Protocol for the recovery and detection of Escherichia coli in environmental water samples.

To achieve active management of bathing areas and to reduce risk associated with the presence of fecal pollution, tests capable of rapid on-site assessment of microbiological water quality are required. A protocol for the recovery and detection of fecal pollution indicator bacteria, E. coli, using β-glucuronidase (GUS) activity was developed. The developed protocol involves two main steps: sample preparation and GUS activity measurement. In the sample preparation step, syringe filters were used with a dual purpose, for the recovery and pre-concentration of E. coli from the water matrix and as μL reactors for bacteria lysis and GUS extraction. Subsequently, GUS activity was measured using a continuous fluorometric method developed previously. The optimum GUS recovery conditions for the sample preparation step were found to be 100 μL PELB (supplemented with 1 mg mL-1 lysozyme and 20 mM DTT) at 37 °C for 30 min. The protocol was evaluated on environmental samples (fresh and seawater) against an establish GUS assay method (Coliplage® ). GUS activities corresponding to samples containing as low as 26 MPN E. coli 100 mL-1 were detected for the seawater sample and as low as 110 MPN E. coli 100 mL-1 for the freshwater samples. By comparison with the Coliplage® method, this protocol offered an improvement in the measured GUS activities of 3.1 fold for freshwater samples and 4.1 fold for seawater samples. Furthermore, the protocol developed here, has a time-to-result of 75 min, and successfully addresses the requirement for tests capable of rapid assessment of microbiological water quality.