🦠 Clostridium perfringens in food

Microbiology PN-EN ISO 7937

Overview

STATUS (as of 02.11.2026). PN-EN ISO 7937:2005 — Polish version, published on 18.11.2005, adopted EN ISO 7937:2004 and ISO 7937:2004 [IDT]; WITHDRAWN on 09.05.2024 and replaced by PN-EN ISO 15213-2:2024-05 (English version, published on 09.05.2024, adopts EN ISO 15213-2:2023 and ISO 15213-2:2023 [IDT]). ISO 7937:2004 was withdrawn on 16.11.2023; ISO 15213-2:2023 (first edition, 16.11.2023) is titled “Microbiology of the food chain — Horizontal method for the detection and enumeration of Clostridium spp. — Part 2: Enumeration of Clostridium perfringens by colony-count technique”. The changes are considered major (ISO 17468) — they affect the performance characteristics: the scope was extended to samples from the primary production stage; the heat treatment of 10 min at 80 °C is optional (with high background flora or for counting only spores); the SC medium was renamed TSC without changes in the formulation; confirmation was modified according to ISO 14189; criteria for the performance testing of media, performance characteristics and molecular methods distinguishing pathogenic strains were added. The description gives the content of ISO 15213-2:2023.

SCOPE (ISO 15213-2, clause 1). Enumeration of C. perfringens by a colony-count technique 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. The method was validated in an interlaboratory study for ready-to-eat and ready-to-reheat meat products, eggs and egg products, processed fruits and vegetables, infant formula and infant cereals, multi-component foods, pet food and animal feed and environmental samples; no performance characteristics were obtained for primary production. The technique is suitable for samples giving at least 10 colonies on a plate — above 10 cfu/ml for liquid samples or 100 cfu/g for solid samples; with duplicate plates (sum of at least 10 colonies) — above 5 cfu/ml or 50 cfu/g.

SIGNIFICANCE (introduction). C. perfringens is a Gram-positive, anaerobic, spore-forming bacterium found predominantly in soil, but also in the intestinal tract of humans and animals — high numbers in food can indicate inadequate preparation or handling. High numbers in ready-to-eat food can cause illness, mainly diarrhoea; food poisonings are caused by strains producing C. perfringens enterotoxin (CPE), released during sporulation in the small intestine. The spores are heat resistant and can germinate and multiply in ready-to-eat food after the cooking process.

COLONIES AND DEFINITIONS (clause 3, 4.3, 9.5). Presumptive C. perfringens are spore-forming bacteria forming countable typical colonies in a selective medium under obligate anaerobic conditions; confirmed — those which in addition possess acid phosphatase; human pathogenic — confirmed strains carrying the cpe gene of the enterotoxin (chromosomal or plasmid). Typical colonies on TSC agar are black or grey to yellow-brown (even if the colour is faint) — the colour comes from iron(II) sulfide formed from sulfide ions and iron(III) of the medium. Plates are counted within 30 min of removal from the anaerobic atmosphere, because the colour rapidly fades on exposure to oxygen; longer incubation can result in excess blackening, and hydrogen-producing anaerobes can blacken the whole medium.

TSC MEDIUM (composition according to ISO 7937:2004, 5.2; ISO 15213-2 only renamed the SC medium to TSC, without changes in the formulation). Base medium per 1 000 ml: enzymatic digest of protein 15.0 g, enzymatic digest of soya 5.0 g, yeast extract 5.0 g, disodium disulfite (Na₂S₂O₅, anhydrous) 1.0 g, ammonium iron(III) citrate (at least 15 % iron) 1.0 g, agar 9–18 g; pH 7.6 ± 0.2 at 25 °C. Immediately before use, 1 ml of D-cycloserine solution (4.0 g in 100 ml of water, sterilized by filtration) is added to each 100 ml of base medium cooled to 44–47 °C. The former SC medium was called “egg-yolk-free TSC”.

REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026) contains no criteria for Clostridium perfringens. In water intended for human consumption Clostridium perfringens (including spores) is tested by the reference method PN-EN ISO 14189 (Journal of Laws 2026, item 748) — a separate Knowledge Base entry.

