A Method of Detecting Total Viable Organisms in Water Based on Solid-Phase Microextraction and Fluorescent Signal Detection
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Microorganisms are monitored as a key indicator of water quality. Measurement of “total viable organisms” (TVO, similar to “heterotrophic plate count”, HPC) is used to indicate the general hygiene level of the water, and is often used to assess water treatment. Automated, instrumental TVO detection in water samples using an enzyme reaction method shortens the detection time compared to conventional HPC methods. A method that can detect TVO without interference in water samples by monitoring enzyme activities through fluorescent signals in small custom sample cells was developed. In this method, fluorogenic substrates were cleaved by specific enzymes to make fluorescent products, which then partitioned into a non-polar siloxane polymer film on the bottom of the sample cell. A miniature spectrometer monitored fluorescence in the polymer to give a present/absent result for bacteria. There is no interference from sample color and turbidity because the light never passes through the sample matrix. Eight substrates that can be converted by six different enzymes were characterized. These had fluorescent products coupled to glucose, glucuronic acid, galactose, phosphate, sulfate and alanine. Four different products were produced, providing detection of some enzymes simultaneously but independently, depending on the substrates chosen. Candidate fluorescent products were first tested with different siloxane polymers and two dimethyl-siloxane polymers were identified to detect all the products separately. Fifteen laboratory bacteria strains (including E. coli, Klebsiella pneumonia, and Pseudomonas aeruginosa) were added individually to a substrate solution containing minimal nutrients and incubated. All bacteria types were successfully detected by at least one substrate, with most detected by several substrates. Sterility of water samples from lake water, treated lake water and tap water were tested with those eight substrates separately or in combination. The lake water had the highest bacteria level and included coliforms and possibly E. coli. Treated lake water and tap water were E. coli and coliforms free, and were safe for drinking. Treated lake water had higher TVO levels than tap water, indicating a lower hygiene level. The results of combined substrate tests matched the corresponding single substrate tests, suggesting specific bacteria strains can be distinguished in a single test.