🦠 Clostridium perfringens in food
Enumeration of Clostridium perfringens in food by pour plating on TSC (Tryptose Sulfite Cycloserine) agar with incubation under anaerobic conditions at 37°C for 20 ± 2 h.
Overview
Clostridium perfringens is an obligate anaerobe, Gram-positive, spore-forming bacterium, widely distributed in the environment (soil, dust, human and animal digestive tract). It is one of the most common causes of bacterial food poisoning — CDC estimates approximately 1 million cases annually in the USA. Poisoning is caused by CPE enterotoxin (Clostridium perfringens enterotoxin), produced during bacterial sporulation in the intestine.
Typical C. perfringens poisoning occurs after consuming large amounts of bacteria (>10⁶ CFU/g) in improperly stored meat dishes (stew, meat sauce, roast), poultry and legumes. Symptoms (watery diarrhea, abdominal cramps) appear 8–22 h after consumption and resolve within 24 h. C. perfringens spores survive cooking and germinate during slow cooling of dishes (40–50°C), achieving generation time of only 8–10 min under optimal conditions.
The PN-EN ISO 7937 standard describes a horizontal method for enumerating C. perfringens on TSC (Tryptose Sulfite Cycloserine Agar) medium. The medium contains sodium sulfite — reduction of sulfite to sulfide by C. perfringens causes precipitation of iron(II) sulfide and formation of black colonies. D-cycloserine inhibits growth of accompanying bacteria. Incubation is under anaerobic conditions.
Confirmation includes nitrate reduction test, motility test (C. perfringens is non-motile) and gelatin liquefaction. The method is used in quality control of ready meals, catering dishes, meat and meat products.
Method principle
Sample is pour plated on TSC (Tryptose Sulfite Cycloserine Agar) medium with optional egg yolk emulsion addition. C. perfringens reduces sodium sulfite to sulfide, which reacts with iron(II) ions forming insoluble black iron sulfide — colonies are black. D-cycloserine inhibits growth of accompanying bacteria (especially Gram-positive aerobes). If egg yolk emulsion added — opalescent halo appears around C. perfringens colonies (lecithinase activity). Incubation under strictly anaerobic conditions at 37°C for 20 ± 2 h. Confirmation: nitrate reduction (+), motility (−), gelatin liquefaction (+).
Applications
- Quality control of ready meals and catering dishes
- Meat and meat products testing (stew, roast, sauce)
- Catering establishments and canteen monitoring
- Food control in institutions (hospitals, schools, care homes)
- Legumes and dishes prepared from them testing
- Epidemiological investigation in food poisoning outbreaks
- Cooling and reheating procedures verification (HACCP)
Key parameters
| Parameter | Value |
|---|---|
| Incubation temperature | 37 ± 1°C |
| Incubation time | 20 ± 2 h |
| Atmosphere | Strictly anaerobic |
| Medium | TSC Agar (Tryptose Sulfite Cycloserine Agar) |
| Colony type | Black, diameter 1–5 mm (sulfite reduction to iron sulfide) |
| Confirmation | Nitrate reduction (+), motility (−), gelatinase (+) |
Standard
- Standard number
- PN-EN ISO 7937:2005
- Title (PL)
- Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby Clostridium perfringens — Metoda liczenia kolonii
- Title (EN)
- Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of Clostridium perfringens — Colony-count technique
Step-by-step procedure
1. Sample preparation
Weigh 10 g sample, add 90 mL BPW, homogenize in stomacher for 60–120 s. Prepare decimal dilutions.
2. TSC medium preparation
Dissolve TSC Agar Base in distilled water. Sterilize in autoclave (121°C/15 min). Cool to 45–50°C, add D-cycloserine aseptically (and optionally egg yolk emulsion). Maintain in water bath at 47°C.
3. Pour plating
Transfer 1 mL of each dilution to two sterile Petri dishes. Pour approx. 15 mL TSC medium (44–47°C). Mix gently. Leave to solidify.
4. Overlay
After solidification, apply additional layer of approx. 10 mL TSC agar without supplements. This limits oxygen access and colony spreading.
5. Anaerobic incubation
Place plates in anaerobic jar (bottom up). Insert anaerobic atmosphere generator (AnaeroGen) and anaerobiosis indicator. Close jar and incubate at 37 ± 1°C for 20 ± 2 h.
6. Colony reading
Count black colonies (diameter 1–5 mm). If egg yolk added — typical colonies surrounded by opalescent halo (lecithinase+). Count plates with 15–150 colonies.
7. Isolate confirmation
Select min. 5 typical colonies. Subculture on blood agar under anaerobiosis (37°C/24 h). Perform: nitrate reduction (+ with Griess reagent), motility in SIM medium (−), gelatin liquefaction in gelatin broth (+, 37°C/48 h).
8. Calculations and reporting
Calculate number of C. perfringens taking into account percentage of confirmed colonies. Express result in CFU/g. Report result as "Clostridium perfringens".
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Anaerobic system (jars + generators) | Oxoid AnaeroJar 2.5 L + AnaeroGen, Merck Anaerocult A | 500–2,000 PLN (jar) + 300–500 PLN/10 gen. |
| Laboratory incubator 37°C | Binder BD 56, Memmert INB 200, POL-EKO CLN 53 | 5,000–18,000 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 |
| Water bath 44–47°C | Memmert WNB 14, Julabo TW 12 | 3,000–10,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65 | 15,000–45,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| TSC Agar Base (Tryptose Sulfite Cycloserine) | 7681-57-4 | Tryptose, soy peptones, yeast extract, sodium sulfite, ferric ammonium citrate, agar; 500 g package |
| D-Cycloserine (selective supplement) | 68-41-7 | Antibiotic inhibiting growth of accompanying bacteria; concentration 400 mg/L; 10 vials package |
| Egg yolk emulsion (optional) | — | Detects C. perfringens lecithinase activity — opalescent halo around colonies; 50 mL/L |
| Sodium sulfite | 7757-83-7 | Reduced by C. perfringens to sulfide; TSC component |
| Ferric ammonium citrate | 1185-57-5 | Provides iron ions reacting with sulfide — black colonies; TSC component |
| Anaerobic atmosphere generators | — | Oxoid AnaeroGen 2.5 L or Merck Anaerocult A; 10 pcs package |
| Gelatin broth and Griess reagent | — | For confirmation tests: gelatinase and nitrate reduction |
| Buffered peptone water (BPW) | — | For sample dilutions, 500 g package |
Health and safety (OHS)
- C. perfringens — BSL-2 pathogen producing toxins and spores, use full PPE
- Anaerobic conditions — caution when opening anaerobic jars (sudden oxygen influx)
- D-Cycloserine — antibiotic, neurotoxic in large doses, avoid inhalation and skin contact
- Work in class II laminar flow cabinet mandatory
- Autoclave used materials (121°C/30 min) — spores require longer sterilization
- Sodium sulfite — may release SO₂, room ventilation