Recently, the β-galactosidase assay has become a key component in the development of assays and biosensors for the detection of enterobacteria and E. coli in water quality monitoring. The assay has often performed below its maximum potential, mainly due to a poor choice of conditions. In this study we establish a set of optimal conditions and provide a rough estimate of how departure from optimal values reduces the output of the assay potentially decreasing its sensitivity. We have established that maximum response for detecting low cell concentrations requires an induction of the samples using IPTG at a concentration of 0.2 mM during 180 min. Permeabilization of the samples is mandatory as lack of it results in an almost 60% reduction in assay output. The choice of enzyme substrate is critical as different substrates yield products with different extinction coefficients or fluorescence yields. The concentration of substrate used must be high enough (around 3 to 4 times Km) to ensure that the activity measured is not substrate limited. Finally, as the color/fluorescence of the reaction products is highly dependent on pH, care must be taken to ensure that pH at the time of reading is high enough to provide maximum signal.
Keywords: Assay optimization; Cell lysis; Cell permeabilization; Coliform detection; E. coli detection; β-galactosidase.
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