WHAT WE DO NOT GIVE HERE. The available text of ISO 15213-2:2023 ends at 9.6.1.2 (subculture of colonies on blood agar). We do not give the course of the acid phosphatase test and of the test on SIM medium, the calculation, the composition of the media of Annex B (except TSC — according to ISO 7937:2004), the molecular methods of Annex D or the performance characteristics.

Method principle

A specified quantity of the liquid sample or the initial suspension and further decimal dilutions is transferred into empty Petri dishes and mixed with the molten selective medium (TSC), and after solidification an overlay of the same medium is poured to prevent spreading colonies on the surface (4.1, 4.3). The plates are incubated anaerobically at 37 °C for 20 h and typical colonies — black or grey to yellow-brown — are counted; selected colonies are confirmed and the result is calculated as the number of confirmed C. perfringens (4.3–4.4). To count only spores, the dilutions are heated for 10 min at 80 °C before plating (4.1).

Applications

• Products intended for human consumption and for the feeding of animals (ISO 15213-2, clause 1)
• Environmental samples in the area of food and feed production and handling, and samples from the primary production stage (clause 1)
• Validated categories: ready-to-eat and ready-to-reheat meat products, eggs and egg products, processed fruits and vegetables, infant formula and infant cereals, multi-component foods, pet food and animal feed, environmental samples (clause 1, note)
• Counting only spores — with the treatment of 10 min at 80 °C (9.3)

Key parameters

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 and dilutions

   The initial suspension and a single decimal dilution series are prepared according to ISO 6887 and the standard for the product concerned (9.2); the whole procedure — Annex A (9.1).

2. Spores (optional)

   To count only spores, heat the dilution series in a water bath for (10 ± 1) min at 80 °C, at the latest 15 min after preparing the suspension (or keep it in melting ice for up to 2 h); monitor the temperature in a reference bottle, start timing when it reaches 80 °C (heating up not longer than 5 min), then cool immediately to about 20 °C (9.3).

   🌡 Temperature: 80 °C

3. Inoculation

   Transfer 1 ml of the liquid sample or initial suspension (10⁻¹) into each of two sterile 90 mm dishes and 1 ml of the next dilution into one dish; further dilutions if necessary, each with a new pipette; for low numbers — 10 ml of suspension on three 140 mm dishes (9.4.1–9.4.3).

4. Pouring and overlay

   Pour 12–15 ml (90 mm) or 30–35 ml (140 mm) of TSC agar at 44–47 °C, mix by rotating the dishes and allow to solidify on a cool horizontal surface; then pour an overlay of about 5 ml (90 mm) or 12 ml (140 mm) of the same medium (9.4.5–9.4.7).

   🌡 Temperature: 44–47 °C

5. Incubation

   Invert the plates and incubate anaerobically at 37 °C (9.4.8).

   🌡 Temperature: 37 ± 1 °C

6. Counting

   After (20 ± 2) h count typical colonies — black or grey to yellow-brown — within 30 min of removing the plates from the anaerobic atmosphere; take plates with fewer than 150 colonies (90 mm) or 365 (140 mm) and choose at random 5 colonies from each for confirmation (9.5).

7. Subculture

   Streak each selected colony on Columbia blood agar (or another nutrient-rich medium) and incubate anaerobically for (20 ± 2) h at 37 °C; with several colony morphologies — one of each; after incubation the plates can be kept at 5 °C for up to 48 h (9.6.1).

   🌡 Temperature: 37 °C

8. Confirmation

   Freshly grown, well-isolated colonies are confirmed by the acid phosphatase test or by the test on SIM medium; alternative methods — after validation according to ISO 16140-4 or ISO 16140-6 (9.6.1.2).

9. Result

   Number of confirmed C. perfringens in cfu per 1 g, 1 ml, 1 cm² or sampling device (3.4, 4.4).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Tests for the enumeration of Clostridium perfringens 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 ISO 15213-2)
• The standard does not address all safety aspects — establishing safety and health practices is the responsibility of the user (warning of ISO 15213-2)

Frequently asked questions