SIST EN ISO 15213-1:2023
(Main)Microbiology of the food chain - Horizontal method for the detection and enumeration of Clostridium spp. - Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-count technique (ISO 15213-1:2023)
Microbiology of the food chain - Horizontal method for the detection and enumeration of Clostridium spp. - Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-count technique (ISO 15213-1:2023)
This document specifies the detection of Clostridium perfringens. This part of ISO 15213 is applicable to:
• products intended for human consumption;
• products intended for animal feeding;
• environmental samples in the area of food and feed production, handling, and
• samples from the primary production stage.
This method is applicable when the number sought is expected to be below 100 per ml or per g of the test sample.
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur Zählung von Clostridium spp. - Teil 1: Zählung von sulfitreduzierenden Clostridium spp. durch Koloniezählverfahren (ISO 15213-1:2023)
Dieses Dokument legt die Zählung von sulfitreduzierenden Clostridium spp. mit dem Koloniezählverfahren fest.
Dieses Dokument gilt für:
Produkte, die für den menschlichen Verzehr vorgesehen sind;
Produkte für das Füttern von Tieren;
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 Spek-trum 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 Lebensmittel-kette 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 die erforderlichen Änderungen so gering sind, dass sie nicht zu einer signifikanten Abweichung von diesem horizontalen Verfahren führen.
Dieses Verfahren ist für die Zählung von mindestens 10 Mikroorganismen-Kolonien auf einer Platte in Unter-suchungsproben geeignet, aber nicht darauf beschränkt. Dies entspricht einem erwarteten Kontaminations-grad, der bei flüssigen Proben höher als 10 KBE/ml und bei festen Proben höher als 100 KBE/g ist.
WARNHINWEIS — Zum Schutz der Gesundheit des Laborpersonals ist es unerlässlich, dass Prüfungen mit der Auszählung sulfitreduzierender Clostridium spp. nur in Laboratorien mit geeigneter Aus-stattung und unter der Leitung eines qualifizierten Mikrobiologen erfolgt und dass bei der Entsorgung allen inkubierten 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 Anwen-dung 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 1: Dénombrement des bactéries Clostridium spp. sulfito-réductices par la technique de comptage des colonies (ISO 15213-1:2023)
Le présent document spécifie le dénombrement des bactéries Clostridium spp. sulfito-réductrices 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; et
— aux échantillons prélevés au 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 ou les composants de repas.
Elle a également été validée pour les 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. - 1. del: Preštevanje Clostridium spp., ki reducirajo sulfit, s tehniko štetja kolonij (ISO 15213-1:2023)
Ta dokument določa zaznavanje bakterije Clostridium perfringens. Ta del standarda ISO 15213 se uporablja za:
• proizvode, namenjene za prehrano ljudi;
• proizvode, namenjene za živalsko krmo;
• okoljske vzorce na področju proizvodnje in ravnanja s hrano in krmo; ter
• vzorce iz faze primarne proizvodnje.
Ta metoda se uporablja, kadar se pričakuje število manjše kot 100 na mililiter ali gram preskusnega vzorca.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 15213-1:2023
01-maj-2023
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Clostridium spp. - 1. del: Preštevanje Clostridium spp., ki
reducirajo sulfit, s tehniko štetja kolonij (ISO 15213-1:2023)
Microbiology of the food chain - Horizontal method for the detection and enumeration of
Clostridium spp. - Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-
count technique (ISO 15213-1:2023)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Clostridium spp. - Teil 1: Zählung von sulfitreduzierenden Clostridium spp.
durch Koloniezählverfahren (ISO 15213-1:2023)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Clostridium spp. - Partie 1: Dénombrement des bactéries Clostridium
spp. sulfito-réductices par la technique de comptage des colonies (ISO 15213-1:2023)
Ta slovenski standard je istoveten z: EN ISO 15213-1:2023
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 15213-1:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 15213-1:2023
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SIST EN ISO 15213-1:2023
EN ISO 15213-1
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2023
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for the
detection and enumeration of Clostridium spp. - Part 1:
Enumeration of sulfite-reducing Clostridium spp. by
colony-count technique (ISO 15213-1:2023)
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 1: Dénombrement des Clostridium spp. - Teil 1: Zählung von
bactéries Clostridium spp. sulfito-réductices par la sulfitreduzierenden Clostridium spp. durch
technique de comptage des colonies (ISO 15213- Koloniezählverfahren (ISO 15213-1:2023)
1:2023)
This European Standard was approved by CEN on 24 December 2022.
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 15213-1:2023 E
worldwide for CEN national Members.
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SIST EN ISO 15213-1:2023
EN ISO 15213-1:2023 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 15213-1:2023
EN ISO 15213-1:2023 (E)
European foreword
This document (EN ISO 15213-1:2023) 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 August 2023, and conflicting national standards shall
be withdrawn at the latest by August 2023.
