Detection procedure A is designed for samples with low number and/or stressed Campylobacter with a low non-target background microflora, such as cooked or frozen products. Procedure A uses a 1:10 dilution of the sample in Bolton broth, which utilizes a cocktail of cefoperazone, vancomycin, trimethoprim and amphotericin B to select for Campylobacter spp. The sample is incubated at 37 degrees Celsius for four to six hours, followed by a 44-hour incubation at 41.5 degrees Celsius. All incubation is in a microaerobic atmosphere. The lower temperature pre-incubation is to allow for resuscitation of stressed cells prior to the more selective higher temperature. The prolonged 44-hour enrichment is to allow for sufficient multiplication of a low number of target cells.
Procedure B is designed for samples with a low number of Campylobacter in the presence of a high level of non-target background microflora, such as raw meat or milk. Procedure B uses a 1:10 dilution of the sample in Preston broth, which utilizes a cocktail of polymyxin B, rifampicin, trimethoprim and amphotericin B to select for Campylobacter spp. The sample is incubated at 41.5 degrees Celsius for 24 hours in a microaerobic atmosphere. The main difference between Bolton and Preston broths is that Bolton is formulated to better cope with the resuscitation of stressed microorganisms, whereas Preston broth is more selective to deal with a high background challenge.
Procedure C does not utilize an enrichment step and is a direct plating method for products with high numbers of Campylobacter, such as poultry caecal content. Procedure C can be used with the second part of ISO 10272 concerned with enumeration of Campylobacter in the test material.
As previously mentioned, the plating medium of choice in all protocols is mCCD agar, which, unlike both Preston and Bolton broths, is a blood-free medium. In the formulation, blood is replaced by charcoal, ferrous sulfate, and sodium pyruvate to aid in the recovery and growth of Campylobacter. The agar is made selective with the addition of the bile salt sodium deoxycholate, the third generation cephalosporin antibiotic cefoperazone and the antifungal amphotericin B.
Typical colonies of Campylobacter on mCCD agar are grey/white, often with a metallic sheen, and are flat and moist. Further confirmation is done by examination of morphology and motility, presence of oxidase, and absence of aerobic growth.
A very similar organism that is often mistaken for Campylobacter is Arcobacter. Like Campylobacter, Arcobacter is also a member of the family Campylobacteraceae and exhibits a very similar morphology on mCCD agar. A way to differentiate the two, however, is by testing duplicate plates both aerobically and microaerobically. Arcobacter can tolerate the aerobic environment and will grow, but Campylobacter cannot. The clinical significance of Arcobacter is debatable; however, in this case, it is a cause of false positive results and/or incorrect counts.
Preston and Bolton broths have been favored by ISO as the enrichment media of choice, although there are other media that have been shown to be highly effective at selectively enriching Campylobacter from other sample types. Exeter broth has been used successfully to enrich Campylobacter from samples such as poultry house boot socks used for monitoring levels of the organism in and around the poultry house. Similar to Preston, Exeter uses a nutrient broth base, supplemented with lysed horse blood, but instead uses trimethoprim, rifampicin, polymyxin, cefoperazone, and amphotericin b as selective antibiotics. Again plating is done using mCCD agar, but reduction of background microflora can be achieved with the use of a 0.45-micrometer disk filter. By placing a filter on the agar surface and placing 100 microliters of enriched sample on top, the Campylobacter can pass through whilst most other enteric microorganisms are retained. The filter can then be discarded and the plates incubated as normal.
Whilst traditional microbiology methods remain at the forefront of Campylobacter testing, rapid methods are being further more utilized as a detection tool. ISO guidelines are available regarding the detections of pathogens using polymerase chain reaction (PCR)—ISO 22174:2005 and ISO 20838:2006—and many commercial products are available for Campylobacter that can give a result in around 90 minutes after 24 hour pre-enrichment. Unique DNA targets are amplified to create millions of copies that can be analyzed to give a result. Real-time PCR can be used to amplify and simultaneously detect amplified target by using florescent-labeled probes that generate reporter molecules. These molecules are excited by light and detected by the machine. This process allows for much quicker results without further analysis.