Quantification of microbial cells using fluorescence

Among the parameters analysed by microbiologists, cell number or biomass is one of the most critical. Many techniques have been developed for direct and indirect cell enumeration in pure cultures and environmental samples. The time requirements, cost, and sensitivity of these methods vary widely. A microtitre assay has been developed recently for the sensitive detection of microbial cells in aqueous samples, using SYBR Green I in combination with a fluorometric microplate reader. The purpose of the present study was to confirm the sensitivity and utility of this method by quantifying purified λ phage DNA, as well as bacterial cells from cultures, industrial samples, and environmental samples. The lower detection limit for viral DNA was found to be 0.1 pg mL-1, and for lab-cultured E. coli, 2.5 x 104 cells mL-1. These values were consistent with those published previously. In addition, SYBR Green I was used to quantify cells in industrial water before and after treatment by UV irradiation. These results were compared to those obtained using the Live/Dead® BacLight™ Bacterial Viability Kit. In both cases, higher fluorescence occurred in UV-treated samples, contrary to expectations. Overall, the method tested here is relatively fast, simple and economical, and may be useful in the estimation of cell numbers or biomass in microbial cell suspensions or cultures. It also appears to have some utility in the quantification of microbes in environmental fresh water. However, its utility in viability assays is uncertain and may be affected by the presence of unknown organic compounds. This is the first reported use of this method to analyse samples of environmental or industrial water without the use of preliminary enrichments.