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PHE updates Campylobacter and Salmonella methods guidance

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By Joe Whitworth+

Last updated on 22-Jul-2014 at 12:19 GMT

Salmonella and Campylobacter detection and enumeration guidance published
Salmonella and Campylobacter detection and enumeration guidance published

Public Health England (PHE) has published standard methods for the detection and enumeration of Campylobacter and Salmonella species.

Performance of a standard method depends on the quality of reagents, equipment, commercial and in-house test procedures and labs should ensure they have been validated and shown to be fit for purpose.  

In using Standard Methods, laboratories should take account of local requirements and it may be necessary to undertake additional investigations, said PHE.

Campylobacter method

Detection of Campylobacter in a 25g sample of ready to eat food is considered unsatisfactory and potentially injurious to health.

The detection of Campylobacter species in food and environmental samples involves enrichment in a selective liquid medium at 37oC for five hours.

Then microaerobic incubation at 41.5oC for 44 hours to allow recovery and growth, sub-culture onto selective solid media, and examination for colonies considered typical of Campylobacter species.

Confirmation of the colonies is performed using morphological, biochemical and growth property tests.

The enrichment culture method uses Bolton Broth which allows resuscitation and recovery of injured organisms and is based on BS EN ISO 10272-1:20063 .

The mathematical lower limit of enumeration is one or two colony forming units (CFU) per millilitre (mL) of liquid samples, or 10 or 20 CFU per gram (g) of other food products.

Internationally recognised methods

The method described is based on BS EN ISO 10272-1:20063 (detection) and BS EN ISO/TS 10272-2:20064 (enumeration). 

Modified Cefperazone Charcoal Deoxycholate Agar (mCCDA) isolation medium is used and Campylobacter species form greyish, flat and moist colonies, with a metallic sheen and a tendency to spread.

Food manufacturing processes such as heating, freezing or chilling can cause sub-lethal injury to Campylobacter species, resulting in increased sensitivity to antibiotics and lower resistance to elevated temperatures.

The enumeration of Campylobacter species involves inoculation of the surface of a selective agar media with a defined volume of an appropriate decimal dilution of the test sample.

Agar plates are incubated microaerobically at 41.5 °C for up to 48 hours and calculation of the number of CFU per g or mL of sample is determined from the number of typical colonies on the selective media, and confirmed by morphological, biochemical and growth property tests.

PHE describes the different methods for sample preparation, inoculation and incubation for detection and dilutions and for enumeration.

The agency also identifies the procedure for recognition and counting colonies from the enumeration method. 

Salmonella method

The presence of Salmonella species in ready-to-eat food is considered to be unsatisfactory regardless of the level of contamination.

PHE’s method is based on BS EN ISO 6579:2002 + A1:2007 .

It uses Rappaport Vassiliadis Soya Peptone (RVS) and Muller Kauffmann Tetrathionate Novobiocin (MKTTn) broth for the isolation of serotypes of Salmonella that are inhibited by the constituents of RVS broth.

The method includes the use of Selenite Cystine (SC) broth for samples in which S. Typhi and S.Paratyphi are specifically sought. 

Xylose Lysine Deoxycholate (XLD) and brilliant green agar (BGA) are isolation media specified and if S.Typhi or S. Paratyphi are being sought Hynes, Deoxycholate Citrate Agar (DCA) should also be used.

Detection in food and environmental samples from the food production environment involves pre-enrichment in a non-selective liquid medium with adjustments to enhance recovery from certain food types, enrichment in two selective liquid media, sub-culture onto two different selective solid media, and examination for colonies considered to be typical of Salmonella.

Confirmation of the colonies as Salmonella is performed using serological and biochemical tests.

PHE covers sample preparation and pre-enrichment, selective enrichment, subculture to selective media, recognition of colonies and confirmatory tests.

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