🔴 Campylobacter in food
Detection of Campylobacter spp. in food and feed by selective enrichment in Bolton or Preston broth, isolation on mCCDA agar and biochemical/molecular confirmation.
Overview
Campylobacter is the most common bacterial cause of gastroenteritis (campylobacteriosis) in the European Union — over 200,000 cases are registered annually. The main pathogenic species are C. jejuni (approx. 90% of cases) and C. coli (approx. 10%). The source of infection is most often poultry meat (especially chickens), unpasteurized milk and contaminated water. The infectious dose is very low — only 500–800 cells are sufficient to cause disease.
The PN-EN ISO 10272-1 standard describes a horizontal method for detecting thermophilic Campylobacter in food, feed and environmental samples. The method includes three detection procedures adapted to sample type: Procedure A (Bolton broth) — for samples with low Campylobacter numbers and low accompanying microflora (frozen food, cooked food); Procedure B (Preston broth) — for samples with low Campylobacter numbers but high accompanying microflora (raw meat, raw milk); Procedure C — direct plating for samples with high Campylobacter numbers.
Campylobacter are microaerophilic bacteria — they require atmosphere with reduced oxygen content (5% O₂, 10% CO₂, 85% N₂) and elevated incubation temperature (41.5°C). They are fastidious, sensitive to oxygen, desiccation and low pH, making their cultivation technically demanding.
Testing for Campylobacter presence is required by Regulation (EC) No 2073/2005 for broiler chicken carcasses. Process hygiene criterion is 1000 CFU/g in carcass after chilling. This is a priority pathogen in food safety monitoring programs.
Method principle
The method involves selective enrichment of Campylobacter in Bolton broth (37°C/4–6 h, then 41.5°C/44 h) or Preston broth (41.5°C/24 h) under microaerophilic conditions. Antibiotics in broths (cefoperazone, trimethoprim, vancomycin, cycloheximide) inhibit accompanying flora. After enrichment, sample is plated on selective solid medium — mCCDA (modified Charcoal Cefoperazone Deoxycholate Agar) agar and second selective medium. Charcoal in mCCDA adsorbs toxic metabolites. Campylobacter colonies are gray, flat, moist, with metallic sheen and tendency to spread. Confirmation includes biochemical tests (oxidase+, catalase+, no growth at 25°C) or PCR.
Applications
- Poultry meat control — broiler chicken carcasses (Regulation (EC) 2073/2005)
- Raw milk and unpasteurized dairy product testing
- Poultry slaughterhouse production line monitoring
- Drinking water and water for food purposes control
- Ready-to-eat (RTE) food testing
- Epidemiological investigation in campylobacteriosis outbreaks
- Salmonella/Campylobacter control programs monitoring in poultry
- Environmental sample control in meat plants
Key parameters
| Parameter | Value |
|---|---|
| Enrichment temperature | 37°C (4–6 h) → 41.5°C (44 h) — Procedure A / 41.5°C (24 h) — Procedure B |
| Atmosphere | Microaerophilic: 5% O₂, 10% CO₂, 85% N₂ |
| Enrichment broth | Bolton Broth (Procedure A) or Preston Broth (Procedure B) |
| Isolation medium | mCCDA (modified Charcoal Cefoperazone Deoxycholate Agar) |
| Colony type | Gray, flat, moist, metallic sheen, spreading |
| Confirmation | Oxidase+, catalase+, morphology (spirals), no growth 25°C or PCR |
Standard
- Standard number
- PN-EN ISO 10272-1:2017
- Title (PL)
- Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania 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 preparation
Weigh 25 g (or 10 g) sample into stomacher bag. Add 225 mL (or 90 mL) Bolton broth with antibiotic supplement and lysed horse blood (5%).
2. Enrichment — phase I (Procedure A)
Homogenize and incubate in microaerophilic atmosphere: first 37 ± 1°C for 4–6 h (resuscitation phase), then raise temperature to 41.5 ± 1°C.
3. Enrichment — phase II
Continue incubation at 41.5 ± 1°C in microaerophilic atmosphere for additional 44 ± 4 h (total 48 h from start).
4. Isolation on mCCDA
From enriched broth, streak onto mCCDA plate and second selective medium. Incubate in microaerophilic atmosphere at 41.5 ± 1°C for 44 ± 4 h.
5. Colony reading
Look for typical Campylobacter colonies — gray, flat, moist, with metallic sheen and tendency to spread. May also be pinpoint, round, shiny colonies.
6. Subculture on non-selective agar
Select min. 1 typical colony from each plate. Subculture on Columbia blood agar with sheep blood (5%). Incubate 24–48 h at 41.5°C in microaerobic atmosphere.
7. Confirmation tests
Perform: oxidase test (+), catalase test (+), microscopic preparation (spirals), growth test at 25°C (no growth) and in aerobic conditions at 41.5°C (no growth). Or confirm by PCR method.
8. Interpretation and reporting
Report result as: "Campylobacter spp. detected/not detected in X g sample". If species identification performed — state: C. jejuni or C. coli.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator with microaerophilic atmosphere | Binder CB 60/160 with gas control, or anaerobic jars + generators | 15,000–50,000 PLN |
| Microaerophilic atmosphere generators | Oxoid CampyGen, bioMérieux GENbag microaer, Anaerocult C | 200–500 PLN / 10 pcs |
| Anaerobic jars | Oxoid AnaeroJar 2.5 L, Merck Anaerocult | 500–1,500 PLN |
| Class II laminar flow cabinet | Thermo Scientific MSC-Advantage, ESCO Airstream AC2 | 25,000–60,000 PLN |
| Stomacher / homogenizer | Seward Stomacher 400 Circulator | 12,000–25,000 PLN |
| Optical microscope | Olympus CX23, Zeiss Primo Star, Nikon Eclipse Si | 5,000–20,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65 | 15,000–45,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Bolton Broth | — | Selective enrichment broth: meat peptone, lactose, sodium pyruvate, α-ketoglutarate, sodium metabisulfite, sodium carbonate; 500 g package + antibiotic supplements |
| Bolton Supplement (antibiotics) | 62893-20-3 | Cefoperazone, trimethoprim, vancomycin, cycloheximide; 10 vials package |
| mCCDA Agar (modified Charcoal Cefoperazone Deoxycholate Agar) | 302-95-4 | Charcoal, sodium deoxycholate, ferrous sulfate, sodium pyruvate, agar; 500 g package |
| mCCDA Supplement (cefoperazone + amphotericin B) | 62893-20-3 | Selective antibiotics, 10 vials package |
| Lysed horse blood (5%) | — | Addition to Bolton broth — growth factor, neutralizes inhibitors; 100 mL package |
| Oxidase reagent (Kovács) | — | For confirmation test — Campylobacter oxidase-positive; strips or drops |
| Buffered peptone water (BPW) | — | For initial suspension preparation, 500 g package |
Health and safety (OHS)
- Campylobacter — BSL-2 pathogen with very low infectious dose (500 cells), use full PPE
- Work exclusively in class II laminar flow cabinet — special caution when opening jars
- Microaerophilic atmosphere — gas generators may cause local O₂ drop, room ventilation
- Autoclave used materials before disposal (121°C/20 min)
- Antibiotics in supplements — avoid skin contact, use gloves
- Surface disinfection with 70% ethanol or 0.5% sodium hypochlorite