ENVIRONMENT AGENCY The Microbiology of Drinking Water (2002)

DD1The enumeration of coliform bacteria and Escherichia coli by a defined substratemost probable number techniqueIntroductionTests for coliform bacteria and Escherichia coli (E. coli) are the most importantroutine microbiological examinations carried out on drinking water. They provide themost sensitive means for detecting faecal contamination, for assessing theeffectiveness of water treatment and disinfection, and for monitoring water quality indistribution. The significance of E. coli and coliform bacteria in water treatment andsupply are described elsewhere (1) in this series.D2ScopeThis method comprises a most probable number (MPN) technique and is suitable forthe examination of drinking waters, including samples from all stages of treatmentand distribution, and those source waters of moderate to high turbidity.Users wishing to employ this method, or similar methods from other manufacturers,should verify the performance under their own laboratory conditions (2) . Details ofevaluation trials are reported elsewhere (3) .D3DefinitionsDefined substrate media are chemically defined formulations containing substrates forthe specific detection of diagnostic enzymes associated with a particular group oforganisms.In the context of this method, organisms which are oxidase-negative and produceβ-galactosidase, as demonstrated by the production of a yellow colour through theenzymatic cleavage of ortho-nitrophenyl-β-D-galactopyranoside in a defined substratemedium, are regarded as coliform bacteria. In addition, organisms which produceβ-glucuronidase, as demonstrated by the production of a yellow colour and blue-whitefluorescence (under long wavelength ultra-violet illumination) through the enzymaticcleavage of 4-methylumbelliferyl-β-D-glucuronide in a defined substrate medium, areregarded as E. coli.Coliform bacteria are considered to be members of genera or species within theFamily Enterobacteriaceae, capable of growth at 37 °C, that possess β-galactosidase.This definition includes anaerogenic (ie non-gas producing) strains. The followinggenera have been commonly isolated in routine practice: Citrobacter, Enterobacter,Escherichia, Hafnia, Klebsiella, Serratia, Yersinia, Buttiauxella, Leclercia.For the purposes of water examination E. coli have historically been regarded asmembers of the Family Enterobacteriaceae which ferment lactose or mannitol at 44 °Cwith the production of acid within 24 hours, and which produce indole fromtryptophan. Most strains produce β-glucuronidase. Strains which possess thesecharacteristics at 37 °C but do not express them at 44 °C may also be E. coli. When44