🦠 Enumeration of sulfite-reducing bacteria growing under anaerobic conditions in food, feed and environmental samples from food production — 1 ml of the liquid sample or initial suspension and of further decimal dilutions (two plates each) is mixed with iron sulfite agar cooled to 44–47 °C, overlaid after solidification with 5–10 ml and incubated in anaerobic jars at 37 ± 1 °C for 24–48 h (if thermophilic bacteria are suspected, a second set at 50 ± 1 °C); black colonies (iron(II) sulfide) are counted on plates with fewer than 150 typical and fewer than 300 total colonies; optional heat treatment of the suspension gives the number of spores, PN-ISO 15213

Microbiology PN-ISO 15213

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 15213:2003 (English text, first edition of 01.05.2003): title page, foreword, introduction, Clauses 1–11 and the heading of Annex A (normative, “Diagram of procedure”) — the sample ends on page 5, before the diagram itself. Numerical values (temperatures, times, volumes, medium composition) fall outside the sentences in the raw text — we assign them according to the page layout (pdftotext -layout). Part of a sentence in 9.5 is covered by a watermark in the sample. We read the successor in the iTeh sample of SIST EN ISO 15213-1:2023 (contents, foreword with the list of changes, introduction).

ACCORDING TO THE TEXT OF ISO 15213:2003. The standard was prepared by ISO/TC 34 “Food products”, Subcommittee SC 9 “Microbiology”. Introduction: a horizontal method may not suit some products in every detail — then, for justified technical reasons, specific methods may be used. Scope (1): enumeration of sulfite-reducing bacteria growing under anaerobic conditions in products intended for human consumption and animal feeding and in environmental samples from food production and handling. References (2): ISO 6887-1:1999, ISO 8261 (milk), ISO 7218:1996 with Amd 1:2001, ISO/TS 11133-1. Definition (3.1): bacteria forming countable typical colonies under the conditions of this standard. Principle (4): two plates (or tubes) of iron sulfite agar with a specified quantity of liquid sample or initial suspension and two with decimal dilutions; anaerobic incubation at 37 ± 1 °C for 24–48 h (final reading after 48 h), possibly at 50 °C if thermophilic bacteria are suspected; typical black colonies are counted — the blackening of colonies and surrounding zones is iron(II) sulfide from the reaction of sulfide ions with the iron(III) of the medium; result per millilitre or gram. Medium (5.1): enzymatic digest of casein 15 g, pancreatic digest of soya 5 g, yeast extract 5 g, disodium disulfite (Na2S2O5) 1 g, ammonium iron(III) citrate 1 g, agar 9–18 g (depending on gel strength), water 1000 ml; pH after sterilization 7,6 ± 0,2 at 25 °C; portions of 250 ml in 500 ml flasks, for tubes 20 or 25 ml; sterilization 15 min at 121 °C; deaeration just before use; diluent — peptone saline according to ISO 6887-1:1999, 5.2.1. Apparatus (6): homogenizer, water bath 44–47 °C, anaerobic jars with a system for checking anaerobic conditions, incubator 37 ± 1 °C and if necessary 50 ± 1 °C, tubes 16 mm × 160 mm, 500 ml flasks. Sampling (7): not part of the method. Preparation (8, 9.2): according to ISO 6887-1, ISO 8261 or specific standards; heat treatment of the initial suspension is sometimes needed to eliminate vegetative forms — from a pasteurizing effect with moderate heat activation (e.g. 75 °C for 20 min) to boiling for a few minutes; the result can then be reported as the number of spores. Inoculation (9.3): 1 ml of liquid sample or initial suspension onto two plates, 1 ml of the first decimal dilution (10−1 for a liquid sample, 10−2 for the initial suspension — as in the text) onto two more, and further dilutions; agar cooled in the bath to 44–47 °C is poured into each plate — the sample text reads “approximately 15 min of iron sulfite agar” (the volume unit written as “min”); the time between inoculation and pouring should not exceed 15 min; after solidification an overlay of 5–10 ml of the same medium; in tubes — 1 ml into two tubes of medium at 44–47 °C, solidified in a cold bath, overlay 2–3 ml. Incubation (9.4): plates in anaerobic jars at 37 ± 1 °C for 24–48 h, if thermophilic bacteria are suspected a second set at 50 ± 1 °C; tubes need not be placed in anaerobic jars. Counting (9.5): reading after 24 and 48 h; black colonies, possibly with a black zone, are counted as sulfite-reducing bacteria; Note 1 — diffuse, non-specific blackening of the medium occurs, especially in tubes, and anaerobes producing only hydrogen (not H2S) may also reduce sulfite and blacken the medium; plates with fewer than 150 typical and fewer than 300 total colonies are counted; in tubes only those with clearly separated colonies; Note 2 — the standard can be used to count only Clostridium, confirming the genus on five colonies from each plate (e.g. respiratory tests, spore formation). Expression of results (10): according to Amd 1 to ISO 7218:1996. Test report (11): including the method with incubation temperature, use of tubes and heat treatment.

