SIST-TS CEN ISO/TS 15213-3:2024
(Main)Microbiology of the food chain - Horizontal method for the detection and enumeration of Clostridium spp. - Part 3: Detection of Clostridium perfringens (ISO/TS 15213-3:2024)
Microbiology of the food chain - Horizontal method for the detection and enumeration of Clostridium spp. - Part 3: Detection of Clostridium perfringens (ISO/TS 15213-3:2024)
This document specifies the detection of Clostridium (C.) perfringens.
This document is applicable to:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.
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.
NOTE Interlaboratory studies with a small number of participating laboratories (<10) were conducted for the following food categories:
— ready-to-eat, ready-to-reheat meat products;
— eggs and egg products (derivates);
— ready-to-eat, ready-to-reheat fishery products;
— processed fruits and vegetables;
— infant formula and infant cereals (with probiotics);
— multi-component foods or meal components.
It has also been validated with a small number of participating laboratories for the following other category:
— environmental samples (food or feed production).
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. The method has not been validated for the category ‘pet food and animal feed’, as the test samples used for the interlaboratory study were already naturally contaminated with C. perfringens. Given the limited number of participating laboratories in the interlaboratory studies, the calculated performance characteristics can be used as indicative values of the method performance. For detailed information on the validation, see Clause 11 and Annexes C to F.
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur Zählung von Clostridium spp. - Teil 3: Nachweis von Clostridium perfringens (ISO/TS 15213-3:2024)
Dieses Dokument legt den Nachweis von Clostridium (C.) perfringens fest.
Dieses Dokument ist anwendbar für:
- Produkte, die für den menschlichen Verzehr vorgesehen sind;
- Produkte, die als Futtermittel vorgesehen sind;
- Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln;
- Proben aus dem Bereich der Primärproduktion.
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.
ANMERKUNG Ringversuche mit einer geringen Anzahl von teilnehmenden Laboren (< 10) wurden für die folgenden Lebensmittelkategorien durchgeführt:
- verzehrfertige, aufwärmfertige Fleischprodukte;
- Eier und Eiprodukte (Derivate);
- verzehrfertige, aufwärmfertige Fischereiprodukte;
- verarbeitetes Obst und Gemüse;
- Säuglingsnahrung und Getreideprodukte für Kleinkinder (mit Probiotika);
- Mehrkomponenten-Lebensmittel bzw. Mahlzeitkomponenten.
Außerdem wurde eine Validierung mit einer geringen Anzahl von teilnehmenden Laboren für die folgende weitere Kategorie durchgeführt:
- Umgebungsproben (Lebensmittel- oder Futtermittelproduktion).
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. Für die Kategorie „(Heim )Tierfuttermittel“ wurde das Verfahren nicht validiert, da die für den Ringversuch verwendeten Untersuchungsproben bereits eine natürliche Kontamination mit C. perfringens aufwiesen. Angesichts der begrenzten Anzahl der Labore, die an dem Ringversuch teilgenommen haben, können die berechneten Leistungsmerkmale als indikative Werte für die Verfahrensleistung genutzt werden. Abschnitt 11 und Anhang C bis Anhang F enthalten detaillierte Informationen zur Validierung.
WARNUNG — Zum Schutz der Gesundheit des Laborpersonals ist es unerlässlich, dass Prüfungen zum Nachweis 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 3 : Recherche de Clostridium perfringens (ISO/TS 15213-3:2024)
Le présent document définit la recherche de Clostridium (C.) perfringens.
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.
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’entraînent pas de déviation significative de cette méthode horizontale.
NOTE Des études interlaboratoires avec un petit nombre de laboratoires participants (<10) ont été réalisées pour les catégories d’aliments suivants:
— les produits à base de viande prêts à consommer et prêts à réchauffer;
— les œufs et ovoproduits (dérivés);
— les produits à base de poisson prêts à consommer et prêts à réchauffer;
— les fruits et légumes transformés;
— les préparations et céréales pour nourrissons (avec probiotiques);
— les aliments composés et les composants de repas.
La méthode a également été validée avec un petit nombre de laboratoires participants pour la catégorie suivante:
— les échantillons environnementaux (production d’aliments ou d’aliments pour animaux).
