also applied to quantify viable bacterial populations. The two DVC methods optimised in this study, successfully quantified "Living" (Viable culturable and VBNC) bacterial populations in natural waters. NADVCs and INTDVCs were consistently lower than total direct counts and also consistently higher than standard heterotrophic plate counts. Results obtained for cultured bacteria using both DVC methods were similar. However, for mixed bacterial populations, the counts were always higher with the INT method than with the NA method. Combining both DVC methods yielded a statistically significant increase in numbers of INT active cells compared with the INT method used alone. Substrate-enhanced INT method when combined with digitised image analysis, allowed for the identification of different bacterial morphological types and the quantification of the overall physiological states of individual bacterial cells in samples of raw domestic sewage.