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

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Edition: Vol. - No. Published: 11/02/2003 Number of Pages: 19File Size: 1 file , 610 KB