🔴 Campylobacter in food

Microbiology PN-EN ISO 10272-1

Overview

STATUS (as of 02.11.2026). PN-EN ISO 10272-1:2017-08 — English version, published on 23.08.2017, adopts EN ISO 10272-1:2017 and ISO 10272-1:2017 [IDT]; it replaced PN-EN ISO 10272-1:2007 (Polish version) with correction Ap1:2008. AMENDMENT PN-EN ISO 10272-1:2017-08/A1:2023-08 (English version, published on 04.08.2023) adopts ISO 10272-1:2017/Amd 1:2023 (25.01.2023): a new definition of Campylobacter, molecular confirmation and identification of thermotolerant species (Annexes D and E), a growth supplement in Preston broth and changes in the performance testing of culture media. The 2017 edition added samples from the primary production stage to the scope, Preston broth as a second enrichment broth and direct plating on mCCD, and replaced the confirmation by microaerobic growth at 25 °C and aerobic growth at 41.5 °C with a test for aerobic growth at 25 °C.

SCOPE (clause 1, 3.1 as amended by A1). Detection of Campylobacter by enrichment or direct plating in products intended for human consumption and for the feeding of animals, in environmental samples in the area of food and feed production and handling, and in samples from the primary production stage (animal faeces, dust, swabs). The standard targets thermotolerant species relevant to human health — most often C. jejuni and C. coli, but also C. lari, C. upsaliensis and others. Campylobacter forms characteristic colonies on solid selective media, such as mCCD agar, in a microaerobic atmosphere at 41.5 °C. According to chapter 7 of the BAM manual (FDA), C. jejuni, C. coli and C. lari account for more than 99 % of human isolates (C. jejuni 90 %), the infective dose of C. jejuni ranges from 500 to 10 000 cells depending on the strain, cell damage and host susceptibility, and infections have been linked to, among others, unpasteurised milk, meat, poultry, shellfish, fruit and vegetables.

THREE PROCEDURES (4.1, 9.1). A — enrichment in Bolton broth: samples with low numbers of Campylobacter and a low level of background flora or with stressed cells, e.g. cooked or frozen products; B — enrichment in Preston broth: low numbers of Campylobacter and a high level of background flora, e.g. raw meats (including poultry) and raw milk; C — direct plating: high numbers of Campylobacter, e.g. faeces, poultry caecal contents, raw poultry meat (it can be combined with ISO 10272-2 for counting). When little is known about the sample, procedure C is run in parallel with A and/or B. Preston broth suppresses flora resistant to third-generation β-lactams, such as cefoperazone, which is present in Bolton broth and in mCCD agar; according to amendment A1 Preston broth can be too selective for some strains (particularly C. coli) — the growth supplement improves their recovery.

ATMOSPHERE AND SAMPLES (6.5, clause 7, 9.5.1). Microaerobic atmosphere: oxygen (5 ± 2) %, carbon dioxide (10 ± 3) %, optionally hydrogen up to 10 %, balance nitrogen — in gastight jars with gas-generating kits (strictly according to the manufacturer) or by filling the jar or incubator with a gas mixture. Campylobacter is very sensitive to freezing but survives best at low temperatures — samples are not frozen, are stored at 3 °C, analysed as rapidly as possible and protected from drying; in air it rapidly loses culturability, so confirmation is done without delay.

REGULATIONS. The process hygiene criterion for Campylobacter in Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026) concerns enumeration, not detection: broiler carcases after chilling, n = 50 samples from 10 consecutive sessions, c = 10 from 01.01.2025, m = M = 1 000 cfu/g, reference method EN ISO 10272-2 (item 2.1.9). Part 1 is not the reference method of any criterion of this regulation.

WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends at 9.5.1. We do not give the selection of colonies for confirmation, the course of the examination of morphology and motility, the subculture on blood agar, the oxidase test and the test for aerobic growth at 25 °C (9.5.2–9.5.5), species identification beyond Table 2 of amendment A1, or the composition of Bolton broth, mCCD agar and the second medium (Annex B).

