EN ISO 15213-2:2023

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 (ISO 15213-2:2023)

This document specifies the enumeration of Clostridium (C.) perfringens by colony-count technique.
This document is applicable to:
—    products intended for human consumption;
—    products for feeding animals;
—    environmental samples in the area of food and feed production and handling;
—    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 (derivates);
—     processed fruits and vegetables;
—     infant formula and infant cereals;
—     multi-component foods or meal components.
It has also been validated for the following other categories:
—     pet food and animal feed;
—     environmental samples (food or feed production).
As this method has been validated for at least five food categories, this method is applicable for a broad range of food. For detailed information on the validation, see Clause 11 and Annex C. Since the method is not commonly used for samples in the primary production stage, this category was not included in the interlaboratory study. Therefore, no performance characteristics were obtained for this category.
This horizontal method was originally developed for the examination of all samples belonging to the food chain. Based on the information available at the time of publication of this document, this method is considered to be fully suited to the examination of all samples belonging to the food chain. However, because of the large variety of products in the food chain, it is possible that this horizontal method is not appropriate in every detail for all products. Nevertheless, it is expected that the required modifications are minimized so that they do not result in a significant deviation from this horizontal method.
This technique is suitable for, but not limited to, the enumeration of microorganisms in test samples with a minimum of 10 colonies counted on a plate. This corresponds to a level of contamination that is expected to be higher than 10 cfu/ml for liquid samples or higher than 100 cfu/g for solid samples.

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur Zählung von Clostridium spp. - Teil 2: Zählung von Clostridium perfringens durch Koloniezählverfahren (ISO 15213‑2:2023)

Dieses Dokument legt die Zählung von Clostridium (C.) perfringens mit dem Koloniezählverfahren fest.
Dieses Dokument ist anwendbar für:
   Produkte, die für den menschlichen Verzehr vorgesehen sind;
   Produkte, die als Futtermittel verwendet werden;
   Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln;
   Proben aus dem Bereich der Primärproduktion.
ANMERKUNG   Dieses Verfahren wurde in einem Ringversuch für die folgenden Lebensmittelkategorien validiert:
   verzehrfertige, aufwärmfertige Fleischprodukte;
   Eier und Eiprodukte (Derivate);
   verarbeitetes Obst und Gemüse;
   Säuglingsnahrung und Getreideprodukte für Kleinkinder;
   Mehrkomponenten-Lebensmittel bzw. Mahlzeitkomponenten.
Es wurde außerdem für die folgenden anderen Kategorien validiert:
   (Heim )Tierfuttermittel;
   Umgebungsproben (Lebensmittel- oder Futtermittelproduktion).
Da dieses Verfahren für mindestens fünf Lebensmittelkategorien validiert wurde, ist es für ein breites Spektrum von Lebensmitteln anwendbar. Abschnitt 11 und Anhang C enthalten detaillierte Informationen zur Validierung. Da das Verfahren üblicherweise nicht für Proben in der Primärproduktion verwendet wird, wurde diese Kategorie nicht in den Ringversuch aufgenommen. Daher wurden für diese Kategorie keine Leistungsmerkmale ermittelt.
Dieses horizontale Verfahren wurde ursprünglich für die Untersuchung aller Proben aus der Lebensmittelkette entwickelt. Basierend auf den zum Zeitpunkt der Veröffentlichung dieses Dokuments verfügbaren Informationen wird dieses Verfahren als für die Untersuchung aller Proben aus der Lebensmittelkette uneingeschränkt geeignet angesehen. Aufgrund der großen Produktvielfalt in der Lebensmittelkette ist es jedoch möglich, dass dieses horizontale Verfahren nicht bis ins Detail für alle Produkte geeignet ist. Dennoch wird erwartet, dass erforderliche Änderungen so gering gehalten werden, dass sie nicht zu einer signifikanten Abweichung von diesem horizontalen Verfahren führen.
Dieses Verfahren eignet sich unter anderem für die Zählung von Mikroorganismen in Untersuchungsproben mit mindestens 10 gezählten Kolonien auf einer Platte. Dies entspricht einem erwarteten Kontaminationsgrad von mehr als 10 KBE/ml bei flüssigen Proben bzw. mehr als 100 KBE/g bei festen Proben.
WARNHINWEIS — Zum Schutz der Gesundheit des Laborpersonals ist es unerlässlich, dass Prüfungen mit Zählung von Clostridium perfringens nur in Laboren mit geeigneter Ausstattung und unter der Leitung eines qualifizierten Mikrobiologen erfolgen und dass bei der Entsorgung allen bebrüteten Materials mit äußerster Vorsicht vorgegangen wird. Personen, die dieses Dokument anwenden, sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument erhebt nicht den Anspruch, alle gegebenenfalls zutreffenden Sicherheitsaspekte im Zusammenhang mit seiner Anwendung zu behandeln. Es liegt in der Verantwortung des Anwenders, angemessene Sicherheits- und Schutzmaßnahmen zu treffen.

