🦠 Campylobacter spp. in food — enumeration on mCCDA agar in a microaerobic atmosphere, 41.5 °C
In short
0. 1 ml of the initial suspension (or 1 ml on a large plate for low numbers) and of the dilutions is inoculated by the spread-plate technique onto modified charcoal cefoperazone deoxycholate agar (mCCDA agar), incubated for 44 h ± 4 h at 41. The test is performed according to PN-EN ISO 10272-2:2017-10 (ISO 10272-2:2017; poprawka ISO 10272-2:2017/Amd 1:2023); Medium: modified charcoal cefoperazone deoxycholate agar (mCCDA agar); composition and performance testing — annex B. The procedure comprises 6 steps; it is used for: Process hygiene criterion for broiler carcases after chilling (neck skin) — 1000 cfu/g, the reference method EN ISO 10272-2 (Regulation 2073/2005, item 2.1.9), Poultry meat and other raw meat, raw milk, Samples from primary production — faeces, dust, swabs on poultry farms (biosecurity).
At a glance
- Standard: PN-EN ISO 10272-2:2017-10 (ISO 10272-2:2017; poprawka ISO 10272-2:2017/Amd 1:2023)
- Category: Microbiology
- Procedure steps: 6
- Medium: modified charcoal cefoperazone deoxycholate agar (mCCDA agar); composition and performance testing — annex B
- Incubation: 41.5 °C ± 1 °C, 44 h ± 4 h, microaerobic atmosphere (O₂ 5 ± 2 %, CO₂ 10 ± 3 %, optionally H₂ ≤ 10 %, the remainder N₂)
- Inoculum: 0.1 ml per 90 mm plate; 1.0 ml per 140 mm plate or 3 × 90 mm (duplicates) for low numbers
Overview
ISO 10272-2:2017 specifies a horizontal method for the enumeration of Campylobacter spp. in products intended for human consumption and for the feeding of animals, in environmental samples from the production and the distribution of food and feed and in samples from primary production (animal faeces, dust, swabs). The first edition replaced ISO/TS 10272-2:2006; the changes (regarded as minor): the addition of samples from primary production, the plating of the dilutions singly instead of in duplicate (in accordance with ISO 7218), the replacement of the tests of microaerobic growth at 25 °C and of aerobic growth at 41.5 °C by a test of aerobic growth at 25 °C, the addition of the performance testing of the media (annex B) and of the characteristics of the method (annex C). Amendment Amd 1:2023 introduced molecular methods of confirmation and identification of thermotolerant Campylobacter and changes in the performance testing of the media. Campylobacter are defined as micro-organisms forming characteristic colonies on solid selective media in a microaerobic atmosphere at 41.5 °C, with a characteristic morphology and motility and with characteristic biochemical and growth properties; the standard covers the thermotolerant species that are of importance for health — most often C. jejuni and C. coli, and also C. lari and C. upsaliensis. Regulation (EC) No 2073/2005 (process hygiene criterion 2.1.9) names EN ISO 10272-2 as the reference method for Campylobacter spp. in broiler carcases after chilling: a limit of 1000 cfu/g, n = 50, c = 10 from 1 January 2025 (previously 20, then 15); in the case of unsatisfactory results — improvement of the hygiene of slaughter and of the biosecurity on the holdings. The tests may be carried out only in appropriately equipped laboratories under the supervision of a microbiologist.
Method principle
The determination requires three successive stages (annex A): the preparation of decimal dilutions in accordance with ISO 6887 (a single series); the plating onto a solid selective medium — modified charcoal cefoperazone deoxycholate agar (mCCDA agar) — of a specified amount of the liquid sample or of the initial suspension and of the dilutions, incubation at 41.5 °C in a microaerobic atmosphere and assessment after 44 h with the recording of the number of suspect colonies; the confirmation — examination of the morphology and of the motility under the microscope, subculture onto non-selective blood agar, the oxidase test and a test of aerobic growth at 25 °C (optionally identification of the species by biochemical and/or molecular tests). 0.1 ml of the suspension is plated onto a plate of mCCDA agar; for low numbers — 1.0 ml onto a 140 mm plate or onto three 90 mm plates (in duplicate: two large ones or six ordinary ones); if only the initial suspension is used, a duplicate is prepared. The inoculum is spread quickly with a sterile spreader; the drying of the plates is critical for countability and every laboratory uses its own standardised way of doing it. The microaerobic atmosphere: 5 % ± 2 % O₂, 10 % ± 3 % CO₂, optionally ≤ 10 % H₂, the remainder nitrogen — in gas-tight jars with gas-generating kits or by filling the jar or the incubator with a mixture of gases. Typical colonies on mCCDA agar are greyish, often with a metallic sheen, flat and moist, with a tendency to spread (less so on a drier agar); the recognition of the colonies depends to a great extent on experience. A nickel-chromium loop is not suitable for the oxidase test. Campylobacter is very sensitive to freezing and survives best at low temperatures — do not freeze the samples, store them at 3 °C, examine them as soon as possible and protect them from drying out.
