ENVIRONMENT AGENCY The Microbiology of Drinking Water (2002)

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D9.3 Reading of resultsAfter incubation, the pouch is examined and the number of wells that have a sufficientyellow colour, compared against the manufacturer’s comparator, is recorded. Thepouch is then re-examined under an ultra-violet long wavelength lamp and the numberof wells, that produce a blue-white fluorescence of sufficient intensity comparedagainst the manufacturer’s comparator, is recorded. If the pouch is examined beforethe completion of 22 hours incubation and this examination reveals borderlineresponses, then it should be returned to the incubator for the remaining incubationperiod. After 22 hours incubation, the pouch is removed and re-examined as before.D9.4 Confirmation testsThis method is reported to be highly specific for coliform bacteria (yellow colorationin the wells) and E. coli (blue-white fluorescence in the wells). Hence, confirmationtests are not usually required. Should there be any doubt as to the type of organismand response detected, then wells showing a positive response should be sub-culturedand confirmatory tests undertaken.D10CalculationsD10.1 Confirmed coliform bacteria and E. coliThe MPN of coliform bacteria is determined by reference to appropriate probabilitytables, see for example table D1. This is derived from the number of wells showing apositive, yellow coloration. For example, if there are 31 wells showing a yellowcoloration in the reaction pouch, then from table D1, the MPN of coliform bacteria is48 per 100 ml of sample, or diluted sample, examined. Any dilution needs to be takeninto account.The MPN of E. coli is determined by reference to the same probability table. This isderived from the number of wells showing a positive blue-white fluorescence. Forexample, if there are 12 wells showing a blue-white fluorescing in the reaction pouchthen, from table D1, the MPN of E. coli is 14 per 100 ml of sample, or diluted sampleexamined. Any dilution needs to be taken into account.D11Expression of resultsConfirmed coliform bacteria and E. coli counts are expressed as MPN counts pervolume of sample. For drinking water, the volume is typically 100 ml.D12Quality assuranceNew batches of media should be tested with appropriate reference strains of targetbacteria (for example, E. coli and Enterobacter aerogenes) and non-target bacteria(for example, Aeromonas hydrophila and Pseudomonas aeruginosa). Pouches shouldbe incubated for 18 - 22 hours at 37 °C. Further details are given elsewhere (2) in thisseries.47
D13References1. Standing Committee of Analysts, The Microbiology of Drinking Water (2002)- Part 1 - Water Quality and Public Health. Methods for the Examination of Watersand Associated Materials, in this series, Environment Agency.2. Standing Committee of Analysts, The Microbiology of Drinking Water (2002)- Part 3 - Practices and Procedures for Laboratories. Methods for the Examination ofWaters and Associated Materials, in this series, Environment Agency.3. Standing Committee of Analysts, Evaluation trials for two media for thesimultaneous detection and enumeration of Escherichia coli and coliform organisms1998, Methods for the Examination of Waters and Associated Materials, in this series,Environment Agency.4. The Control of Substances Hazardous to Health Regulations 1999, StatutoryInstrument 1999 No. 437.5. IDEXX Laboratories, Milton Court, Churchfield Road, Chalfont St Peter,Buckinghamshire, SL9 9EW.48
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ENVIRONMENT AGENCY The Microbiology of Drinking Water (2002)

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