🦠 Sulfite-reducing Clostridium spp. in food, feed, environmental samples and samples from primary production — pour plate in iron sulfite agar with overlay, anaerobic incubation at 37 °C for 48 h, confirmation of colonies, optionally spores only after 10 min at 80 °C, PN-EN ISO 15213-1

Microbiology PN-EN ISO 15213-1

Overview

WHAT THE STANDARD COVERS. Scope from the PKN card, in full (our translation): "This document specifies the enumeration of sulfite-reducing anaerobes and of sulfite-reducing bacteria of the genus Clostridium spp. by the colony-count technique. The standard is applicable to: — products intended for human consumption; — products intended for feeding animals; — environmental samples in the area of food and feed production and handling, and — samples from the primary production stage. NOTE This method has been validated in an interlaboratory study for the following food categories: — ready-to-eat, ready-to-reheat meat products; — eggs and egg products (derivatives); — processed fruits and vegetables; — infant formula and infant cereals; — multi-component foods or meal components. It has also been validated for other categories of products, i.e.: — pet food and animal feed; — environmental samples (taken from the food or feed production environment). This method has been validated for at least five food categories, therefore it is applicable to a broad range of food products. Detailed information on the validation is given in Clause 11 and Annex C. Since this method is not commonly used for samples from the primary production stage, this category was not included in the validation studies. Therefore, no corresponding values of validation parameters were obtained for this category. The method presented is suitable (but not exclusively) for the enumeration of microorganisms in test samples and is based on counting at least 10 colonies on a plate. This corresponds to a level of contamination expected to be higher than 10 cfu/ml for liquid samples or higher than 100 cfu/g for solid samples." The English ISO text speaks in the first sentence only of sulfite-reducing Clostridium spp. We have a sample extract of ISO 15213-1:2023 (first edition) from the cover to subclause 9.4.4 (page 5).

ACCORDING TO THE 2023 EDITION (foreword, introduction, Clauses 1–9.4.4). The standard was prepared by ISO/TC 34/SC 9 "Microbiology" in collaboration with CEN/TC 463 (Vienna Agreement); it replaced ISO 15213:2003, and the changes were considered major (according to ISO 17468), with a major impact on the performance characteristics of the method: the scope was extended to samples from primary production; the quantity was changed from "sulfite-reducing bacteria" to "sulfite-reducing Clostridium spp.", so typical colonies are confirmed; the sulfite content of iron sulfite agar was reduced from 1.0 g/l to 0.5 g/l; heating for 10 min at 80 °C became optional (with high background flora or when only spores are counted); inoculation in tubes and incubation at 50 °C for thermophilic sulfite-reducing bacteria were removed; a description of the confirmation, a new flow diagram in Annex A, performance characteristics in Annex C and a special protocol for feed in Annex D were added. Introduction: sulfite-reducing Clostridium spp. are obligate anaerobic, Gram-positive, spore-forming rods — among them C. perfringens, C. bifermentans, C. sporogenes, C. botulinum — indicators of contamination; the enumeration of C. perfringens is described in ISO 15213-2 and its detection in ISO/TS 15213-3. Principle (Clause 4): a specified quantity of the liquid sample or initial suspension is mixed in an empty Petri dish with molten agar (pour plate), after solidification an overlay is poured to prevent spreading colonies; the plates are incubated anaerobically at 37 °C for 48 h and typical colonies are counted — black or grey to yellow-brown, coloured by iron(II) sulfide formed from sulfide ions and iron(III) in the medium; typical colonies are confirmed; NOTE: without confirmation the result can be reported as "anaerobic sulfite-reducing bacteria". When the number is close to the limit of determination, duplicate plates are preferable — the sum of colonies on both at least 10, which corresponds to more than 5 cfu/ml or 50 cfu/g. The result is given in cfu per gram, millilitre, square centimetre or per sampling device. Equipment (Clause 6): anaerobic atmosphere of less than 1 % oxygen and from 9 % to 13 % carbon dioxide (jar, cabinet or gas packs), incubator 37 °C ± 1 °C, pH meter ± 0.1 at 25 °C, refrigerator 5 °C ± 3 °C, pipettes of 10 ml and 1 ml, loops of about 1 µl, Petri dishes of about 90 mm and optionally about 140 mm, water bath 44–47 °C and 80 °C ± 2 °C. Sampling is not part of the method — recommended documents: ISO/TS 17728 (food and feed), ISO 707 (milk), ISO 6887-3, ISO 13307 (primary production), ISO 17604 (carcasses), ISO 18593 (surfaces); preparation according to the ISO 6887 series, for feed according to Annex D. Spores only (9.3): the dilution series is heated in a water bath at 80 °C for 10 min ± 1 min, at the latest 15 min after preparation of the initial suspension (otherwise in melting ice for at most 2 h); the temperature is monitored by a thermometer in a reference bottle; reaching 80 °C takes no longer than 5 min (water at least 4 cm above the sample level, circulation pump), the time is counted from reaching 80 °C in the reference bottle, followed by rapid cooling to about 20 °C; heating also reduces the background flora (e.g. liquid whey, silage). Inoculation (9.4): 1 ml of the liquid sample or initial suspension (10⁻¹) into each of two 90 mm dishes, 1 ml of the next dilution — 10⁻¹ (liquid) or 10⁻² (other) — into another dish; the limit of determination can be lowered tenfold by examining 10 ml of the initial suspension on three 140 mm dishes; dilutions giving 10 to 150 colonies per 90 mm dish or 10 to 360 per 140 mm dish are selected.

