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Modified Charcoal Cefoperazone deoxycholate Agar Base (mCCD agar)
Recommended for selective detection and enumeration of Campylobacter species from food chain as per the specifications laid down in ISO 10272-2:2017.
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Intended use
Recommended for selective detection and enumeration of Campylobacter species from food chain. The composition and performance criteria of this medium are as per the specifications laid down in ISO 10272-1:2017, 10272-2:2017, ISO 17995:2019 and ISO 11133:2014 (E) /Amd.: 2020
Composition
**Formula adjusted, standardized to suit performance parameters
ISO 10272-1 and ISO 10272-2 / ISO 17995:2019 (Ε) Specification - mCCD agar
| Ingredients | g / L |
|---|---|
| Meat extract | 10.000 |
| Enzymatic digest of animal tissues | 10.000 |
| Enzymatic digest of casein | 3.000 |
| Sodium chloride | 5.000 |
| Sodium deoxycholate | 1.000 |
| Iron (II) sulfate, hydrate | 0.250 |
| Sodium pyruvate | 0.250 |
| Activated charcoal | 4.000 |
| Agar | 8.0-18.0 |
| Supplements to be added after autoclaving | |
| Cefoperazone | 0.032 g |
| Amphotericin B | 0.01 g |
| Final pH (at 25°C) | 7.4±0.2 |
mCCD agar M8871
| Ingredients | g / L |
|---|---|
| HM Extract # | 10.000 |
| Peptone ## | 10.000 |
| Tryptone ### | 3.000 |
| Sodium chloride | 5.000 |
| Sodium deoxycholate | 1.000 |
| Iron (II) sulfate, hydrate | 0.250 |
| Sodium pyruvate | 0.250 |
| Activated charcoal | 4.000 |
| Agar | 12.000 |
| Supplements to be added after autoclaving | |
| CCDA Selective Supplement-FD135 (2 vials) | |
| Cefoperazone | 16 mg |
| Amphotericin B | 5mg |
| Final pH (at 25°C) | 7.4±0.2 |
# Equivalent to Meat extract
## Equivalent to Enzymatic digest of animal tissues,
### Equivalent to Enzymatic digest of casein
Directions
Suspend 22.74 gram (the equivalent weight of dehydrated medium per litre) in 500 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C and aseptically add rehydrated contents of 1 vial of CCDA Selective Supplement (FD135). Mix well and pour into sterile Petri plates.
Principle And Interpretation
Campylobacters are carried in the intestinal tract of animal and therefore contaminate foods of animal origin (1). Campylobacter causes intestinal upset or abortion in animals. It is also one of the most important causes of human gastroenteritis, particularly in children. Initially blood was used in the isolation of Campylobacter. But, later it was reported by Bolton et al (2) that charcoal can be effectively used in place of blood. This rules out the variability obtained due to the use of blood. Modified Charcoal Cefoperazone Deoxycholate Agar Base is formulated as per APHA (1) and is also recommended by the ISO Committee (3,4,5) for detection and enumeration of Campylobacter spp. from food and environmental samples in food production area.
Cephalothin in the original formulation was replaced by Cefoperazone as the selective agent since the latter gave better selectivity (7). Campylobacter species are highly resistant to cefoperazone, an antibiotic which effectively suppresses growth of Pseudomonas and Enterobacteriaceae (8-10). Addition of cefoperazone increases the selectivity of the medium. Due to this addition, the medium is also known as Campylobacter Charcoal Differential Agar (CCDA). Charcoal, sodium pyruvate and iron sulfate reduces the aero tolerance of medium by quenching photo chemically generated toxic oxygen derivatives (11). Peptone, Tryptone and HM extract serve as sources of carbon, nitrogen, long chain amino acids and essential nutrients. Sodium chloride maintains osmotic balance. Additional Amphotericin B suppresses the growth of yeast and mold contaminants.
