CCDA Campylobacter Agar ISO

Description

After determining that Campylobacter species grow best on solidified Nutrient Broth No. 2 compared to other media, workers (1983) carried out a systematic survey of alternatives to blood for neutralizing oxygen toxicity. A combination of 0.4% charcoal, 0.25% ferrous sulfate and 0.25% sodium pyruvate proved best.

A further study surveyed the suppressive effect of several inhibitors on the undesirable microbiota, showing deoxycholate and cefazolin as the most effective inhibitory agents. Later, in 1984, Hutchinson and Bolton replaced cefazolin (10 mg/L) with cefoperazone (32 mg/L). This allowed fewer contaminants to grow, and permitted the modified medium (modified CCD Agar or mCCDA) to be used at 37 °C. However, amphotericin B was needed to prevent overgrowth by yeasts able to grow at 37 °C but not at 42 °C.

In 1993 Aspinall et al. developed a modification of mCCDA designed for use at 37 °C to isolate C. upsaliensis as well as the other thermophilic campylobacter species. This medium contains 8 mg/L cefoperazone and 4 mg/L teicoplanin, replacing 32 mg/L cefoperazone in mCCDA. Teicoplanin has an antimicrobial spectrum similar to that of vancomycin, active mainly against Gram-positive bacteria. By comparison with mCCDA, the final formulation of this medium, called CAT Agar, isolated the same numbers of Campylobacter spp. other than C. upsaliensis from faeces and is superior to mCCDA for C. upsaliensis, with slightly higher growth of competing microflora.

Technique

Immediately before use, carefully dry the agar plates, preferably with the lids off and the agar surface downwards, in a drying cabinet, until the agar surface is free of visible moisture (maximum 30 minutes).

Using the culture obtained from the enrichment broth (Bolton Broth), inoculate the medium with a sterile loop. Incubate plates at 41.5 ± 0.5 °C in a microaerobic atmosphere (approximately 5% O₂, 10% CO₂ and 85% N₂ or H₂) for 44 ± 4 hours.

• Campylobacter jejuni strains produce grey, moist, flat and occasionally spreading growth, which may be accompanied by a green hue and/or a metallic sheen.
• Campylobacter coli strains tend to be creamy-grey in colour, moist, and often produce a more discrete type of colony.
• Campylobacter lari strains are more varied and produce both types of colonial morphology.
• Occasionally, contaminating organisms may grow on this medium. These include cefoperazone-resistant Pseudomonas spp., Enterobacteriaceae, and some streptococci and yeasts.

Physical/chemical control

Microbiological control

Spiral spreading: practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity).

Microbiological control according to ISO 11133:2014/Amd 1:2018.

Microaerophilia. Incubation at 41.5 ± 1 °C; reading at 44 ± 4 h.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ASPINALL, S.T., D.R.A. WAREING, P.G. HAYWARD & D.N. HUTCHINSON (1993) Selective medium for thermophilic campylobacters including Campylobacter upsaliensis. J. Clin. Pathol. 46:829-831.
• BAYLIS, C.L., (editor) (2007) Manual of Microbiological Methods for the Food and Drinks Industry. 5th Edition Method 3.3.1:2007. CCFRA. Chipping Campden. U.K.
• BOLTON, F.J. (2000) Methods for isolation of campylobacters from humans, animals, food and water. In "The increasing incidence of human campylobacteriosis" Report and Proceedings of a WHO Consultation of Experts. Copenhagen Denmark 21-25 November 2000, WHO/CDS/CSRAPH 2001. p. 87-93.
• BOLTON, F.J. & D. COATES (1983) Development of a blood-free campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance. J. Appl. Bacteriol. 54:115-125.
• BOLTON, F.J., D. COATES & D.N. HUTCHINSON (1984) The ability of Campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. J. Appl. Bacteriol. 56:151-157.
• CORRY, J.E.L., H. IBRAHIM ATABAY, S.J. FORSYTHE & L.P. MANSFIELD (2003) Culture Media for the isolation of campylobacters, helicobacters and arcobacters. In Handbook of Culture Media for Food Microbiologists. J.E.L. Corry et al. (Eds.) Elsevier Science B.V. Amsterdam.
• CORRY, J.E.L., G.D.W. CURTIS & R.M. BAIRD (2003) Handbook of culture media for food Microbiology. Elsevier Sci. B.V. Amsterdam.
• FDA (Food and Drug Administration) (1998) Bacteriological Analytical Manual. 8th Edition. Rev. A. AOAC International. Gaithersburg, Maryland, USA.
• HUNT, J.M., C. ABEYTA & T. TRAN (1998) Campylobacter. In FDA BAM 8th Edition (rev. A) 7.01-7.027 AOAC International. Gaithersburg, Md, USA.
• HUTCHINSON, D.N. & F.J. BOLTON (1984) Improved blood-free selective medium for the isolation of Campylobacter jejuni from faecal specimens. J. Clin. Pathol. 37:956-957.
• ISO 10272-1 Standard (2017) Microbiology of the food chain - Horizontal Method for detection and enumeration of Campylobacter spp. - Part 1: Detection method.
• ISO 10272-2 Standard (2017) Microbiology of the food chain - Horizontal Method for detection and enumeration of Campylobacter spp. - Part 2: Colony count technique.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• STERN, N.J., J.E. LINE & H.C. CHEN (2001) Campylobacter In "Compendium of methods for the Microbiological Examination of Foods" 4th Ed. F.P. Downes & K. Ito (Eds.) APHA, Washington DC. USA.