STATUS (state of the cards on 30 September 2026). The card of PN-EN ISO 15213-1:2023-08 (English version) has no "Withdrawn" header — the standard is CURRENT: published 08-08-2023, 34 pages, price group Q, Food, Agriculture and Forestry Sector, KT 3 for Microbiology of the Food Chain, ICS 07.100.30, "Introduces: EN ISO 15213-1:2023 [IDT], ISO 15213-1:2023 [IDT]", "Replaces: PN-ISO 15213:2005 - Polish version", corrigendum Ap1:2024-02. The national corrigendum PN-EN ISO 15213-1:2023-08/Ap1:2024-02 (approved on 22 February 2024, file from the card read) added PN-ISO 15213:2005 to the "Replaces" field on the title page and the sentence "This standard replaces PN-ISO 15213:2005" to the national foreword. The card of PN-ISO 15213:2005 "Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of sulfite-reducing bacteria growing under anaerobic conditions" — "Withdrawn and replaced by PN-EN ISO 15213-1:2023-08": published 29-06-2005, "Withdrawal date: 23-02-2024", 12 pages, introduced ISO 15213:2003, replaced PN-A-86034-12:1993. The PKN search for "PN-EN ISO 15213" (30 September 2026): PN-EN ISO 15213-1:2023-08 and PN-EN ISO 15213-2:2024-05 without labels, PN-ISO 15213:2005 — "Withdrawn and replaced by", and standards PN-EN 15213-1…5 from another field (transport systems).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation-scope database, data loaded up to 17 September 2026, read on 30 September 2026). All notations of number 15213-1: 11 records at 10 laboratories; in the current PCA documents the number stands in 13 cells — AB 924 has three entries in issue No. 25 of 16.09.2026, the copy one. The notation "PN-EN ISO 15213-1:2023-08" — 8 laboratories, including with corrigendum Ap1:2024-02 — AB 285, AB 313, AB 370, AB 819, AB 924 (AB 285 as "PN_EN ISO 15213-1:2023-08/Ap1:2024-02"); AB 400 writes "PN-EN ISO 15213-1" and in a second entry "PN-ISO 15213-1", AB 1179 — "PN-ISO 15213-1:2023-08" (a form that does not exist in the PKN catalogue); AB 370 cites the standard "excluding clause 9.6" (confirmation). The "Laboratories" tab looks for notations that BEGIN with "PN-EN ISO 15213-1" and will show 9 laboratories — it will not show AB 1179; checked with the tab's query on the production server labcoda.pl on 30 September 2026: 9 (under "PN-ISO 15213-1" — 2). Current scopes on the PCA website (read on 30 September 2026, issues from 30.10.2025 to 16.09.2026): the entries with this standard are not suspended (AB 1179 has another part of its scope suspended).

QUANTITY AND ITEMS. Name of the quantity: "Count of sulfite-reducing Clostridium spp." — 7 laboratories (AB 285, AB 313, AB 400, AB 452, AB 462, AB 924, AB 1415); "Count of anaerobic sulfite-reducing bacteria" — AB 819; "Count of spores of anaerobic sulfite-reducing bacteria" — AB 370; "Count of sulfite-reducing bacteria growing under anaerobic conditions" — AB 1179 (wording of the title of the withdrawn PN-ISO 15213:2005). Technique: plate method, pour plate — 9 laboratories, AB 452 — "horizontal method". Items: food — milk and milk products, meat and meat products, fish, fruits and vegetables and their products, concentrates, confectionery, cereal products, eggs, frozen food, ready meals (at AB 285 also apple juice); AB 400 — food in a flexible scope. The entries have no numerical ranges.

