🦠 Clostridium perfringens in food and feed — pour plates with an overlay on TSC agar (37 °C, 20 h, anaerobically)

Microbiology PN-EN ISO 15213-2

Overview

ISO 15213-2:2023 specifies the enumeration of Clostridium perfringens by the colony-count technique. It is used for products intended for human consumption, for feed, for environmental samples from the production and the distribution of food and feed, and for samples from primary production. The method has been validated in an interlaboratory study for meat products ready to eat and to be reheated, for eggs and egg products, for processed fruit and vegetables, for infant formulae and cereals and for multi-component food, as well as for pet food, feed and environmental samples — since it covered at least five categories, it applies to a wide range of foods (clause 11, annex C). In this edition the heat treatment for 10 min at 80 °C has become optional (where the accompanying microflora is abundant); annex D describes a method for the molecular differentiation of the strains of C. perfringens that are pathogenic for humans from the non-pathogenic ones.

Where the expected number of cfu is close to the limit of quantification, duplicate plates are recommended (the sum of the colonies on both ≥ 10, which corresponds to a contamination of > 5 cfu/ml for liquids or > 50 cfu/g for solid products). The pour plate with an overlay is particularly useful for products in which spreading colonies obscuring the target colonies are to be expected. In Poland it is in force as PN-EN ISO 15213-2:2024-05 (English version). Tryptose sulfite agar (TSC) with the D-cycloserine supplement is available in Poland as a commercial medium (powder, bottles, plates).

Method principle

A specified amount of the liquid sample or of the initial suspension (for other products) is placed in an empty Petri dish and mixed with the molten agar medium (a pour plate); further plates are prepared from the decimal dilutions according to the ISO 6887 series. Once it has set, an overlay of the same agar is applied in order to prevent the development of spreading colonies. If only the spores are to be counted, the series of dilutions is heated before the inoculation for 10 min ± 1 min at 80 °C (in a water bath, within 15 min of the preparation of the suspension, otherwise the tubes are kept in melting ice for up to 2 h; the time taken to reach 80 °C ≤ 5 min; after the heating it is cooled immediately to about 20 °C). Into two dishes (about 90 mm) 1 ml of the liquid sample or of the initial suspension is transferred (where a small number is expected the limit may be lowered tenfold by examining 10 ml), into a further one 1 ml of the 10⁻¹ dilution (liquid product) or of the 10⁻² dilution (other products) and so on, choosing at least two successive critical dilution levels. 12–15 ml (90 mm plates) or 30–35 ml (140 mm) of TSC agar, molten and tempered to 44–47 °C, is poured in and mixed with a rotary movement, and once it has set about 5 ml (90 mm) or 12 ml (140 mm) of TSC agar is applied as an overlay. The plates are incubated inverted in an anaerobic atmosphere at 37 °C for 20 h ± 2 h; after they have been removed from the anaerobic atmosphere the typical colonies are counted within 30 min — black or grey to yellowish brown ones, whose colour, together with the surrounding zone, comes from the iron(II) sulfide formed from the sulfide ions and the iron(III) present in the medium — on the plates containing < 150 presumptive colonies (90 mm). Confirmation: from each plate 5 colonies are subcultured onto non-selective blood agar (or nutrient agar) and incubated anaerobically at 37 °C for 20 h ± 2 h (the plates may be cooled at 5 °C for up to 48 h before the reading), after which the acid phosphatase test is carried out (the colonies are rubbed onto filter paper with the reagent; a purple colour within 3–4 min = positive) together with the test in sulfite indole motility agar (SIM, 22 h ± 2 h at 37 °C: reduction of sulfite, indole, motility). The result is calculated as the number of confirmed C. perfringens per volume of sample; annex D makes it possible to distinguish the strains pathogenic for humans by molecular means.

Applications

• Meat products ready to eat and to be reheated, multi-component dishes, catering — an indicator of food poisoning
• Eggs and egg products, processed fruit and vegetables, infant formulae and cereals (the validated categories)
• Feed and pet food, environmental samples from the production of food and feed, primary production
• Counting of the spores only after the heat treatment of 80 °C/10 min (e.g. assessment of the effectiveness of the heat treatment)
• Differentiation of the strains pathogenic for humans from the non-pathogenic ones (the molecular method of annex D)

Key parameters

Standard

Standard number

PN-EN ISO 15213-2:2024-05 (ISO 15213-2:2023)

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania i oznaczania liczby Clostridium spp. — Część 2: Oznaczanie liczby Clostridium perfringens metodą liczenia kolonii

Title (EN)

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

Step-by-step procedure

1. Test portion, suspension and dilutions

   Prepare the initial suspension and the decimal dilutions according to the ISO 6887 series.

2. Optional selection of the spores

   If you are counting the spores only: heat the series of dilutions in a bath at 80 °C for 10 min ± 1 min (start once 80 °C has been reached in the reference sample; reaching it in ≤ 5 min), within 15 min of the preparation of the suspension (otherwise store it in melting ice for up to 2 h); cool it immediately to about 20 °C.

3. Inoculation

   Into two 90 mm dishes transfer 1 ml each of the liquid sample or of the initial suspension (where a small number is expected — 10 ml, the limit tenfold lower); into a further dish 1 ml of the 10⁻¹ dilution (liquid) or of the 10⁻² dilution (other products); repeat with the further dilutions using a new pipette, choosing at least two successive critical levels.

4. Pour plate and overlay

   Pour in 12–15 ml (90 mm) or 30–35 ml (140 mm) of TSC agar tempered to 44–47 °C, mix it with a rotary movement and leave it to set; then apply about 5 ml (90 mm) or 12 ml (140 mm) of TSC agar as an overlay.

5. Anaerobic incubation

   Invert the plates and incubate them in an anaerobic atmosphere at 37 °C for 20 h ± 2 h.

6. Counting of the typical colonies

   Within 30 min of their removal from the anaerobic atmosphere count the black or grey to yellowish brown colonies on the plates with < 150 presumptive colonies.

7. Confirmation

   From each plate choose 5 colonies, plate them onto blood agar (or nutrient agar) and incubate them anaerobically at 37 °C for 20 h ± 2 h (read them directly after the incubation; the plates may be cooled at 5 °C for up to 48 h). Carry out the acid phosphatase test (a purple colour within 3–4 min = positive) and the SIM test (22 h ± 2 h, 37 °C).

8. Result

   Calculate the number of confirmed C. perfringens per g or ml of sample; where necessary apply the method of annex D for the differentiation of the strains pathogenic for humans.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• C. perfringens produces toxins and is a cause of food poisoning — aseptic work, autoclaving of the cultures
• The bath at 80 °C and the molten agar at 44–47 °C — risk of burns
• Anaerobic systems (gas generators, catalysts) — use them according to the manufacturer's instructions
• D-cycloserine (an antibiotic) in the medium — avoid inhaling it and contact with the skin during the preparation

Frequently asked questions