Method principle

Detection requires three successive stages (Annex A): enrichment in a selective liquid medium or direct plating, isolation on a solid selective medium and confirmation (4.1). In procedure A the test portion is added to Bolton broth and incubated microaerobically for 4–6 h at 37 °C, then for 44 h at 41.5 °C; in procedure B — to Preston broth, 24 h at 41.5 °C (4.2). From the culture mCCD agar is inoculated (in procedure A also a second selective medium with different selective agents), and in procedure C the test portion is plated directly on mCCD; the plates are incubated microaerobically at 41.5 °C and examined after 44 h (4.3). Suspect colonies are examined under the microscope (morphology and motility), subcultured on a non-selective blood agar and confirmed by oxidase and a test for aerobic growth at 25 °C; the species can optionally be identified by biochemical tests or molecular methods (4.4).

Applications

• Products intended for human consumption and for the feeding of animals (clause 1)
• Environmental samples in the area of food and feed production and handling (clause 1)
• Samples from the primary production stage: animal faeces, dust, swabs (clause 1)
• Cooked or frozen products — procedure A; raw meats (including poultry) and raw milk — procedure B; faeces, poultry caecal contents, raw poultry meat — procedure C (9.1)

Key parameters

Standard

Standard number

PN-EN ISO 10272-1:2017-08 + A1:2023-08

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania obecności i oznaczania liczby Campylobacter spp. — Część 1: Metoda wykrywania

Title (EN)

Microbiology of the food chain — Horizontal method for detection and enumeration of Campylobacter spp. — Part 1: Detection method

Step-by-step procedure

1. Sample

   Samples are not frozen — they are stored at 3 °C, analysed as rapidly as possible and protected from drying (clause 7); the test sample is prepared according to ISO 6887 (clause 8).

2. Initial suspension (A, B)

   The test portion is mixed with the enrichment broth pre-warmed to room temperature in a 1 in 10 ratio — usually 10 g or 10 ml per 90 ml of Bolton broth (A) or Preston broth (B) — and homogenized according to ISO 7218 (9.2.1–9.2.3).

3. Direct plating (C)

   Faeces and caecal contents are brought with a loop or a sterile swab onto the first half of an mCCD plate and streaked out with another loop; other samples are mixed with a liquid (e.g. peptone salt solution or Preston broth, e.g. 1 in 2) and streaked with a loop or spread over the plate (9.2.4).

4. Enrichment

   Procedure A: microaerobically 4–6 h at 37 °C, then (44 ± 4) h at 41.5 °C; procedure B: microaerobically (24 ± 2) h at 41.5 °C (9.3).

   🌡 Temperature: 37 °C → 41.5 °C

5. Isolation

   With a 10 µl loop inoculate the culture onto mCCD agar, and in procedure A also onto the second selective medium; incubate the plates microaerobically at 41.5 °C (9.4.1–9.4.3).

   🌡 Temperature: 41.5 °C

6. Reading

   After (44 ± 4) h look for typical or suspect colonies: greyish, often with a metallic sheen, flat and moist, with a tendency to spread (less on drier agar); other forms of colonies may occur (9.4.3).

7. Confirmation

   Without delay, because Campylobacter rapidly loses culturability in air: morphology and motility under the microscope, subculture on non-selective blood agar, oxidase and aerobic growth at 25 °C (4.4, 9.5.1); as control strains the standard gives C. jejuni WDCM 00005 (positive) and E. coli WDCM 00013 (negative). Instead of or in addition to these tests — PCR, serological methods, MALDI-TOF after verification of suitability (9.5.1; PCR — amendment A1, Annex D).

8. Identification (optional)

   Catalase, hydrolysis of hippurate and of indoxyl acetate according to Table 2 as amended by A1: C. jejuni +/+/+, C. coli +/−/+, C. lari +/−/−, C. upsaliensis − or weak/−/+; indoxyl acetate — blue colour within 5–10 min, if the result is unclear — read again after a further 20 min; instead of biochemical tests — molecular methods of Annex E (9.6, amendment A1).

9. Result

   Presence or absence of Campylobacter in a defined quantity of product (3.2).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Tests for Campylobacter are only undertaken in properly equipped laboratories, under the control of a skilled microbiologist, with great care in the disposal of all incubated materials (warning of the standard)
• The standard does not address all safety aspects — establishing safety and health practices is the responsibility of the user (warning of the standard)

Frequently asked questions