Cultivation - independent assessment of viability with flow cytometry

Less than 1 % of bacteria in drinking water are typically culturable on conventional growth media with the heterotrophic plate count (HPC) method. In addition, it is believed that some culturable bacteria can enter a state of unculturability when exposed to nutrient-poor environments. Therefore, a need exists for rapid and cultivation-free methods with which the viability/activity of drinking water microbial communities can be assessed. This study investigated cultivation-free viability analysis using fluorescent staining methods coupled with flow cytometric detection/enumeration. The study focussed solely on quantification of cells in the planktonic phase. The results were normalised to flow cytometric total cell counts (TCC), and compared to conventional HPC and total adenosine tri-phosphate (ATP) analysis. Three fluorescent dyes, targeting different aspects of viability, were chosen based on available data in literature and preliminary experiments. These were propidium iodide (targeting membrane integrity), DiBAC3(4) (targeting membrane potential) and CFDA (measuring esterase activity). The results have shown propidiuim iodide in combination with SYBR Green I to be an easy method which can be used to assess severe cell damage as caused by ozonation, chlorination, heat and UV-A treatments. While DiBAC3(4) generally produced similar results to propidium iodide, the esterase activity assay (CFDA) tended to detect significantly less " viability " than the other dyes. ATP analysis was found to be a very sensitive indicator of viability as well, although care should be taken with free-ATP in water samples, especially during drinking water treatment with ozonation. Drinking water samples (non-chlorinated tap water and bottled drinking water) that were assessed showed around 30 – 80 % viability with the cultivation free methods, and about 0.3 – 15 % viability with conventional HPC methods. As such, the viability stains in combination with flow cytometry provide additional tools for the rapid assessment of the general microbial quality of drinking water.