AWWA WQTC58879

One challenging aspect of water analysis can be the detection of a minuscule number of organisms in a large volume of water. Traditionally, membrane filtration followed by growth on a selective medium is used for the detection of indicator microorganisms. Visible colonies are presumptively identified based on pigmentation with final identification involving microscopic morphology and biochemical analysis. This entire process can take between 24 to 72 hours. Such methods are not only time consuming but uncertainties exist due to high variability in differing environmental conditions as well as the ability to capture such minute numbers in water systems. Since September 11th, 2001, concerns over intentional contamination of public water systems have been rampant. Recreational and drinking water industries are now more focused in pursuing near-real time technologies that have a high degree of specificity as well as sensitivity. The result of a sample analysis today is crucial in the event of a threat or actual contamination event. This study adapted the peptide nucleic acid chemiluminescent in situ hybridization (PNA CISH) technique, to simultaneously detect, identify, and enumerate bacteria within 6 to 8 hours in beach sand and water. Bacteria are extracted by membrane filtration, followed by incubation at 37C for 5 hours. Microcolonies are then fixed to a membrane for 5 minutes, followed by hybridization using species-specific probes for 30 minutes at 50C. Visualization of the hybridized probes was achieved by placing the membrane filter for 2 minutes in a mixture of luminol enhancer and stable peroxidase, followed by a 10-minute exposure on Polariod film. The specificity of the assay was evaluated using environmental and reference bacteria including species of Vibrio, Shigella, Salmonella, Acetobacter, Enterobacter, and Citrobacter, for which the golden standard was plate count method (PCM). Matrix interference was minimized using the buffer extraction procedure. The method is particularly sensitive, being able to detect up to one colony-forming unit per 100ml of sample. In all samples tested, levels of Eubacteria, Escherichia coli, and Staphylococcus aureus were comparable for PNA CISH and PCM. Pseudomonas aeruginosa counts tended to be higher with PNA CISH than PCM. This was probably due to PNA CISH being able to detect micro-colonies unlike PCM. Overall data analysis showed the average bacterial density in seawater at 2.27 log units/ml, with wet sand and dry sand increasing by magnitudes of 2.5 and 4.5 respectively (p=0.01). The significance of this finding is that sand may act as a reservoir for microorganisms, re-inoculating overlying water during storm and tidal events. This phenomenon is not unlikely in drinking water distribution systems in which periodic release of viable bacteria may occur during pressure fluctuation events. Includes 55 references, table, figures.