Methods

The EU Food Hygiene Regulations specify the use of a 5-tube 3-dilution Most Probable Number (MPN) test for the enumeration of faecal coliforms or Escherichia coli in the classification of shellfish harvesting areas. Additionally, they specify ISO TS 16649-3 'Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of ß-glucuronidase-positive Escherichia coli - Part 3: Most probable number technique using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide.' as the reference method for such testing. In the UK, this method is currently stipulated for all testing for the purpose of classification of harvesting areas.

The method includes an initial resuscitation step in Minerals Modified Glutamate Broth followed by confirmation of E. coli on a chromogenic agar detecting beta-glucuronidase activity.

ISO TS 16649-3 does not contain MPN tables and cross-refers to ISO 7218 for these. ISO 7218:2007 contains tables for the 5-tube 3-dilution format; however, earlier versions do not.

Additional advice for sampling officers and testing laboratories has been produced, for details please view the following documents: Recommendations for the collection and transport of bivalve molluscs for the classification of bivalve mollusc harvesting areas under regulation (EC) No 854/2004 and Recommendations for the laboratory testing of bivalve molluscs for the classification of bivalve mollusc harvesting areas under regulation (EC) No 854/2004. These documents are currently under review.

Regulation (EC) No 2073/2005 on the microbiological criteria for foodstuffs recommends the use of EN ISO 6579 for the detection of Salmonella in bivalve molluscan shellfish. However, because this method has not been thoroughly validated with respect to Salmonella Typhi and Salmonella Paratyphi A, B and C, laboratories testing samples specifically for these organisms may wish to consider supplementing EN ISO 6579 with the method for S. Typhi given in Donovan et al. 1998, the reference for which is given below:

Donovan TD, et al. (1998) Modification of the standard UK method for the enumeration of Escherichia coli in live bivalve molluscs. Communicable Disease and Public Health 1, 188-196. For details please view this document, (reproduced with permission from the PHLS Communicable Disease Surveillance Centre).

Laboratories undertaking official testing of shellfish for microbiological parameters in the UK must be accredited for these specific methods by an appropriate body (e.g. UKAS ), must take part in the HPA/Cefas Shellfish EQA Scheme, and any relevant ring trials organised by the NRL.