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 (ISO 15213-2:2023)

Le présent document spécifie le dénombrement de Clostridium (C.) perfringens par la technique de comptage des colonies.
Le présent document s’applique:
—    aux produits destinés à la consommation humaine;
—    aux produits destinés à l’alimentation animale;
—    aux échantillons environnementaux prélevés dans les secteurs de la production et de la distribution des aliments;
—    aux échantillons issus du stade de la production primaire.
NOTE            Cette méthode a été validée dans le cadre d’une étude interlaboratoires pour les catégories d’aliments suivantes:
—     les produits à base de viande prêts à consommer et prêts à réchauffer;
—     les œufs et ovoproduits (dérivés);
—     les fruits et légumes transformés;
—     les préparations et céréales pour nourrissons;
—     les aliments composés et les composants de repas.
Elle a également été validée pour les autres catégories suivantes:
—     les produits destinés à l’alimentation animale et les aliments pour animaux;
—     les échantillons environnementaux (production d’aliments ou d’aliments pour animaux).
Cette méthode ayant été validée pour au moins cinq catégories d’aliments, elle s’applique à un large éventail d’aliments. Pour des informations détaillées sur la validation, voir l’Article 11 et l’Annexe C. Étant donné que cette méthode n’est pas couramment utilisée pour les échantillons au stade de la production primaire, cette catégorie n’a pas été incluse dans l’étude interlaboratoires. Par conséquent, aucune caractéristique de performance n’a été déterminée pour cette catégorie.
Cette méthode horizontale a été initialement développée pour l’examen de tous les échantillons provenant de la chaîne alimentaire. Sur la base des informations disponibles au moment de la publication du présent document, cette méthode est considérée comme parfaitement adaptée à l’examen de tous les échantillons provenant de la chaîne alimentaire. Cependant, en raison de la grande diversité des produits de la chaîne alimentaire, il est possible que cette méthode horizontale ne soit pas appropriée dans ses moindres détails à tous les produits. Néanmoins, il est attendu que les modifications requises soient réduites au minimum afin qu’elles n’entrainent pas de déviation significative de cette méthode horizontale.
Cette technique est adaptée, sans toutefois s’y limiter, au dénombrement des micro-organismes dans les échantillons d’essai avec un minimum de 10 colonies dénombrées par boîte. Cela correspond à un niveau de contamination attendu supérieur à 10 UFC/ml pour les échantillons liquides ou supérieur à 100 UFC/g pour les échantillons solides.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila Clostridium spp. - 2. del: Preštevanje Clostridium perfringens s tehniko štetja kolonij (ISO 15213-2:2023)