ALONGSIDE THIS STANDARD. The withdrawn PN-ISO 15213:2005 is present in the current scopes of three laboratories: AB 285 (two entries — anaerobic sulfite-reducing bacteria and their spores — next to the entry according to the new standard, issue No. 28 of 13.01.2026), AB 418 (spores of anaerobes, including sulfite-reducing ones, in wastes) and AB 1866 (count of anaerobic sulfite-reducing microorganisms in plant cultivation aids, issue No. 4 of 09.06.2026). The qualitative test for the "presence of anaerobic spore-forming bacteria and anaerobic sulfite-reducing bacteria" according to PN-A-82055-12 is carried out in the current scopes by 5 laboratories (AB 400, AB 462, AB 483, AB 510, AB 1415; the copy also has AB 437, whose issue of 10.09.2026 no longer contains this entry). In water, spores of sulfite-reducing clostridia are enumerated by membrane filtration according to PN-EN 26461-2, and the count of Clostridium perfringens in food according to PN-EN ISO 15213-2 (both have separate Knowledge Base entries).

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. First — with or without confirmation: the result "sulfite-reducing Clostridium spp." requires confirmation of typical colonies; without it the standard allows reporting only "anaerobic sulfite-reducing bacteria" — AB 370 excludes clause 9.6 and names the quantity "spores of anaerobic sulfite-reducing bacteria". Second — spores or vegetative cells: heating for 10 min at 80 °C is optional; the result after heating concerns only spores and is not comparable with a result without heating — the report must state this. Third — old and new method: the 2023 edition has a medium with a lower sulfite content (0.5 g/l instead of 1.0 g/l), no incubation at 50 °C and no inoculation in tubes — results according to the withdrawn PN-ISO 15213:2005 are not interchangeable with results according to this standard. Fourth — limit: counting requires at least 10 colonies on a plate — more than 10 cfu/ml or 100 cfu/g; with two plates the sum must be at least 10 — more than 5 cfu/ml or 50 cfu/g; lower levels require plating 10 ml of the suspension on 140 mm dishes. Fifth — anaerobiosis: an atmosphere below 1 % oxygen for the whole incubation time; with a leaking jar there will be fewer colonies.

WHAT WE DO NOT GIVE. The extract ends at subclause 9.4.4: we do not give the further course of inoculation and pouring of the overlay (9.4.5 onwards), the counting of typical colonies (9.5), confirmation (9.6), expression of results (10), validation data (11, Annex C), the composition of the media (Annex B) or the protocol for feed (Annex D).

Method principle

The liquid sample or the initial suspension and its dilutions are mixed in Petri dishes with molten iron sulfite agar, covered with an overlay after solidification and incubated anaerobically at 37 °C for 48 h. Sulfite-reducing Clostridium spp. form black or grey to yellow-brown colonies (iron(II) sulfide); typical colonies are counted and confirmed, and the result is given in cfu/g, cfu/ml, cfu/cm² or per sampling device. When only spores are counted, the dilutions are heated for 10 min at 80 °C.

Applications

• Food — milk and milk products, meat, fish, fruits and vegetables, concentrates, confectionery, cereal products, eggs, frozen food — in the scopes of 10 laboratories
• Feed and pet food, environmental samples from food and feed production, samples from primary production — according to the scope of the standard

Key parameters

Standard

Standard number

PN-EN ISO 15213-1:2023-08 (wersja angielska; EN ISO 15213-1:2023, ISO 15213-1:2023 IDT) z poprawką PN-EN ISO 15213-1:2023-08/Ap1:2024-02

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania i oznaczania liczby Clostridium spp. — Część 1: Oznaczanie liczby Clostridium spp. redukujących siarczany(IV) metodą liczenia kolonii

Title (EN)

Microbiology of the food chain — Horizontal method for the detection and enumeration of Clostridium spp. — Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-count technique

Step-by-step procedure

1. Sample and dilutions

   Preparation according to the ISO 6887 series (feed — Annex D); a single series of decimal dilutions. PN-EN ISO 15213-1:2023-08 current (state of the card on 30 September 2026).

2. Spores only (option)

   Heat the dilutions in a water bath at 80 °C for 10 min ± 1 min, at the latest 15 min after preparation of the suspension; reaching 80 °C ≤ 5 min; cool to about 20 °C.

3. Pour plate

   1 ml of the liquid sample or the 10⁻¹ suspension into each of two 90 mm dishes, 1 ml of the next dilution into another; for low levels 10 ml of the suspension on three 140 mm dishes; iron sulfite agar and overlay.

4. Incubation

   Anaerobic (< 1 % O₂, 9–13 % CO₂), 37 °C, 48 h.

5. Counting, confirmation, result

   Black or grey to yellow-brown colonies on plates with 10–150 colonies (90 mm); confirmation of typical colonies (9.6 — NOT GIVEN); without confirmation the result as "anaerobic sulfite-reducing bacteria".

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Cultures may contain pathogenic Clostridium species (C. perfringens, C. botulinum) — tests only in properly equipped laboratories, under the supervision of a microbiologist, with safe disposal of incubated materials (warning of the standard)
• SUBSTANTIVE NOTE: without confirmation of colonies the result is "anaerobic sulfite-reducing bacteria", not Clostridium spp.; results according to the withdrawn PN-ISO 15213:2005 are not interchangeable with results according to this standard

Frequently asked questions