Type of specimen
Food samples, Water samples
Specimen Collection and Handling:
Processing: (3,4,5)
Test portion and initial suspension:
- Selective Enrichment A : To prepare the initial suspension, combine a quantity of 10g or 10ml of the test portion with 90ml of the enrichment medium Bolton Broth (M1592) from cooked and frozen food. Incubate the initial suspension in a microaerobic atmosphere at 37°C for 4 to 6 hours and then at 41.5°C for 44 hours ± 4 hours.
- Selective Enrichment B: To prepare the initial suspension, combine a quantity of 10g or 10ml of the test portion with 90ml of the enrichment medium Preston Broth (M8991) from raw meats or raw milk. Incubate the initial suspension in a microaerobic atmosphere at 41.5°C for 24 hours ± 2 hours.
Plating out :
Using the culture obtained in the enrichment medium, inoculate with a sterile 10 µl loop on the surface of mCCD Agar. Incubate the plates at 41.5°C in a microaerobic atmosphere for 44 hours ± 4 hours.
Selective Enrichment C:
Directly swab or streak on mCCD agar from caecal or faecal samples by using a loop or a sterile swab and second medium can be optional. Incubate the plates at 41.5°C in a microaerobic atmosphere for 44 hours ± 4 hours.
Confirmation:
Biochemical and serological tests are performed for confirmation.
Warning and Precautions
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
- Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
- Further biochemical tests must be carried out for confirmation.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.
Quality Control
Appearance
Grey to black homogeneous free flowing powder
Gelling
Firm, comparable with 1.2% Agar gel
Colour and Clarity of prepared medium
Black coloured, opaque gel forms in Petri plates
Reaction
Reaction of 4.55% w/v aqueous solution at 25°C. pH: 7.4±0.2
pH
7.20-7.60
Cultural Response
Productivity:
Cultural characteristics observed with added CCDA Selective Supplement V (FD135),after an incubation at 41.5°C±1°C for 44 ± 4 hours under micro-aerobic atmosphere. Recovery is considered as 100% on Reference medium - Blood Agar.
Selectivity:
Cultural characteristics observed with added CCDA Selective Supplement V (FD135),after an incubation at 41.5°C±1°C for 44 ± 4 hours under micro-aerobic atmosphere.
| Organism | Inoculum (CFU) | Growth | Recovery | Characteristic reaction |
|---|---|---|---|---|
| Productivity | ||||
| Campylobacter coli ATCC 33559 (00004*) | 50-100 | good-luxuriant | >=50% | Greyish, flat and moist colonies, sometimes with metallic sheen |
| Campylobacter jejuni ATCC 33291 (00005*) | 50-100 | good-luxuriant | >=50% | Greyish, flat and moist colonies, sometimes with metallic sheen |
| Campylobacter jejuni ATCC 29428 (00156*) | 50-100 | good-luxuriant | >=50% | Greyish, flat and moist colonies, sometimes with metallic sheen |
| Selectivity | ||||
| Escherichia coli ATCC 25922 (00013*) | >=104 | total or partial inhibition | No characteristic colonies | |
| Escherichia coli ATCC 8739 (00012*) | >=104 | total or partial inhibition | No characteristic colonies | |
| Escherichia coli ATCC 11775 (00090*) | >=104 | total or partial inhibition | No characteristic colonies | |
| Escherichia coli (00179*) | >=104 | total or partial inhibition | No characteristic colonies | |
| Staphylococcus aureus subsp. aureus ATCC 25923 (00034*) | >=104 | total or partial inhibition | No characteristic colonies | |
| Staphylococcus aureus subsp. aureus ATCC 6538 (00032*) | >=104 | total or partial inhibition | No characteristic colonies | |
Key:
(*) Corresponding WDCM numbers
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (11,12).
| Product Name | Modified Charcoal Cefoperazone deoxycholate Agar Base (mCCD agar) |
|---|---|
| SKU | M887I |
| Product Type | Regular |
| Physical Form | Powder |
| Origin | Animal |
| Packaging type | HDPE |
| References | 1. Downes F. P. and Ito K., (Eds.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th Ed.,APHA, Washington, D.C. |
| Customized Product Available | No |
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