Ta dokument določa števila bakterije Clostridium (C) perfringens s tehniko štetja kolonij.
Ta dokument se uporablja za:
– proizvode, namenjene za prehrano ljudi;
– proizvode za živalsko krmo;
– okoljske vzorce v območju proizvodnje in ravnanja z živili in krmo;
– vzorce iz faze primarne proizvodnje.
OPOMBA: Ta metoda je bila validirana z medlaboratorijsko študijo za naslednje kategorije živil:
– mesni izdelki, pripravljeni za uživanje, pripravljeni za pogrevanje;
– jajca in jajčni izdelki (stranski proizvodi);
– predelano sadje in zelenjava;
– formula za dojenčke in žitne kašice za dojenčke;
– večkomponentna živila ali deli obrokov.
Prav tako je izvedena validacija za naslednje kategorije:
– hrana za domače živali in živalska krma;
– okoljski vzorci (proizvodnja živil ali krme).
Ker je ta metoda validirana za vsaj pet kategorij živil, je metoda uporabna za širok obseg vrst živil. Za podrobne informacije o validaciji glej točko 11 in dodatek C. Ker se ta metoda ne uporablja pogosto za vzorce v primarni fazi proizvodnje, ta kategorija ni bila vključena v medlaboratorijsko študijo.
Tako za to kategorijo niso bile pridobljene zmogljivostne lastnosti.
Ta horizontalna metoda je bila prvotno razvita za preiskovanje vseh vzorcev, ki pripadajo prehranski verigi. Na podlagi informacij, ki so bile na voljo v času objave tega dokumenta, ta metoda
velja za povsem primerno za preiskovanje vseh vzorcev, ki pripadajo prehranski verigi. Vseeno pa zaradi velike raznolikosti proizvodov v prehranski verigi morda ta horizontalna metoda v vseh podrobnostih ni v celoti primerna za vse proizvode. Vseeno gre pričakovati, da je število zahtevanih sprememb manjše, s tem pa ni bistvenih odstopanj od te horizontalne metode.
Ta tehnika je med drugim primerna za ugotavljanje števila mikroorganizmov v preskusnih vzorcih z najmanj 10 preštetimi kolonijami na pladnju. To ustreza stopnji kontaminacije, ki je pričakovano
večja od 10 cfu/ml za tekoče vzorce oziroma večja od 100 cfu/g za trdne vzorce.

General Information

• Status: Published
• Publication Date: 21-Nov-2023
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463 - Microbiology of the food chain
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 22-Nov-2023
• Completion Date: 22-Nov-2023
• Directive: 2017/625 - Regulation (EU) 2017/625 of the European Parliament and of the Council of 15 March 2017 on official controls and other official activities performed to ensure the application of food and feed law, rules on animal health and welfare, plant health and plant protection products
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: SIST EN ISO 15213-2:2024 - 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 (ISO 15213-2:2023)

Relations

• Replaces: EN ISO 7937:2004 - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of Clostridium perfringens - Colony-count technique (ISO 7937:2004)
• Effective Date: 19-Jan-2023

Overview

EN ISO 15213-2:2023 (CEN adoption) is the international horizontal method for the enumeration of Clostridium perfringens by colony-count technique in the food chain. The standard specifies procedures for sample preparation, dilution, inoculation, incubation, colony counting and confirmation to determine C. perfringens levels in products for human consumption, animal feed, environmental samples from food/feed production and samples from primary production. It replaces ISO 7937:2004 and is intended for routine food microbiology and food safety testing.

Key Topics and Requirements

• Scope and applicability: Validated for multiple food categories including ready-to-eat/meat products, eggs and egg products, processed fruits and vegetables, infant formula and cereals, multi-component foods; also applicable to pet food, animal feed and environmental samples. Primary production samples are included in scope but were not part of the interlaboratory validation.
• Principle: Enumeration by colony-count on selective agar after appropriate dilutions; technique suited to samples yielding at least 10 colonies per plate (≈ >10 cfu/mL liquid or >100 cfu/g solid).
• Selective medium: Use of tryptose sulfite cycloserine (TSC) agar (formerly sulfite-cycloserine, no formulation change).
• Heat treatment: Optional heat shock (historically 10 min at 80 °C) to select for spores-now optional when high background flora is present or when spores-only enumeration is required.
• Confirmation: Procedures updated in line with ISO 14189; includes biochemical tests (acid phosphatase, SIM agar) and optional molecular differentiation between pathogenic and non‑pathogenic strains (Annex D).
• Validation and performance: Method validated by interlaboratory study; performance characteristics and validation data provided in Clause 11 and Annex C. Annex B includes culture media performance criteria.
• Reporting and QA: Requirements for expression of results, test reporting and quality assurance suitable for laboratory SOPs and accreditation.