Applications
- Process hygiene criterion for broiler carcases after chilling (neck skin) — 1000 cfu/g, the reference method EN ISO 10272-2 (Regulation 2073/2005, item 2.1.9)
- Poultry meat and other raw meat, raw milk
- Samples from primary production — faeces, dust, swabs on poultry farms (biosecurity)
- Environmental samples from slaughterhouses and cutting plants
- Quantitative testing in risk assessment and in the assessment of the effectiveness of interventions (chilling, washing of the carcases)
Key parameters
| Parameter | Value |
|---|---|
| Medium | modified charcoal cefoperazone deoxycholate agar (mCCDA agar); composition and performance testing — annex B |
| Incubation | 41.5 °C ± 1 °C, 44 h ± 4 h, microaerobic atmosphere (O₂ 5 ± 2 %, CO₂ 10 ± 3 %, optionally H₂ ≤ 10 %, the remainder N₂) |
| Inoculum | 0.1 ml per 90 mm plate; 1.0 ml per 140 mm plate or 3 × 90 mm (duplicates) for low numbers |
| Confirmation | morphology and motility (microscope, preferably phase contrast), subculture onto blood agar, oxidase +, absence of aerobic growth at 25 °C; optionally identification of the species (biochemistry, PCR — Amd 1:2023) |
| Legal criterion | broiler carcases: 1000 cfu/g, n = 50, c = 10 (from 1 January 2025) |
| Storage of the samples | without freezing, 3 °C ± 2 °C, analysis as soon as possible, protection from drying out |
| Result | cfu/g, cfu/ml, cfu/cm² or per sampling device |
Standard
- Standard number
- PN-EN ISO 10272-2:2017-10 (ISO 10272-2:2017; poprawka ISO 10272-2:2017/Amd 1:2023)
- Title (PL)
- Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania obecności i oznaczania liczby Campylobacter spp. — Część 2: Metoda liczenia kolonii
- Title (EN)
- Microbiology of the food chain — Horizontal method for detection and enumeration of Campylobacter spp. — Part 2: Colony-count technique
Step-by-step procedure
-
Sample
Take it in accordance with ISO/TS 17728 (food), ISO 13307 (primary production), ISO 17604 (carcases) or ISO 18593 (surfaces); do not freeze it, store it at 3 °C, examine it quickly. Prepare the initial suspension and a single series of decimal dilutions in accordance with ISO 6887.
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Plating
Transfer 0.1 ml of the initial suspension (or of the liquid sample) and of the successive dilutions onto plates of mCCDA agar; for low numbers — 1.0 ml onto a 140 mm plate or onto three 90 mm plates, in duplicate; where only the initial suspension is used, prepare a duplicate. Spread it quickly with a sterile spreader without touching the edges; dry the plates in a standardised way.
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Microaerobic incubation
Place the plates in a jar with a gas-generating kit (strictly in accordance with the manufacturer's instructions) or in an incubator with a mixture of gases and incubate for 44 h ± 4 h at 41.5 °C.
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Counting of the suspect colonies
Record the number of typical/suspect colonies: greyish, often with a metallic sheen, flat and moist, spreading; other forms are possible.
-
Confirmation
Examine the selected colonies under the microscope (morphology, motility), subculture them onto blood agar, carry out the oxidase test (a loop other than Ni-Cr) and a test of aerobic growth at 25 °C (Campylobacter does not grow); optionally identify the species by biochemical or molecular tests.