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. La méthode n’a pas été validée pour la catégorie «aliments pour animaux», car les échantillons pour essai utilisés dans l’étude interlaboratoires étaient déjà naturellement contaminés par C. perfringens. Étant donné le nombre limité de laboratoires participants dans les études interlaboratoires, les caractéristiques de performance calculées peuvent être utilisées comme valeurs indicatives pour la performance de la méthode. Pour de plus amples informations sur la validation, voir l’Article 11 et les Annexes C à F.
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila Clostridium spp. - 3. del: Ugotavljanje prisotnosti Clostridium perfringens (ISO/TS 15213-3:2024)
Ta dokument določa ugotavljanje prisotnosti bakterije Clostridium (C.) perfringens. Ta del standarda ISO 15213 se uporablja za:
• proizvode, namenjene za prehrano ljudi;
• proizvode, namenjene za živalsko krmo;
• okoljske vzorce na področju proizvodnje hrane in krme; ter
• vzorce iz faze primarne proizvodnje.
Ta tehnika se uporablja, kadar se pričakuje več kot 10 kolonij na mililiter ali gram preskusnega vzorca.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-julij-2024
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Clostridium spp. - 3. del: Ugotavljanje prisotnosti Clostridium
perfringens (ISO/TS 15213-3:2024)
Microbiology of the food chain - Horizontal method for the detection and enumeration of
Clostridium spp. - Part 3: Detection of Clostridium perfringens (ISO/TS 15213-3:2024)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Clostridium spp. - Teil 3: Nachweis von Clostridium perfringens (ISO/TS
15213-3:2024)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Clostridium spp. - Partie 3 : Recherche de Clostridium perfringens
(ISO/TS 15213-3:2024)
Ta slovenski standard je istoveten z: CEN ISO/TS 15213-3:2024
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
CEN ISO/TS 15213-3
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
May 2024
TECHNISCHE SPEZIFIKATION
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for the
detection and enumeration of Clostridium spp. - Part 3:
Detection of Clostridium perfringens (ISO/TS 15213-
3:2024)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche et le dénombrement de Verfahren zum Nachweis und zur Zählung von
Clostridium spp. - Partie 3: Recherche de Clostridium Clostridium spp. - Teil 3: Nachweis von Clostridium
perfringens (ISO/TS 15213-3:2024) perfringens (ISO/TS 15213-3:2024)
This Technical Specification (CEN/TS) was approved by CEN on 6 February 2024 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.
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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 15213-3:2024 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (CEN ISO/TS 15213-3:2024) 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.
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 has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
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 announce this Technical Specification: 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/TS 15213-3:2024 has been approved by CEN as CEN ISO/TS 15213-3:2024 without any
modification.
Technical
Specification
ISO/TS 15213-3
First edition
Microbiology of the food chain —
2024-05
Horizontal method for the
detection and enumeration of
Clostridium spp. —
Part 3:
Detection of Clostridium perfringens
Microbiologie de la chaîne alimentaire — Méthode horizontale
pour la recherche et le dénombrement de Clostridium spp. —
Partie 3: Recherche de Clostridium perfringens
Reference number
ISO/TS 15213-3:2024(en) © ISO 2024
ISO/TS 15213-3:2024(en)
© ISO 2024
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
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO/TS 15213-3:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 2
4 Principle . 3
4.1 General .3
4.2 Enrichment in selective liquid medium . .3
4.3 Isolation on selective solid medium .3
4.4 Confirmation .3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure . 5
9.1 General .5
9.2 Test portion and initial suspension .5
9.3 Selective enrichment .5
9.4 Isolation .5
9.5 Confirmation of C. perfringens .5
9.5.1 Selection of colonies for confirmation .5
9.5.2 Acid phosphatase test .6
9.5.3 Sulfite indole motility (SIM) agar test .6
9.5.4 Differentiation between human pathogenic and non-pathogenic C. perfringens
strains (optional) .6
9.5.5 Interpretation .7
10 Expression of results . 7
11 Indicative performance characteristics of the method. 7
11.1 Validation based on principles of ISO 17468 .7
11.2 Indicative performance characteristics .7
12 Test report . 9
13 Quality assurance . 10
Annex A (normative) Flow diagram of the procedure .11
Annex B (normative) Culture media and reagents .12
Annex C (informative) Indicative performance characteristics of the method using TSC
isolation agar and acid phosphatase confirmation test .21
Annex D (informative) Indicative performance characteristics of the method using TSC
isolation agar and SIM agar test .24
Annex E (informative) Indicative performance characteristics of the method using LENA
isolation agar and acid phosphatase confirmation test .27
Annex F (informative) Indicative performance characteristics of the method using LENA
isolation agar and SIM agar confirmation test .30
Annex G (informative) Molecular differentiation between pathogenic and non-pathogenic
Clostridium perfringens .33
Bibliography .34
iii
ISO/TS 15213-3:2024(en)
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 document 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).
