Fisheries Research Services
The Classification of Shellfish Production Areas Using E.coli
How are shellfish production areas classified?
Production areas are grouped into four different categories:
Category A
Less than
230 E. coli/100g
shellfish flesh
May go for human
consumption if End
Product Standard* met
Category B
Less than
4,600 E. coli/100g
shellfish flesh in
90% of samples
Must be depurated,
heat-treated or relayed
to meet Category A
requirement
Category C
Less than
Must be relayed for long
46,000 E. coli/100g periods (at least two
shellfish flesh
months) whether or not
combined with
purification, or after
intensive purification
to meet Category A or B
Microscopic appearance of E. coli (magnification x 100)
What are E. coli?
Escherichia coli (E. coli) are bacteria.
Where are E. coli found?
E. coli are found in large numbers in the intestines of
humans and warm-blooded animals. They are generally
harmless, but when passed in excrement they can enter
seawater from sewage or farm animal run-off. Once in
seawater, they can be concentrated by filter-feeding
shellfish.
Can the presence of E. coli in shellfish tell us anything?
Yes, if E. coli are found in shellfish, it is likely that the
shellfish have been exposed to faecal contamination.
Many bacterial pathogens, for example Salmonella,
Campylobacter and E. coli 0157, found in the faeces of
warm-blooded animals, also infect humans. Therefore an
indicator of both human and animal faecal contamination
(such as E. coli) is required.
What does the EU legislation say?
EU Directive 91/492/EEC, and associated UK legislation
states that it is a requirement of the competent authority
to classify shellfish production areas according to the
amount of microbiological contamination found there.
The Directive also states that indicators of faecal bacteria,
such as E. coli, should be used, and that the recommended
laboratory method to determine levels of E. coli present
in shellfish samples is a five tube, three dilution Most
Probable Number (MPN) test.
More than
Unsuitable for production
46,000 E. coli/100g
shellfish flesh
* A requirement to be met before a product can be marketed
Who carries out the Most Probable Number (MPN) tests?
The Food Standards Agency Scotland (FSAS) inherited
the role of competent authority in Scotland, from the
Scottish Executive, to commission testing for E. coli in
shellfish in April 2000. FSAS commissions this work from
Fisheries Research Services (FRS) Marine Laboratory. FRS
Marine Laboratory has United Kingdom Accreditation
Service (UKAS) accreditation for the EU method of E. coli
detection in shellfish.
What is the MPN test?
To comply with the Directive, the test used at FRS Marine
Laboratory is a two-stage method devised to distinguish
between characteristic features of E. coli and other bacteria
which do not originate from human or animal faeces.
Fisheries Research Services is an agency of the Scottish Executive
FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen AB11 9DB UK
tel +44 (0)1224 876544 fax +44 (0)1224 295511
enquiries@marlab.ac.uk http://www.frs-scotland.gov.uk
Fisheries Research Services
MPN test: Stage One
The first stage of this test involves a resuscitation step.
Resuscitation allows the recovery of injured E. coli cells.
This is necessary, as they may not initially be culturable
since they have been taken from the marine environment
(cold, salty water).
In the resuscitation stage of the test, tubes containing
minerals modified glutamate broth (MMGB) are inoculated
with dilutions made from an homogenate of shellfish
flesh. These are incubated at 37° C for 24 hours. Tubes
showing the presence of acid (as determined by colour
change from purple to yellow) are then examined for the
presence of E. coli.
Stage One: Resuscitation of E. coli in MMGB broth.
determines the concentration of E. coli in the shellfish
flesh.
This method is recommended by the UK National Reference Laboratory (The Centre for Environment, Fisheries
and Aquaculture Science, CEFAS, Weymouth) which was
designated in 1999 to monitor and ensure the uniformity
of microbiological testing for shellfish in the UK.
Why do we need to ensure uniformity in testing?
To ensure uniformity in results between different
laboratories it is essential that the same sampling and
methodology procedures are used. Whilst there are
different methods available for the detection of E. coli,
these tend to be tailored to specific sample types. Clinical
samples (faeces, urine), dairy products and water samples
may all be tested using slightly different procedures.
Routine detection methods for E. coli in clinical samples
do not include the resuscitation step, and will not recover
damaged E. coli cells. This may lead to an underestimation
of the levels of E. coli in shellfish.
E. coli positive
E. coli negative
Stage Two: Confirmation of E. coli.
MPN test: Stage Two
The presence of E. coli is confirmed by sub-culture to a
chromogenic medium at 44° C for 24 hours. This medium
visibly differentiates between E. coli and other bacteria
with blue/green colonies denoting the presence of E. coli.
Referring the number of tubes (mentioned at Stage One)
yielding a positive result to published standard tables
Tests used to detect contamination in other foodstuffs
are often based upon the need to detect coliform
organisms. In general these include E. coli but also other
bacteria such as Klebsiella, Enterobacter and Citrobacter
which are not necessarily of faecal origin. Using this type
of test for shellfish could lead to an overestimation of the
levels of E. coli in shellfish.
Are quality assurance (QA) measures in place?
To address the possibility of different testing laboratories
obtaining different results, the Public Health Laboratory
Service (PHLS) run an External Quality Assurance (EQA)
scheme in conjunction with the National Reference
Laboratory (CEFAS). Twice a year, samples are sent to FRS
Marine Laboratory to be analysed following the normal
testing routine. These samples are artificially contaminated
with a variety of bacteria, including known levels of
E. coli, and are used to simulate raw shellfish from
harvesting sites. FRS Marine Laboratory has consistently
detected the correct levels of E. coli in these samples. In
addition, the United Kingdom Accreditation Service (UKAS)
has accredited FRS Marine Laboratory’s methods for the
detection of E. coli in shellfish.
AE04|02|04
Fisheries Research Services is an agency of the Scottish Executive
FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen AB11 9DB UK
tel +44 (0)1224 876544 fax +44 (0)1224 295511
enquiries@marlab.ac.uk http://www.frs-scotland.gov.uk
© Crown copyright