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Result
From the number of confirmed typical colonies per plate calculate the number of Campylobacter in cfu/g, ml, cm² or per sampling device; compare it with the criterion of 1000 cfu/g for broiler carcases.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubators 25 °C ± 1 °C, 37 °C ± 1 °C and 41.5 °C ± 1 °C | aerobic growth at 25 °C — the confirmatory test | — |
| Gas-tight jars with gas-generating kits or an incubator with a mixture of gases | microaerobic atmosphere 5 % O₂, 10 % CO₂ | — |
| Phase-contrast microscope | morphology and motility of Campylobacter | — |
| Sterile Petri dishes of 90 mm and optionally of 140 mm (preferably vented) | — | |
| Loops of 10 µl and 1 µl (not Ni-Cr for the oxidase test), inoculating needle, water bath 37 °C ± 1 °C, refrigerators 3 °C ± 2 °C and 5 °C ± 3 °C | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| mCCDA agar (modified charcoal cefoperazone deoxycholate agar) — basal medium with the antibiotic supplement (annex B) | — | selective colony count |
| Blood agar (non-selective) | — | pure cultures for the confirmation |
| Reagent for the oxidase test | — | confirmation (oxidase positive) |
| Diluents in accordance with ISO 6887 | — | initial suspension and a single series of dilutions |
| Control strains and kits for biochemical/molecular identification (optionally) | — | performance testing of the media, identification of the species |
Health and safety (OHS)
- Campylobacter is a human pathogen — testing only in appropriately equipped laboratories under the supervision of a microbiologist, careful disposal of the materials after the incubation
- Gas-generating kits and mixtures with hydrogen (≤ 10 %) — handling in accordance with the manufacturer's instructions, gas-tight jars
- Cefoperazone and the other antibiotics in the supplements — avoid raising dust and contact
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 10272-2:2017-10 (ISO 10272-2:2017; poprawka ISO 10272-2:2017/Amd 1:2023) — “Microbiology of the food chain — Horizontal method for detection and enumeration of Campylobacter spp. — Part 2: Colony-count technique”.
How does this method work?
The determination requires three successive stages (annex A): the preparation of decimal dilutions in accordance with ISO 6887 (a single series); the plating onto a solid selective medium — modified charcoal cefoperazone deoxycholate agar (mCCDA agar) — of a specified amount of the liquid sample or of the initial suspension and of the dilutions, incubation at 41.5 °C in a microaerobic atmosphere and assessment after 44 h with the recording of the number of suspect colonies; the confirmation — examination of the morphology and of the motility under the microscope, subculture onto non-selective blood agar, the oxidase test and a test of aerobic growth at 25 °C (optionally identification of the species by biochemical and/or molecular tests).
What is the measuring range and accuracy?
Medium: modified charcoal cefoperazone deoxycholate agar (mCCDA agar); composition and performance testing — annex B; Incubation: 41.5 °C ± 1 °C, 44 h ± 4 h, microaerobic atmosphere (O₂ 5 ± 2 %, CO₂ 10 ± 3 %, optionally H₂ ≤ 10 %, the remainder N₂); Inoculum: 0.1 ml per 90 mm plate; 1.0 ml per 140 mm plate or 3 × 90 mm (duplicates) for low numbers; Confirmation: morphology and motility (microscope, preferably phase contrast), subculture onto blood agar, oxidase +, absence of aerobic growth at 25 °C; optionally identification of the species (biochemistry, PCR — Amd 1:2023).
How long does the test take?
The procedure comprises 6 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Incubators 25 °C ± 1 °C, 37 °C ± 1 °C and 41.5 °C ± 1 °C, Gas-tight jars with gas-generating kits or an incubator with a mixture of gases, Phase-contrast microscope, Sterile Petri dishes of 90 mm and optionally of 140 mm (preferably vented), Loops of 10 µl and 1 µl (not Ni-Cr for the oxidase test), inoculating needle, water bath 37 °C ± 1 °C, refrigerators 3 °C ± 2 °C and 5 °C ± 3 °C.
Where is this test used?
Process hygiene criterion for broiler carcases after chilling (neck skin) — 1000 cfu/g, the reference method EN ISO 10272-2 (Regulation 2073/2005, item 2.1.9); Poultry meat and other raw meat, raw milk; Samples from primary production — faeces, dust, swabs on poultry farms (biosecurity); Environmental samples from slaughterhouses and cutting plants; Quantitative testing in risk assessment and in the assessment of the effectiveness of interventions (chilling, washing of the carcases).
What safety precautions apply?
Campylobacter is a human pathogen — testing only in appropriately equipped laboratories under the supervision of a microbiologist, careful disposal of the materials after the incubation; Gas-generating kits and mixtures with hydrogen (≤ 10 %) — handling in accordance with the manufacturer's instructions, gas-tight jars; Cefoperazone and the other antibiotics in the supplements — avoid raising dust and contact.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 10272-2.