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.
iv
ISO/TS 15213-3:2024(en)
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 related to C. perfringens are mostly 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 detection 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 and ISO 15213-2 describes the method for
the enumeration of C. perfringens. 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.
v
Technical Specification ISO/TS 15213-3:2024(en)
Microbiology of the food chain — Horizontal method for the
detection and enumeration of Clostridium spp. —
Part 3:
Detection of Clostridium perfringens
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for the
detection 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 detection of Clostridium (C.) perfringens.
This document is applicable to:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.
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.
NOTE Interlaboratory studies with a small number of participating laboratories (<10) were conducted for the
following food categories:
— ready-to-eat, ready-to-reheat meat products;
— eggs and egg products (derivates);
— ready-to-eat, ready-to-reheat fishery products;
— processed fruits and vegetables;
— infant formula and infant cereals (with probiotics);
— multi-component foods or meal components.
It has also been validated with a small number of participating laboratories for the following other category:
— environmental samples (food or feed production).
ISO/TS 15213-3:2024(en)
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. The method has
not been validated for the category ‘pet food and animal feed’, as the test samples used for the interlaboratory study
were already naturally contaminated with C. perfringens. Given the limited number of participating laboratories in
the interlaboratory studies, the calculated performance characteristics can be used as indicative values of the method
performance. For detailed information on the validation, see Clause 11 and Annexes C to F.
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
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 typical colonies on 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 on the specified selective medium under obligate anaerobic
conditions and either possess the enzyme acid phosphatase, or are able to produce sulfite, are not able to
produce indole and are not motile (SIM agar)
3.3
human pathogenic C. perfringens
human pathogenic Clostridium perfringens
confirmed C. perfringens (3.2) strains which possess the ability to produce C. perfringens enteroxin (CPE),
encoded by the cpe gene
Note 1 to entry: The cpe gene can be located either chromosomally or on large plasmids. These isolates are able to
produce CPE in the small intestine on sporulation and cause human illness.
ISO/TS 15213-3:2024(en)
3.4
detection of C. perfringens
detection of Clostridium perfringens
determination of confirmed C. perfringens (3.2) in a particular mass, volume of product, on a surface area or
object, when a specified test is conducted
Note 1 to entry: Specified tests are given in Clause 9.
4 Principle
4.1 General
The detection of C. perfringens requires three successive stages as specified in Annex A.
4.2 Enrichment in selective liquid medium
A selective culture medium (at ambient temperature) is inoculated with a specified quantity of the test
sample if the initial product is liquid, or a specified quantity of an initial suspension in the case of other
products. The inoculated selective medium is incubated at 46 °C for 18 h.
4.3 Isolation on selective solid medium
From the cultures obtained in 4.2, two selective plating media are inoculated. The plates are incubated at
37 °C and at 46 °C respectively for 24 h anaerobically.
4.4 Confirmation
Confirmatory tests are carried out. The result is expressed as C. perfringens detected or not detected per
sample volume. Additionally, the method mentioned in Annex G can be used for molecular differentiation
between non-pathogenic and human pathogenic C. perfringens strains.
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 apparatus (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 Drying cabinet or oven, ventilated, capable of operating between 25 °C and 50 °C.
ISO/TS 15213-3:2024(en)
6.4 Freezers, capable of operating at -20 °C ± 2 °C and below -70 °C.
6.5 Incubator(s), capable of operating at 37 °C ± 1 °C, 46 °C ± 1 °C.
6.6 pH-meter, having an accuracy of calibration of ±0,1 pH unit at 25 °C.
6.7 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.8 Sterile bottles, flasks or tubes, of appropriate capacity. Bottles, flasks or tubes with non-toxic
metallic of plastic screw-caps may be used.
