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INTERNATIONAL ISO
STANDARD 15213-2
First edition
2023-11
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
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Clostridium spp. —
Partie 2: Dénombrement de Clostridium perfringens par la technique
de comptage des colonies
Reference number
© ISO 2023
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ii
Contents Page
Foreword .iv
Introduction . vi
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 2
4 Principle . 3
4.1 General . 3
4.2 Preparation of dilutions . 3
4.3 Enumeration . 3
4.4 Confirmation . 3
5 Culture media and reagents .4
6 Equipment and consumables .4
7 Sampling . 4
8 Preparation of test sample . 5
9 Procedure .5
9.1 General . 5
9.2 Test portion, initial suspension and dilutions . 5
9.3 Heat treatment to select spores . 5
9.4 Inoculation and incubation. 6
9.5 Enumeration of typical colonies . 6
9.6 Confirmation of C. perfringens . 7
9.6.1 Selection of colonies for confirmation . 7
9.6.2 Acid phosphatase test . 7
9.6.3 Sulfite indole motility (SIM) agar test . 7
9.6.4 Differentiation between human pathogenic and non-pathogenic
C. perfringens strains (optional) . 8
9.6.5 Interpretation . 8
10 Expression of results . 8
11 Validation of the method . 8
11.1 Interlaboratory study . 8
11.2 Performance characteristics . 8
12 Test report . 9
13 Quality assurance .10
Annex A (normative) Flow diagram of the procedure .11
Annex B (normative) Culture media and reagents .13
Annex C (informative) Method validation studies and performance characteristics .18
Annex D (informative) Molecular differentiation between pathogenic and non-pathogenic
C. perfringens .21
Bibliography .43
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
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electrotechnical standardization.
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described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
This first edition of ISO 15213-2 cancels and replaces ISO 7937:2004, which has been technically
revised.
The main changes are as follows:
— the Scope has been expanded to samples from the primary production stage;
— the heat treatment of 10 min at 80 °C has been made optional, in the case of high background flora
or for the enumeration of only spores of Clostridium (C.) perfringens present in the sample;
— the selective medium has been re-named from sulfite-cycloserine agar (SC) to tryptose sulfite
cycloserine agar (TSC agar) without changes in the formulation;
— the confirmation methods described have been modified according to ISO 14189;
— the flow diagram in normative Annex A giving a short description of the procedure has been revised;
— in Annex B, criteria for the performance testing of culture media have been added;
— in Annex C (informative), the performance characteristics have been added;
— in Annex D (informative), two molecular methods have been added for differentiation between
pathogenic and non-pathogenic C. perfringens and one molecular method for the differentiation of
C. perfringens type A strains carrying a chromosomally encoded cpe gene or a plasmid encoded cpe
gene.
iv
A list of all parts in the ISO 15213 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
Introduction
Clostridium (C.) perfringens is a Gram-positive, anaerobic, spore-forming bacterium. As a ubiquitous
bacterium, C. perfringens is predominantly found in soil, but also in the intestinal tract of humans and
animals. Therefore, the presence of C. perfringens in high numbers can be an indication of inadequate
preparation or handling of food.
High numbers of C. perfringens in ready-to-eat-food can cause human illness, mainly diarrhoea. The
strains are classified into toxin types, depending on the ability to produce different so called “major”
and “minor” toxins. Food poisonings are caused by C. perfringens isolates with the ability to produce
C. perfringens enterotoxin (CPE).
A characteristic feature is the heat resistance of the spores; they have the ability to germinate and
multiply in ready-to-eat food after the cooking process. Ingestion of contaminated food is followed
by gastrointestinal disease, when enzyme-resistant C. perfringens enterotoxins are set free during
sporulation in the small intestine. The strains are classified into different types.
This document describes the horizontal method for the enumeration of C. perfringens in food,
feed, environmental samples and samples from the primary production stage. The method for the
enumeration of sulfite-reducing Clostridium spp. is described in ISO 15213-1. The method for the
detection of C. perfringens is described in ISO/TS 15213-3. These three parts are published as a series
of International Standards because the methods are closely linked to each other. These methods are
often conducted in association with each other in a laboratory and the media and their performance
characteristics can be similar.
The main technical changes listed in the Foreword, introduced in this document compared with
ISO 7937:2004, are considered as major (see ISO 17468).
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