Applications and Users

Who uses EN ISO 15213-2:2023:

• Food microbiology laboratories and QC/analytical labs
• Food and feed manufacturers (HACCP and shelf-life testing)
• Public health and regulatory laboratories (surveillance, outbreak investigation)
• Environmental monitoring teams in production and processing sites
• Research labs studying Clostridium perfringens contamination and spore survival

Practical uses:

• Routine enumeration of C. perfringens for food safety compliance
• Environmental swabbing and process hygiene monitoring
• Support for risk assessments and corrective actions in production
• Differentiation of pathogenic strains where required for epidemiology

Related Standards

• ISO 15213 series (other parts)
• ISO 14189 (confirmation methods)
• Supersedes ISO 7937:2004

This standard is essential for accurate, standardized colony-count enumeration of C. perfringens in the food chain, enabling consistent results across laboratories and supporting food safety management and regulatory compliance.

Standards Content (Sample)

SLOVENSKI STANDARD
01-marec-2024
Nadomešča:
SIST EN ISO 7937:2005
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Clostridium spp. - 2. del: Preštevanje Clostridium perfringens
s tehniko štetja kolonij (ISO 15213-2:2023)
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 (ISO 15213-2:2023)
Mikrobiologie der Nahrungskette - Horizontales Verfahren zum Nachweis und zur
Aufzählung von Clostridium spp. - Teil 2: Zählung von Clostridium perfringens durch
Koloniezählverfahren (ISO 15213-2:2023)
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 (ISO 15213-2:2023)
Ta slovenski standard je istoveten z: EN ISO 15213-2:2023
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 15213-2
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2023
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 7937:2004
English Version
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 (ISO 15213-2:2023)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Nahrungskette - Horizontales
horizontale pour la recherche et le dénombrement de Verfahren zum Nachweis und zur Aufzählung von
Clostridium spp. - Partie 2: Dénombrement de Clostridium spp. - Teil 2: Zählung von Clostridium
Clostridium perfringens par la technique de comptage perfringens durch Koloniezählverfahren (ISO 15213-
des colonies (ISO 15213-2:2023) 2:2023)
This European Standard was approved by CEN on 12 September 2023.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15213-2:2023 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 15213-2:2023) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2024, and conflicting national standards shall be
withdrawn at the latest by May 2024.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 7937:2004.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 15213-2:2023 has been approved by CEN as EN ISO 15213-2:2023 without any
modification.
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 15213-2:2023(E)
ISO 15213-2:2023(E)
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
ISO 15213-2:2023(E)
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
ISO 15213-2:2023(E)
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
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
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).
ISO draws attention to the possibility that the implementation of this document may involve the use
of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed
patent rights in respect thereof. As of the date of publication of this document, ISO had not received
notice of (a) patent(s) which may be required to implement this document. However, implementers are
cautioned that this may not represent the latest information, which may be obtained from the patent
database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all
such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
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
ISO 15213-2:2023(E)
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
ISO 15213-2:2023(E)
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).
These changes have a major impact on the performance characteristics of the method.
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INTERNATIONAL STANDARD ISO 15213-2:2023(E)
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
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
enumeration of Clostridium perfringens are only undertaken in properly equipped laboratories,
under the control of a skilled microbiologist, and that great care is taken in the disposal of all
incubated materials. Persons using this document should be familiar with normal laboratory
practice. This document does not purport to address all of the safety aspects, if any, associated
with its use. It is the responsibility of the user to establish appropriate safety and health
practices.
1 Scope
This document specifies the enumeration of Clostridium (C.) perfringens by colony-count technique.
This document is applicable to:
— products intended for human consumption;
— products for feeding animals;