6.9 Sterile graduated pipettes or automatic pipettes, of nominal capacities of 10 ml and 1 ml.
6.10 Sterile loops, of approximate diameter of 3 mm (10 µl volume) and of 1 µl volume, or inoculation
needle or wire.
6.11 Sterile Petri dishes, with a diameter of approximately 90 mm.
6.12 Thermostatically controlled water bath, capable of operating at 44 °C to 47 °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.
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.
ISO/TS 15213-3:2024(en)
9 Procedure
9.1 General
The procedure as given in Annex A shall be followed.
9.2 Test portion and initial suspension
Follow the procedures in accordance with the ISO 6887 series and the specific International Standard
dealing with the product concerned.
Prepare the initial suspension in the case the product of concern is not liquid. Add 1 ml (6.9) of the liquid
sample or 1 ml (6.9) of the initial suspension (0,1 g product) to 9 ml of rapid perfringens medium (RPM, B.2).
Alternatively, 10 ml (6.9) of the liquid sample or of the initial suspension (1 g product) is added to 90 ml of
RPM (B.12).
It is possible to composite or pool samples of the same type, to reduce workload when a large number of
samples are required to be examined. This can be necessary to reflect microbiological quality of a large
batch of product of environmental samples or required by regional legislation.
Similarly, a number of test portions may be pooled and examined together in larger quantities of media, or
the (pre)enrichment cultures from the test portions may be pooled and carried out as a single test. These
pooling procedures are described in ISO 6887-1. Whether it is possible to pool samples of a certain type shall
be verified according to the protocol described in ISO 6887-1.
NOTE Validation of this method can be conducted according to the appropriate documents in ISO 16140 (all parts).
9.3 Selective enrichment
Incubate the selective enrichment broth RPM in closed tubes or bottles (9.2) at 46 °C (6.5) for 18 h ± 2 h.
9.4 Isolation
Allow the selective plating media (B.3 and B.4) to equilibrate at room temperature if they were stored at a
lower temperature. If necessary, dry the surfaces of the plates (see ISO 11133) in a drying cabinet or oven
(6.3) before use.
From the selective enrichment obtained at 9.3, inoculate by means of a 10 µl loop (6.10) the surface of a
Petri dish (6.11) containing the selective medium tryptose sulfite cycloserine agar (TSC agar, B.3) and a
Petri dish (6.11) containing the selective medium Lactose egg-yolk neomycin agar (LENA, B.4).
Incubate the TSC agar plates anaerobically (6.1) in an incubator (6.5) at 37 °C for 24 h ± 2 h. Incubate the
LENA plates anaerobically (6.1) in an incubator (6.5) at 46 °C for 24 h ± 2 h.
NOTE After inoculation of the TSC agar plates an overlay of TSC agar can be used to prevent the development of
spreading colonies on the surface of the medium. Pour about 5 ml of the TSC medium (see B.3) as overlay and allow to
solidify by leaving the Petri dishes standing on a cool horizontal surface.
9.5 Confirmation of C. perfringens
9.5.1 Selection of colonies for confirmation
9.5.1.1 Typical colonies on TSC agar are black or grey to yellow-brown staining, even if the colour is faint.
Typical colonies on LENA show yellow colour (acid fermentation from lactose) and precipitation (lecithinase
reaction).
Upon removal of the TSC agar plates from the anaerobic atmosphere, plates shall be read within 30 min as
the colour of the colonies can rapidly fade and disappear upon exposure to oxygen. If anaerobic jars are used,
ISO/TS 15213-3:2024(en)
the plates should be checked jar by jar or in small portions if the incubation was performed in an anaerobic
incubator (6.1, 6.5).
For confirmation, take five presumptive C. perfringens colonies from each dish containing typical colonies
(see 9.4). If more than one morphology is present among the colonies, select one of each morphology for
subculture and confirmation.
9.5.1.2 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).
Streak each of the selected colonies with a sterile loop (6.10) onto one non-selective blood agar plate, e.g.
Columbia blood agar (B.5). 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 with or without blood. Several isolates can be
streaked onto identified sectors of a non-selective agar plates. Streaks should obtain well-isolated colonies.
Incubate the plates in an anaerobic atmosphere (6.1) at 37 °C (6.5) 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 (9.5.2) or by the sulfite indole motility (SIM) agar test (9.5.3).