WHAT REPLACED THE STANDARD AND WHAT CHANGED FOR THE RESULT (according to the foreword of ISO 15213-1:2023). The first edition of ISO 15213-1 cancels and replaces ISO 15213:2003, technically revised; it was prepared in collaboration with CEN/TC 463. Main changes: scope extended to samples from primary production; the subject is now “sulfite-reducing Clostridium spp.”, not “sulfite-reducing bacteria” — typical colonies are confirmed; the sulfite concentration in the agar reduced from 1,0 g/l to 0,5 g/l; heat treatment for 10 min at 80 °C is optional (with high background flora or to count spores only); inoculation in tubes and incubation at 50 °C for thermophilic bacteria removed; a description of confirmation added, the diagram in Annex A changed, performance characteristics (Annex C) and a protocol for feed (Annex D) added. According to the introduction the changes are “major” within the meaning of ISO 17468 and have a large impact on method performance. The series also includes ISO 15213-2 (enumeration of C. perfringens) and ISO/TS 15213-3 (detection of C. perfringens).

STATUS (as of 03.10.2026). In the PKN search (term “15213”, 03.10.2026) PN-ISO 15213:2005 (Polish version, “Introduces: ISO 15213:2003 [IDT]”) — “Withdrawn and replaced by PN-EN ISO 15213-1:2023-08 – English version”. Card: publication date 29.06.2005, withdrawal date 23.02.2024, 12 pages, KT 3 Food Chain Microbiology, ICS 07.100.30, “Replaces PN-A-86034-12:1993”. Card of PN-EN ISO 15213-1:2023-08 (English version): publication 08.08.2023, 34 pages, KT 3, introduces EN ISO 15213-1:2023 and ISO 15213-1:2023, “Replaces PN-ISO 15213:2005”, supplementary element PN-EN ISO 15213-1:2023-08/Ap1:2024-02E, no withdrawal note. In the iTeh catalogue (read 03.10.2026) ISO 15213:2003 has the status “Withdrawn”, stage 95.99, revised by ISO 15213-1:2023, ISO 15213-2:2023 and ISO/TS 15213-3:2024.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number “15213” without a part number appears in 4 records at 3 laboratories: AB 285 (2), AB 418, AB 1866. In the current PCA documents (read 03.10.2026; AB 285 issue No. 28 of 13.01.2026, AB 418 issue No. 37 of 26.01.2026, AB 1866 issue No. 4 of 09.06.2026) the number appears in 4 lines at the same 3 laboratories; in each document the number of page markers equals the number of PDF pages, without repetitions; a search of all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. The form is always “PN-ISO 15213:2005”. None of these items is suspended. AB 285 also has an item with PN-EN ISO 15213-1:2023-08 in the same document. The “Laboratories” tab (term “PN-ISO 15213”) shows 5 laboratories — AB 285, AB 418, AB 1866 and AB 400 and AB 1179 — checked by a tab query on the labcoda.pl production server on 03.10.2026; AB 400 and AB 1179 appear there through entries of parts of the new series with the prefix “PN-ISO” (“PN-ISO 15213-1”, “PN-ISO 15213-2” at AB 400; “PN-ISO 15213-1:2023-08” at AB 1179), not through the withdrawn standard.

THE SUCCESSOR IN SCOPES (read 03.10.2026). Part 1 of the new series (“15213-1” in any form) appears in the current PCA documents at 11 laboratories: AB 285, AB 313, AB 370, AB 400, AB 452, AB 462, AB 819, AB 924, AB 1179, AB 1415, AB 1651; in the database copy at 10 (without AB 1651). Part 2 (C. perfringens) — in the database copy at 11 laboratories.