— environmental samples in the area of food and feed production and handling;
— 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 (derivates);
— processed fruits and vegetables;
— infant formula and infant cereals;
— multi-component foods or meal components.
It has also been validated for the following other categories:
— pet food and animal feed;
— environmental samples (food or feed production).
As this method has been validated for at least five food categories, this method is applicable for a broad range of
food. For detailed information on the validation, see Clause 11 and Annex C. Since the method is not commonly
used for samples in the primary production stage, this category was not included in the interlaboratory study.
Therefore, no performance characteristics were obtained for this category.
This horizontal method was originally developed for the examination of all samples belonging to the
food chain. Based on the information available at the time of publication of this document, this method
is considered to be fully suited to the examination of all samples belonging to the food chain. However,
because of the large variety of products in the food chain, it is possible that this horizontal method is not
ISO 15213-2:2023(E)
appropriate in every detail for all products. Nevertheless, it is expected that the required modifications
are minimized so that they do not result in a significant deviation from this horizontal method.
This technique is suitable for, but not limited to, the enumeration of microorganisms in test samples
with a minimum of 10 colonies counted on a plate. This corresponds to a level of contamination that is
expected to be higher than 10 cfu/ml for liquid samples or higher than 100 cfu/g for solid samples.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of the food chain — General requirements and guidance for microbiological
examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
ISO 19036:2019, , Microbiology of the food chain — Estimation of measurement uncertainty for quantitative
determinations
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
presumptive C. perfringens
presumptive Clostridium perfringens
spore-forming bacteria forming countable typical colonies in a specific selective medium under obligate
anaerobic conditions
Note 1 to entry: Presumptive C. perfringens are spore-forming bacteria that are able to produce typical colonies
under the conditions specified in this document.
3.2
confirmed C. perfringens
confirmed Clostridium perfringens
bacteria that produce characteristic colonies in the specified selective medium under obligate anaerobic
conditions and possess the enzyme acid phosphatase
3.3
human pathogenic C. perfringens
human pathogenic Clostridium perfringens
confirmed C. perfringens strains (3.2) which possess the ability to produce C. perfringens enterotoxin
(CPE), encoded by the cpe gene
Note 1 to entry: The cpe gene can be located either chromosomally or on plasmids. These isolates are able to
produce CPE in the small intestine on sporulation and cause human illness.
ISO 15213-2:2023(E)
3.4
enumeration of C. perfringens
enumeration of Clostridium perfringens
determination of the number of colony-forming units (cfu) of confirmed C. perfringens (3.2) found per
gram, per millilitre, per square centimetres or per sampling device when a specified test is conducted
Note 1 to entry: Specified tests are given in Clause 9.
4 Principle
4.1 General
A specified quantity of the liquid test sample, or of an initial suspension in the case of other products,
is dispensed into an empty Petri dish and mixed well with a specified molten agar culture medium
to form a poured plate. Additional plates are prepared under the same conditions using decimal
dilutions of the test sample. After solidification of the agar medium, an overlay is used to prevent the
development of spreading colonies on the surface of the medium. If it is the intention to count only
spores, a heat treatment of 10 min at 80 °C is performed before plating. Additionally, a method for
molecular differentiation between human pathogenic and non-pathogenic C. perfringens strains is
described in Annex D.
When the number of cfu is expected to be at or near the limit of determination, the use of duplicate
plates is preferable. If duplicate plates are used, the minimum for the sum of colonies on both plates
should be 10 colonies. In this case, the level of contamination is expected to be higher than 5 cfu/ml for
liquid samples or higher than 50 cfu/g for solid samples.
A pour-plate technique with overlay is especially suited for the enumeration of products expected to
contain spreading colonies that can obscure colonies of the target microorganisms.
The enumeration of C. perfringens requires four successive stages as specified in the normative Annex A.
4.2 Preparation of dilutions
For the preparation of decimal dilutions from the test portion, follow the procedure as specified in the
ISO 6887 series.
4.3 Enumeration
Petri dishes are inoculated with a specified quantity of the test sample if the initial product is liquid,