NOTE Alternative procedures (see ISO 7218) can be used to confirm whether the typical colonies are 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 (6.7) for a maximum of 48 h before reading. For
plates which were incubated anaerobically, maintain the anaerobic atmosphere.
9.5.2 Acid phosphatase test
9.5.2.1 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.
9.5.2.2 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.6) 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.
9.5.2.3 A purplish colour developed within 3 min to 4 min is considered as a positive reaction.
9.5.3 Sulfite indole motility (SIM) agar test
Colonies grown anaerobically on blood or nutrient agar plates are stabbed into SIM agar tubes (B.7). The
tubes are incubated for 22 h ± 2 h at 37 °C (6.5), in an anaerobic atmosphere (6.1) with the caps of the SIM
agar tubes loosened. 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.8) are positive
C. perfringens is positive for sulfite production and negative for indole production and motility.
9.5.4 Differentiation between human pathogenic and non-pathogenic C. perfringens strains
(optional)
Additionally, the method described in Annex G can be used for molecular differentiation between human
pathogenic and non-pathogenic C. perfringens strains.
ISO/TS 15213-3:2024(en)
9.5.5 Interpretation
C. perfringens produces black or grey to yellow-brown staining on TSC agar, even if the colour is faint,
and possesses acid phosphatase, or are positive for sulfite production, negative for indole production and
mobility on SIM agar.
10 Expression of results
In accordance with the interpretation of the results, indicate C. perfringens detected or not detected in a test
portion of x g or x ml of product, or on the surface area swabbed or per sampling device.
11 Indicative performance characteristics of the method
11.1 Validation based on principles of ISO 17468
Using the standardized reference method, an interlaboratory study with a small number of participating
laboratories (<10) was conducted based on the principles of ISO 17468.
The indicative performance characteristics of the method as derived from the interlaboratory study are
described in 11.2.
NOTE In this document, the words “category”, “item”, “matrix” and ”type” are combined with “food” to improve
the clarity of this document. However, the word ”food” is interchangeable with “feed” and the other areas of the food
chain as mentioned in Clause 1 of this document.
11.2 Indicative performance characteristics
The indicative performance characteristics of the method (specificity, sensitivity, LOD ) were determined
in interlaboratory studies. All data are given in Annex C to F. It is possible that the values derived from the
interlaboratory studies are not applicable to (food) categories other than those used in the study.
A summary of the indicative LOD values is given in Tables 1 till 4.
ISO/TS 15213-3:2024(en)
Table 1 — Summary of the indicative LOD values from the interlaboratory study for TSC isolation
agar and acid phosphatase as confirmation method
(Food) category (Food) item LOD Test portion size Strain used in the interlab-
oratory study
in cfu/test portion
Ready-to-eat, ready- Canned meat 2,9 (1,7 – 5,1) 1 gram Clostridium perfringens
a
to-reheat meat prod- CECT 4110
ucts
Eggs and egg products Egg powder 0,9 (0,4 – 2,3) 1 gram Clostridium perfringens NCTC
13170 (WDCM 00201)
Ready-to-eat, ready- Canned fish 3,2 (1,3 – 7,6) 1 gram Clostridium perfringens CECT
to-reheat fishery 4110
products
Processed fruits and Canned 1,0 (0,6 – 1,5) 1 gram Clostridium perfringens NCTC
vegetables 13170 (WDCM 00201)
pineapple
Infant formula and Infant formula 4,9 (3,1 – 7,6) 1 gram Clostridium perfringens CECT
infant cereals with probiotics 4110
Multi-component Instant soup 0,5 (0,2 – 1,4) 1 gram Clostridium perfringens NCTC
foods or meal compo- 13170 (WDCM 00201)
nents
Environmental Swabs 1,7 (0,7 – 4,4) Cloth Clostridium perfringens CECT
samples (food or feed 4110
production)
a
Colección Española de Cultivos Tipo (CECT)