WHAT THE LABORATORIES TEST (items in PCA). AB 285: number of anaerobic sulfite-reducing bacteria in fruit and vegetable, meat and fish products and the number of their spores also in milk and milk products and apple juice; AB 418: spores of anaerobes, including sulfite-reducing ones, in waste (groups I, IV, V, VI, IX, XI and others according to DAB-11), with a footnote referring to the regulation of the Minister of Climate of 24.12.2019 on the conditions for recognising waste as having infectious properties; AB 1866: number of anaerobic (sulfite-reducing) microorganisms in plant growth aids (soil improvers, growing media, microbial fertiliser products). Technique: plate method (pour plate).

WHERE AN APPARENTLY CORRECT RESULT IS EASY TO GET. With the status: all three laboratories cite PN-ISO 15213:2005, withdrawn in PKN on 23.02.2024; the successor counts a different quantity — only Clostridium spp. with confirmation — on a medium with half the sulfite, so the results of the two standards are not interchangeable (foreword and introduction of ISO 15213-1). With the object: the scope of the standard is food, feed and environmental samples from food production (1) — waste (AB 418) and plant growth aids (AB 1866) are not mentioned in it. With reading: blackening of the medium can be non-specific, especially in tubes, and anaerobes producing only hydrogen also reduce sulfite (9.5, Note 1). With spores: heat treatment (e.g. 75 °C, 20 min) changes the measured quantity to the number of spores and must be stated in the report (9.2, 11 c). With counting: only plates with fewer than 150 typical and fewer than 300 total colonies (9.5). With temperature: a second set at 50 °C only if thermophilic bacteria are suspected (9.4) — the successor no longer has this option. With the text: the agar volume in 9.3 is written in the sample as “15 min” — we do not correct the standard, we record the wording.

WHAT WE DO NOT GIVE. We do not quote the diagram of Annex A or the expression of results from Amd 1 to ISO 7218. We have not read ISO 15213-1:2023 beyond the foreword and introduction (media, confirmation, precision data, protocol for feed).

Method principle

The liquid sample or initial suspension and its decimal dilutions are inoculated, 1 ml each, onto two plates, mixed with iron sulfite agar (disodium disulfite and ammonium iron(III) citrate, 1 g/l each) at 44–47 °C, overlaid after solidification and incubated anaerobically at 37 °C for 24–48 h. Sulfite-reducing bacteria produce sulfide, which with the iron(III) of the medium gives black iron(II) sulfide; black colonies are counted on plates with fewer than 150 typical colonies and converted per gram or millilitre. Heat treatment of the suspension narrows the result to spores.

Applications

• Sulfite-reducing bacteria under anaerobic conditions in food, feed and environmental samples from food production (1)
• Number of spores after heat treatment of the suspension; after confirmation — Clostridium (9.2, 9.5)
• Fruit, vegetable, meat, fish and dairy products, waste, plant growth aids — items in the accreditation scopes of 3 laboratories in PCA

Key parameters

Standard

Standard number

PN-ISO 15213:2005

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby bakterii redukujących siarczany(IV) rosnących w warunkach beztlenowych

Title (EN)

Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of sulfite-reducing bacteria growing under anaerobic conditions

Step-by-step procedure

1. Suspension and dilutions

   According to ISO 6887-1, ISO 8261 or a specific standard; possible heat treatment for spores (9.2). PN-ISO 15213:2005 withdrawn in PKN on 23.02.2024 (03.10.2026).

2. Inoculation

   1 ml onto two plates from each level; agar at 44–47 °C poured within 15 min of inoculation; overlay 5–10 ml (9.3).

3. Incubation

   Anaerobically, 37 ± 1 °C, 24–48 h; for thermophiles a second set at 50 ± 1 °C (9.4).

4. Counting

   Black colonies after 24 and 48 h; plates < 150 typical and < 300 total colonies (9.5).

5. Result

   Number per gram or millilitre according to Amd 1 to ISO 7218:1996; the report states temperature, tubes and heat treatment (10, 11).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Work with anaerobic bacteria, including potentially pathogenic Clostridium — microbiological practice according to ISO 7218
• Sterilization in an autoclave at 121 °C; hot medium and water bath

Frequently asked questions