Use of the ISO 9308-1 procedure for the detection of E. coli in water utilizing two incubation temperatures and two confirmation procedures and comparison with defined substrate technology

Abstract

Disinfected and non-disinfected samples have been used to determine the accuracy of the ISO procedure (ISO 9308-1) for detection of E. coli in drinking water. Samples were analysed using the ISO procedure at both 36 and 448C and using the defined substrate technology method -18). Utilizing the confirmation procedure described in ISO 9308-1, large numbers of false positive E. coli results were obtained using the ISO primary isolation procedure at 368C. However, when glucuronidase production was used as the confirmation procedure, the 'confirmed' count of E. coli was lowest with ISO 9308-1 performed at 368C. When TTC medium was incubated at 368C confirmation using production of indole at 448C resulted in 29% more 'E. coli' being recovered than when confirmation was performed using production of glucuronidase. When 448C was used as the primary isolation temperature the difference between the number of 'confirmed' E. coli identified using the two confirmation procedures was less than 1% and was not significant. Identification of isolates which 'confirmed' when using production of indole at 448C as the test method but 8which failed to produce b-D-glucuronidase, showed that the majority of these isolates were Klebsiella oxytoca.

Understanding the cause of false negative β-d-glucuronidase reactions in culture media containing fermentable carbohydrate

“…In recent years, there has been considerable interest in the application of methods utilizing substrates for the enzyme β‐ d ‐glucuronidase in the food and water industries (Frampton and Restaino 1993; Manafi 2000) and methods relying upon detection of the enzyme are becoming more and more widely used. Whilst there has been much discussion on the relative merits of such media, particularly because they use a different definition of E. coli than is used by more traditional methods, Fricker et al. (2008a) demonstrated that not only did such methods tend to be more sensitive than traditional lactose‐based methods, but they were also considerably more specific and less prone to false positive results.…”

“…Furthermore, such media tend to be more sensitive and more specific than media based on fermentation of lactose at elevated temperatures (Niemela et al. 2003; Fricker et al. 2008a).…”

“…Despite the wide distribution of b-d-glucuronidase in nature, media used for the detection of E. coli based on detection of the enzymes b-d-glucuronidase and b-d-galactosidase have become widely used, particularly in water microbiology (Fricker and Fricker 1996). Furthermore, such media tend to be more sensitive and more specific than media based on fermentation of lactose at elevated temperatures (Niemela et al 2003;Fricker et al 2008a).…”

False-negative β-d-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms andEscherichia colifrom water

“…2008). However, it is clear that not all such media perform equally (Fricker and Fricker 1996; Niemela et al 2003; Fricker et al. 2008; Olstadt et al.…”

Equivalency testing of TTC Tergitol 7 agar (ISO 9308-1:2000) with five culture media for the detection of E. coli in water samples in Greece

“…The method defined for enumeration of total coliforms and E. coli is MF on Lactose TTC agar with Tergitol 7 (Chapman 1951) as described in ISO 9308-1:2000 corrigendum to this standard was published recommending the additional use of the b-D-glucuronidase test. Fricker et al (2008) suggest that use of the test for the detection of b-D-glucuronidase as a marker for E. coli gives more accurate results than use of tests for indole production at 448C. Nevertheless, the Directive specifies that "Member States which have recourse to alternative methods shall provide the Commission with all relevant information concerning such methods and their equivalence".…”