or a specified quantity of the initial suspension, in the case of other products. Additional Petri dishes
are inoculated, under the same conditions, using decimal dilutions of the test sample or of the initial
suspension. A selective medium is added (pour-plate technique) and then overlaid with the same
medium.
The plates are incubated under anaerobic conditions at 37 °C for 20 h. After incubation, the number
of typical colonies, which show black or grey to yellow-brown staining, are counted. The colour of the
colonies and the surrounding zone changes due to the formation of iron(II)sulfide as a result of the
reaction between sulfide ions and trivalent iron [Fe(III)] present in the medium.
4.4 Confirmation
Confirmatory tests are carried out. The result is calculated as the colony count of confirmed
C. perfringens per sample volume. Additionally, the method described in Annex D can be used for
molecular differentiation between human pathogenic and non-pathogenic C. perfringens strains.
ISO 15213-2:2023(E)
5 Culture media and reagents
Follow current laboratory practices in accordance with ISO 7218. The composition of culture media
and reagents and their preparation are specified in Annex B. For performance testing of culture media,
follow the procedures in accordance with ISO 11133 and Annex B.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
The usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following shall be
used.
6.1 Appropriate apparatus for achieving an anaerobic atmosphere, a jar that can be hermetically
sealed or any other appropriate equipment which enables anaerobic atmosphere conditions to
be maintained for the total incubation time of the culture medium. Other systems of equivalent
performance, such as anaerobic cabinets, may be used. Follow the manufacturer’s instructions for
installation and maintenance.
The composition of the atmosphere required can be achieved by means of the addition of a gas mixture
(e.g. from a gas cylinder) after evacuation of air from the jar, by displacement of the atmosphere in
a cabinet or by any other appropriate means (such as commercially available gas packs). In general,
anaerobic incubation requires an atmosphere of less than 1 % volume fraction oxygen, 9 % volume
fraction to 13 % volume fraction carbon dioxide.
6.2 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).
6.3 Freezer, capable of operating at −20 °C ± 2 °C and at −70 °C ± 3 °C.
6.4 Incubator, capable of operating at 37 °C ± 1 °C.
6.5 pH-meter, having a maximum permissible error of calibration of ±0,1 pH unit at 25 °C.
6.6 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.7 Sterile bottles, flasks or tubes, of appropriate capacity. Bottles, flasks or tubes with non-toxic
metallic or plastic screw-caps may be used.
6.8 Sterile graduated pipettes or automatic pipettes, of nominal capacities 10 ml, 1 ml and 0,1 ml.
6.9 Sterile loops, of approximate diameter 3 mm (10 μl volume), and of 1 μl volume, or inoculation
needle or wire.
6.10 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
6.11 Thermostatically controlled water bath, capable of operating at 44 °C to 47 °C and 80 °C ± 2 °C.
7 Sampling
Sampling is not part of the method specified in this document. Follow the specific International
Standard dealing with the product concerned. If there is no specific International Standard dealing
with the sampling of the product concerned, it is recommended that the parties concerned come to an
agreement on this subject.
ISO 15213-2:2023(E)
Recommended sampling techniques are given in the following documents:
— ISO/TS 17728 for food and animal feed;
— ISO 707 for milk and milk products;
— ISO 6887-3 for raw molluscs, tunicates and echinoderms from primary production areas;
— ISO 13307 for primary production stage;
— ISO 17604 for carcasses;
— ISO 18593 for surfaces.
It is important that the laboratory receives a sample that is representative of the product under
consideration. The sample should not have been damaged or changed during transport or storage.
8 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International
Standard dealing with the product concerned. Follow the procedures as specified in the ISO 6887 series.
If there is no specific International Standard available, it is recommended that the parties concerned
come to an agreement on this subject.
9 Procedure
9.1 General
The procedure as given in Annex A shall be followed.
9.2 Test portion, initial suspension and dilutions
Follow the procedures in accordance with the ISO 6887 series and the specific International Standard
dealing with the product concerned. Prepare a single decimal dilution series from the test portion if the
product is liquid, or from the initial suspension in the case of other products.
9.3 Heat treatment to select spores
If it is the intention to count only spores, heat the decimal dilution series to 80 °C in a water bath
(6.11) for 10 min ± 1 min. Heat treatment shall be given within 15 min after preparation of the initial
