ISO/NP 9308-2

Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 2: Most probable number method

Scope

This document specifies a method for the enumeration of E. coli and coliform bacteria in water. The method is based on the growth of target organisms in a liquid medium and calculation of the “Most Probable Number” (MPN) of organisms by reference to MPN tables. This method can be applied to arrange of types of water (for example drinking water, groundwater, surface water, recreational water and wastewater), including those containing an appreciable amount of suspended matter and high background counts of heterotrophic bacteria.
When using for the enumeration of E. coli in marine waters, a 1→10 dilution in sterile water is typically required because elevated levels of salts can slow the growth of the target bacteria. However, the method has been shown to work well with some marine waters that have a lower than normal concentration of salts and provided verification of the performance has been undertaken, the procedure can be used for such waters. It should be noted that when elevated levels of salt are present some halophilic vibrios can give a false positive β-D-galactosidase result. In the absence of data to support the use of the method without dilution, a 1→10 dilution should be used.
This method relies upon the detection of E. coli based upon expression of the enzyme β-D-glucuronidase and consequently does not detect many of the enterohaemorhagic strains of E. coli, which do not typically express this enzyme. Additionally, there are a small number of other E. coli strains that do not express β-D-glucuronidase.
The choice of tests used in the detection and confirmation of the coliform group of bacteria, including E. coli, can be regarded as part of a continuous sequence. The extent of confirmation with a particular sample depends partly on the nature of the water and partly on the reasons for the examination. The test described in this part of ISO 9308 provides a confirmed result with no requirement for further confirmation of positive wells.
NOTE While this method describes the use of an enumeration device that is commercially available, the medium described here can also be used in a standard MPN format although verification of such a protocol should be carried out.