Development and evaluation of a colorimetric coliphage assay detection system

A colorimetric coliphage assay detection system (CCADS), composed of a liquid colorimetric presence-absence (LCPA) method and a colorimetric agar-based (CAB) method, was developed to overcome the limitations imposed by the Standard Methods for the Examination of Water and Wastewater agar-based coli phage method (APHA method). Both CCADS methods are based on the induction of p-galactosidase in Escherichia coli and the release of the enzyme through a lytic cell infection. The released enzyme then cleaves a chromogenic substrate, which produces a colored reaction product.

The CCADS was evaluated against the APHA method under laboratory conditions using a common sewage coliphage strain as a model (American Type Culture Collection-13706-B2), and under field conditions using water samples collected from four different sources. During thelaboratory evaluation, both the LCPA and CAB methods were found to be superior to the APHA method in coliphage detection because: 1) the LCPA and CAB methods were easier to read and interpret than the APHA method, 2) the LCPA and CAB methods were not subject to false positive results, 3) the ·LCPA method theoretically detected fewer coliphage particles than the APHA method, and 4) the CAB method detected roughly twice the number of coliphage particles detected with the APHA method. During the field evaluation, the results indicated: 1) the LCPA method was as reliable as either the CAB or APHA method in coliphage detection; 2) the LCPA and CAB methods were easier to read and interpret than the APHA method; 3) neither the LCPA method nor the CAB method were subject to false positive results; 4) the CAB method detected more coliphages than the APHA method under conditions of high fecal pollution, but both methods performed equally well in coliphage detection under conditions of low fecal contamination; and 5) the LCPA and CAB methods were equally as sensitive in coliphage detection as the APHA method.

Finally, the coliphage group proved to be a useful indicator of fecal pollution in nonpotable water supplies exhibiting a high degree of fecal pollution, whereas they were not shown to be useful indicators in potable water supplies exhibiting low levels of fecal contamination. The lack of coliphage detection sensitivity under conditions of low fecal contamination does not appear to be method-limited, but the result of inefficiencies in processing environmental samples using the concentration methods currently available.