Table 2 — Summary of the indicative LOD values from the interlaboratory study for TSC isolation
agar and SIM agar test as confirmation method
(Food) category (Food) item LOD Test portion size Strain used in the interlab-
oratory study
in cfu/test portion
Ready-to-eat, ready- Canned meat 2,9 (1,7 – 5,1) 1 gram Clostridium perfringens CECT
to-reheat meat prod- 4110
ucts
Eggs and egg products Egg powder 2,0 (0,9 – 5,1) 1 gram Clostridium perfringens NCTC
13170 (WDCM 00201)
Ready-to-eat, ready- Canned fish 3,0 (1,2 – 7,2) 1 gram Clostridium perfringens CECT
to-reheat fishery 4110
products
Processed fruits and Canned 1,1 (0,8 – 1,7) 1 gram Clostridium perfringens NCTC
vegetables 13170 (WDCM 00201)
pineapple
Infant formula and Infant formula 2,9 (1,9 – 4,5) 1 gram Clostridium perfringens CECT
infant cereals with probiotics 4110
Multi-component Instant soup 0,5 (0,2 – 1,4) 1 gram Clostridium perfringens NCTC
foods or meal compo- 13170 (WDCM 00201)
nents
Environmental Swabs 2,5 (1,4 – 4,5) Cloth Clostridium perfringens CECT
samples (food or feed 4110
production)
ISO/TS 15213-3:2024(en)
Table 3 — Summary of the indicative LOD values from the interlaboratory study for LENA isolation
agar and acid phosphatase as confirmation method
(Food) category (Food) item LOD Test portion size Strain used in the interlab-
oratory study
in cfu/test portion
Ready-to-eat, ready- Canned meat 3,0 (1,9 – 4,7) 1 gram Clostridium perfringens CECT
to-reheat meat prod- 4110
ucts
Eggs and egg products Egg powder 0,7 (0,3 – 1,9) 1 gram Clostridium perfringens NCTC
13170 (WDCM 00201)
Ready-to-eat, ready- Canned fish 3,5 (2,0 – 6,1) 1 gram Clostridium perfringens CECT
to-reheat fishery 4110
products
Processed fruits and Canned 1,0 (0,6 – 1,6) 1 gram Clostridium perfringens NCTC
vegetables 13170 (WDCM 00201)
pineapple
Infant formula and Infant formula 3,6 (2,5 – 5,2) 1 gram Clostridium perfringens CECT
infant cereals with probiotics 4110
Multi-component Instant soup 0,8 (0,3 – 2,0) 1 gram Clostridium perfringens NCTC
foods or meal compo- 13170 (WDCM 00201)
nents
Environmental Swabs 2,1 (1,2 – 3,8) Cloth Clostridium perfringens CECT
samples (food or feed 4110
production)
Table 4 — Summary of the indicative LOD values from the interlaboratory study for LENA isolation
agar and SIM agar test as confirmation method
(Food) category (Food) item LOD Test portion size Strain used in the interlab-
oratory study
in cfu/test portion
Ready-to-eat, ready- Canned meat 3,0 (1,9 – 4,7) 1 gram Clostridium perfringens CECT
to-reheat meat prod- 4110
ucts
Eggs and egg products Egg powder 1,9 (0,8 – 4,5) 1 gram Clostridium perfringens NCTC
13170 (WDCM 00201)
Ready-to-eat, ready- Canned fish 5,3 (3,0 – 9,3) 1 gram Clostridium perfringens CECT
to-reheat fishery 4110
products
Processed fruits and Canned 1,3 (0,9 – 1,9) 1 gram Clostridium perfringens NCTC
vegetables 13170 (WDCM 00201)
pineapple
Infant formula and Infant formula 3,5 (2,5 – 4,8) 1 gram Clostridium perfringens CECT
infant cereals with probiotics 4110
Multi-component Instant soup 1,3 (0,8 – 2,0) 1 gram Clostridium perfringens NCTC
foods or meal compo- 13170 (WDCM 00201)
nents
Environmental Swabs 2,0 (1,1 – 3,7) Cloth Clostridium perfringens CECT
samples (food or feed 4110
production)
12 Test report
The test report shall specify at least the following:
— the test method used, with reference to this document, i.e. ISO/TS 15213-3:2024;
— the sampling method used, if known;
— the size of the test portion and/or the nature of the subject examined;
ISO/TS 15213-3:2024(en)
— all operating conditions not specified in this document, or regarded as optional or informative (including
informative annexes), together with details of any incidents which can have influenced the test result(s);
— any deviations from this document;
— all information necessary for the complete identification of the sample;
— the test result(s) obtained;
— the date of the test.
13 Quality assurance
The laboratory should have a quality control system to ensure tha
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