Evaluating the potential of flow cytometry for microbial drinking water research
Different methods for the detection of microorganisms and viruses in water samples with flow cytometry were evaluated. We have enumerated T4 bacteriophages with flow cytometry, evaluated the combination of fluorescence in situ hybridisation (FISH) and flow cytometry for mixed bacterial samples, and developed a method based on immunostaining for quantitative specific discrimination of Vibrio cholerae O1 serotype Ogawa biotype Eltor in combination with flow cytometry. In addition, the established methods for bacterial discrimination were subsequently applied for analysis of microbial populations growing on assimilable organic carbon (AOC). Since FISH in combination with flow cytometry did not enable quantitative enumeration of bacterial cells, microscopic analysis was performed to examine the composition of natural bacteria populations growing up during an AOC assay using group specific probes hybridizing to different classes of proteobacteria. The results showed that under different conditions of the assay, different microbial compositions are present. Furthermore, immunostaining of Vibrio cholerae O1 in combination with flow cytometry was applied to study the growth of this pathogen on autoclaved and sterile filtered fresh water on natural AOC. V. cholerae O1 showed extensive growth (up to 2 x 106 cells/mL; starting with 5x103 cells/mL) in river water, lake water and the effluent of a wastewater treatment plant. In these water samples, the AOC concentrations ranged from 60 μg/L up to 800 μg/L, and the results suggest that AOC is one of the key parameters governing growth. The maximum specific growth rates (μmax) of V. cholerae O1 growing on lake water at different temperatures (20 °C, 25 °C, 30 °C) were 0.23 h-1, 0.32 h-1, 0.39 h-1 respectively. The μmax increased with higher temperature, but was always half of the μmax of a similarly growing consortium of natural bacteria, isolated from the sampling site. Concurrently, in a direct batch competition experiment between V. cholerae O1 and the natural consortium at different temperatures, V. cholerae O1 grew up to 10 % of the total population in each case and no significant effect of temperature was observed on the outcome of the competition. Our results conclude that V. cholerae O1 is not only able to survive, but also able to grow in freshwater samples up to significant concentrations and hence to compete successfully with the natural microbial flora for AOC.