suspension to avoid germination of spores. If the tube is not placed in the water bath within 15 min, it
should be placed immediately in melting ice for a maximum of 2 h.
The temperature during heat treatment should be monitored by placing an appropriate thermometer in
a reference bottle of the same size as the sample bottle and containing the same volume of water at the
same initial temperature as the sample being treated (6.7). The tubes should not be hermetically sealed
during the heat treatment. The time taken to reach 80 °C shall not exceed 5 min and can be minimized
by ensuring the water level to be at least 4 cm above the level of the sample and that the water bath is
equipped with a circulating-water pump to maximize heat exchange.
Start the time of heating (10 min) when the temperature of the reference sample has reached 80 °C.
After heat treatment, the samples should be cooled immediately till approximately 20 °C.
Heat treatment should also reduce the competitive flora in some matrices containing a high level of
background flora (e.g. liquid whey, silage).
ISO 15213-2:2023(E)
9.4 Inoculation and incubation
9.4.1 Take two sterile Petri dishes with a diameter of approximately 90 mm (6.10). Transfer to each
dish, by means of a sterile pipette (6.8), 1 ml of the test sample if liquid, or 1 ml of the initial suspension
−1
(10 dilution) in the case of other products. If plates from more than one dilution are prepared, this
may be reduced to one dish (see ISO 7218). When, for certain products, it is necessary to estimate low
numbers of C. perfringens, the limit of enumeration may be lowered by a factor of 10 by examining 10 ml
of the initial suspension in three large (140 mm) Petri dishes (6.10).
9.4.2 Take one other sterile Petri dish (6.10). Use another sterile pipette (6.8) to dispense 1 ml of the
−1 −2
10 dilution (liquid product) or 1 ml of the 10 dilution (other products).
9.4.3 If necessary, repeat the procedure with further dilutions, using a new sterile pipette (6.8) for
each decimal dilution.
9.4.4 If appropriate and possible, select only the critical dilution steps (at least two consecutive
decimal dilutions) for the inoculation of the Petri dishes (6.10) that will give colony counts of between
10 and 150 colonies per plate (on 90 mm Petri dishes) or between 10 and 365 colonies per plate (on
140 mm Petri dishes).
9.4.5 Pour about 12 ml to 15 ml for 90 mm Petri dishes or 30 ml to 35 ml for 140 mm Petri dishes of
the tryptose sulfite cycloserine agar (TSC agar) (see B.2), molten and tempered at 44 °C to 47 °C (6.11),
into each Petri dish (6.10).
9.4.6 Carefully mix the inoculum with the medium by rotating the Petri dishes and allow the mixture
to solidify by leaving the Petri dishes standing on a cool horizontal surface.
9.4.7 After complete solidification, pour about 5 ml of the TSC agar (see B.2) for 90 mm Petri dishes
(6.10) or 12 ml for 140 mm Petri dishes (6.10) as overlay, to prevent the development of spreading
colonies on the surface of the medium. Allow to solidify as specified in 9.4.6.
9.4.8 Invert the plates obtained in 9.4.7 and incubate (6.4) the plates at 37 °C in an anaerobic
atmosphere (6.1).
9.5 Enumeration of typical colonies
9.5.1 After 20 h ± 2 h of incubation, examine the plates (see 9.4.8) for presumptive C. perfringens.
Longer incubation can result in excess blackening of the plates.
Typical colonies, which show black or grey to yellow-brown staining (even if the colour is faint) on the
TSC agar, are counted.
Upon removal of the plates from the anaerobic atmosphere, plates shall be counted within 30 min as
the colour of the colonies can rapidly fade and disappear upon exposure to oxygen. If anaerobic jars are
used, the plates should be checked jar by jar or in small portions if the incubation was performed in an
anaerobic incubator (6.1, 6.4).
NOTE Diffuse, unspecific blackening of the medium can occur. The growth of anaerobic bacteria, which
produce hydrogen (not H S), can also reduce the sulfite present and lead to a general blackening of the medium,
which makes enumeration of typical colonies difficult.
9.5.2 Select the plates (see 9.5.1) containing less than 150 presumptive colonies (for 90 mm Petri
dishes) or less than 365 colonies (for 140 mm Petri dishes); count these colonies and record the
number of presumptive colonies per dish. Then choose, at random, five such colonies from each dish for
confirmation (see 9.6). For enumeration of plates with low or high numbers of presumptive colonies,
see ISO 7218.
ISO 15213-2:2023(E)
9.6 Confirmation of C. perfringens
9.6.1 Selection of colonies for confirmation
9.6.1.1 For confirmation, take five presumptive colonies from each dish retained for enumeration
(see 9.5.2). If more than one morphology is present among the colonies, select one of each morphology
for subculture and confirmation.
9.6.1.2 Streak each of the selected colonies with a sterile loop (6.9) onto one non-selective blood
agar plates, e.g. Columbia blood agar (see B.3). If blood is not available, Columbia agar base or another
nutrient-rich medium (e.g. tryptone soya agar or brain heart infusion agar) can be used.
