Campylobacter Agar Base Blood Free (CCDA) ISO

Campylobacter is considered the main cause of enteric illnesses. Campylobacter spp. can cause mild to severe diarrhea, with loose, watery stools frequently followed by bloody diarrhea. These pathogens are very infective and are transmitted by contaminated food or water.

The medium contains ferrous sulfate, sodium pyruvate and charcoal to promote the growth of Campylobacter species, as they quench the toxic forms of oxygen (hydrogen peroxide), increasing the aerotolerance and enabling the oxygen-sensitive strains to be readily isolated. Sodium deoxycholate partially or completely inhibits Gram-positive organisms, coliforms and Proteus. Enzymatic digest of animal tissue and casein, and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Bacteriological agar is the solidifying agent. Cefoperazone increases selectivity and inhibits the growth of Gram-negative enteric bacilli and some Gram-positive species, whilst amphotericin B suppresses yeasts and fungi growth. The increment of temperature has shown an increase in selectivity.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 22.75 grams of the medium in 500 mL of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 °C for 15 minutes. Cool to 44-47 °C and aseptically add one vial of CCDA Supplement (Campylobacter Blood Free) (LC-6053). Homogenize gently and dispense into Petri dishes.

According to ISO 10272

For the detection and enumeration of Campylobacter spp. according to ISO 10272.

For samples with low numbers of campylobacters and low level of background microflora and/or stressed campylobacters:

1. Inoculate 10 g or 10 mL of the test portion into 90 mL of the enrichment medium Bolton Broth, so as to obtain a 1 in 10 dilution.
2. Incubate in a microaerobic atmosphere at 37 °C for 4 to 6 hours, then at 41.5 °C for 44 ± 4 hours.
3. Using the culture obtained from the enrichment broth (Bolton Broth), inoculate the CCDA medium and another Campylobacter selective medium with a sterile loop.
4. Incubate the CCDA plates at 41.5 °C in a microaerobic atmosphere for 44 ± 4 hours.
5. Confirm the suspect colonies.

For samples with low numbers of campylobacters and high level of background microflora:

1. Inoculate 10 g or 10 mL of the test portion into 90 mL of the enrichment medium Preston Broth, so as to obtain a 1 in 10 dilution.
2. Incubate in a microaerobic atmosphere at 41.5 °C for 14 ± 2 hours.
3. Using the culture obtained from the enrichment broth (Preston Broth), inoculate the CCDA medium with a sterile loop.
4. Incubate the CCDA plates at 41.5 °C in a microaerobic atmosphere for 44 ± 4 hours.
5. Confirm the suspect colonies.

For samples with high numbers of campylobacters:

1. Inoculate the test portion if the product is liquid, or the initial suspension in the case of other products (direct plating), onto CCDA plates.
2. Incubate the plates at 41.5 °C in a microaerobic atmosphere for 44 ± 4 hours.
3. Confirm the suspect colonies.
4. The direct plating method is the adequate procedure to count the colonies of Campylobacter.

If little information is available about the test samples, use one of the two enrichment methods in parallel with the direct plating method.

Typical colonies are greyish, often with a metallic sheen, and are flat and moist, with a tendency to spread.

In order to confirm the suspect colonies, examine the Campylobacter colonies for morphology and motility using a microscope, subculture on a non-selective blood agar, and then confirm by detection of oxidase activity and an aerobic growth test at 25 °C.

The container of this product may suffer deformation due to the high oxygen adsorption capacity of the activated charcoal contained in the formula.

• Bolton F.J., Hutchinson D.N. and Cioste D. (1984) Clin. Microbiol. 19,169-171
• Bolton E.J., Robertson L. (1982) J. Clin. Path. 35, 462-67
• ISO 10272-1:2017. Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method