Allow the plates to equilibrate at room temperature if they were stored at a lower temperature. If
necessary, dry the surface of the plates before use (see ISO 11133).
Several isolates can be streaked onto identified sectors. Streaks should obtain well-isolated colonies.
Incubate the plates in an anaerobic atmosphere (6.1) at 37 °C (6.4) for 20 h ± 2 h. Right after incubation,
select well-isolated freshly grown colonies for confirmation. Confirmation may be done either by the
acid phosphatase test or by the sulfite indole motility (SIM) agar test.
NOTE Alternative procedures (see ISO 7218) can be used to confirm the isolate(s) as C. perfringens, provided
that the suitability of the alternative procedure has been validated (see ISO 16140-4 or ISO 16140-6).
After incubation, these plates can be refrigerated at 5 °C (6.6) for a maximum of 48 h before reading.
For plates which were incubated anaerobically, maintain the anaerobic atmosphere.
9.6.2 Acid phosphatase test
9.6.2.1 It is known that, beside C. perfringens, some other Clostridium strains (e.g. some strains of
C. baratii) can produce acid phosphatase, but this ability is very limited. Therefore, only a very low
percentage of false positives is expected.
9.6.2.2 Colonies grown anaerobically on blood or nutrient agar plates are spread on filter paper
and 2 to 3 drops of the acid phosphatase reagent (B.4) are placed onto the colonies. If a commercially
available test kit is used, follow the manufacturer’s instructions.
NOTE It is possible to drip acid phosphatase reagent on colonies, if no further investigation of the colonies is
needed.
9.6.2.3 A purplish colour developed within 3 min to 4 min is considered as a positive reaction.
9.6.3 Sulfite indole motility (SIM) agar test
Colonies grown anaerobically on blood agar plates or nutrient agar plates are stabbed into SIM tubes
(B.5). The tubes are incubated for 22 h ± 2 h at 37 °C (6.4). After incubation the tubes are read for:
— sulfite production: tubes showing blackening are positive;
— motility: tubes showing growth outside the inoculation stab are positive;
— indole production: tubes giving a red coloured ring directly after adding Kovacs reagent (B.6) are
positive.
C. perfringens is positive for sulfite production and negative for indole production and motility.
ISO 15213-2:2023(E)
9.6.4 Differentiation between human pathogenic and non-pathogenic C. perfringens strains
(optional)
Additionally, the method described in Annex D can be used for molecular differentiation between
human pathogenic and non-pathogenic C. perfringens strains.
9.6.5 Interpretation
C. perfringens produces black or grey to yellow brown colonies on TSC agar, even if the colour is faint,
and possesses acid phosphatase, or is positive for sulfite production, negative for indole production and
motility in SIM agar.
10 Expression of results
For calculation of the results, follow the procedure(s) in accordance with ISO 7218. Calculate and report
the results as the number of confirmed C. perfringens or, if the method of Annex D was also used for
differentiation, of confirmed human pathogenic C. perfringens, in cfu per gram, per millilitre or per
square centimetre. When the sampled area is not known, report as per sampling device, such as a cloth,
sponge swab or stick.
If heat pre-treatment for the selection of spores (9.3) was used, the result is reported as number of
confirmed C. perfringens spores or, if the method of Annex D was also used for differentiation, of
confirmed human pathogenic C. perfringens spores in cfu per gram, per millilitre, per square centimetre
or per sampling device.
In cases when no typical colonies of C. perfringens have been detected, or when no typical colonies are
confirmed as C. perfringens, follow ISO 7218 for the expression of results for special cases.
11 Validation of the method
11.1 Interlaboratory study
Results of the interlaboratory study (step 6 in ISO 17468) to determine the performance characteristics
of the method are described in 11.2.
11.2 Performance characteristics
The performance characteristics of the method (repeatability and reproducibility standard
deviations) were determined in an interlaboratory study. It is possible that the values derived from the
interlaboratory study are not applicable to concentration ranges and (food) categories other than those
used in the study. All data are given in Annex C.
A summary of the interlaboratory repeatability standard deviation (s ) is given in Table 1.
r
ISO 15213-2:2023(E)
Table 1 — Summary of s values from the interlaboratory study
r
(Food) category (Food) item s values of low s values of medi- s values of high
r r r
inoculation level um inoculation inoculation level
level
Ready-to-eat, ready-to-reheat Corned beef 0,292 0,272 0,100
meat products
Ready-to-eat, ready-to-reheat Canned fish 0,204 0,193 0,035
fishery products
Multi-component foods or Instant soup 0,208 0,080 0,070
meal components
Infant formula and infant Powdered infant 0,160 0,200 0,098
cereals formula
Processed fruits and vege
...