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 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 15213-1:2023 has been approved by CEN as EN ISO 15213-1:2023 without any
modification.
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SIST EN ISO 15213-1:2023
INTERNATIONAL ISO
STANDARD 15213-1
First edition
2023-01
Microbiology of the food chain —
Horizontal method for the detection
and enumeration of Clostridium
spp. —
Part 1:
Enumeration of sulfite-reducing
Clostridium spp. by colony-count
technique
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Clostridium spp. —
Partie 1: Dénombrement des bactéries Clostridium spp. sulfito-
réductrices par la technique de comptage des colonies
Reference number
ISO 15213-1:2023(E)
© ISO 2023
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SIST EN ISO 15213-1:2023
ISO 15213-1:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
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SIST EN ISO 15213-1:2023
ISO 15213-1:2023(E)
Contents Page
Foreword .iv
Introduction . vi
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Preparation of dilutions . 3
4.3 Enumeration . 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, initial suspension and dilutions . 5
9.3 Heat treatment to select spores . 5
9.4 Inoculation and incubation. 5
9.5 Enumeration of typical colonies . 6
9.6 Confirmation of sulfite-reducing Clostridium spp. . 6
10 Expression of results . 7
11 Validation of the method . 7
11.1 Interlaboratory study . 7
11.2 Performance characteristics . 7
12 Test report . 8
13 Quality assurance . 8
Annex A (normative) Flow diagram of the procedure . 9
Annex B (normative) Culture media and reagents .11
Annex C (informative) Performance characteristics of the method .14
Annex D (informative) Special protocol for the enumeration of sulfite-reducing
Clostridium spp. in feed .17
Bibliography .21
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SIST EN ISO 15213-1:2023
ISO 15213-1:2023(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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
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 15213-1 cancels and replaces ISO 15213:2003, which has been technically
revised.
The main changes are as follows:
— the Scope has been expanded to include samples from the primary production stage;
— the scope of the method has been changed from “sulfite-reducing bacteria” to “sulfite-reducing
Clostridium spp.”: therefore, typical colonies on the iron sulfite agar plates are confirmed;
— the concentration of sulfite in the iron sulfite agar has been reduced from 1,0 g/l to 0,5 g/l;
— 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 sulfite-reducing Clostridium spp. present in the sample;
— the option for using tubes for inoculation has been removed;
— the option for incubating the samples at 50 °C for the enumeration of thermophilic sulfite-reducing
bacteria has been removed;
— a description of how the confirmation of typical colonies has to be performed has been added;
— the flow diagram in Annex A giving a short description of the procedure has been revised;
— in Annex C, the performance characteristics have been added;
— Annex D has been added to provide a special protocol for the enumeration of sulfite-reducing
Clostridium spp. in feed.
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ISO 15213-1:2023(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.
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Introduction
Sulfite-reducing Clostridium spp. are obligate anaerobic, Gram-positive, spore-forming, rod-shaped
bacteria. The most important species which belong to this group are Clostridium (C.) perfringens,
C. bifermentans, C. sporogenes and C. botulinum. Some species can cause foodborne illness. As ubiquitous
bacteria they are predominantly found in nature. The Clostridium species inhabit soils and the intestinal
tract of animals and humans.
Sulfite-reducing Clostridium spp., including C. perfringens, are widely used as microbial indicators of
clostridial contamination in the manufacturing of foods (e.g. meat production). These have the capacity
to produce heat-resistant spores. Outside the dairy industry, the use of sulfite-reducing Clostridium
spp. as a microbial indicator is limited to a relatively small number of foods. Its current application
in non-dairy foods is either an indication of faecal contamination (especially C. perfringens, see also
ISO 15213-2 and ISO/TS 15213-3) and/or as an indicator of sanitation/process control related to
potential growth and survival of anaerobic spore-forming bacteria.
This document describes the horizontal method for the enumeration of sulfite-reducing Clostridium spp.
in food, feed, environmental samples and samples from the primary production stage. The method
for the enumeration of C. perfringens is described in ISO 15213-2. 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 15213:2003, are considered as major (see ISO 17468).
These changes have a major impact on the performance characteristics of the method.
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SIST EN ISO 15213-1:2023
INTERNATIONAL STANDARD ISO 15213-1:2023(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Clostridium spp. —
Part 1:
Enumeration of sulfite-reducing Clostridium spp. by
colony-count technique
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
the enumeration of sulfite-reducing Clostridium spp. 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 sulfite-reducing Clostridium spp. by the 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
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ISO 15213-1:2023(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
sulfite-reducing Clostridium spp.
genus of microorganisms of the family of Clostridiaceae, usually capable of growth in/on iron sulfite
agar (ISA) under anaerobic conditions, forming typical or less typical colonies, and displaying certain
characteristics with biochemical confirmation tests
Note 1 to entry: The biochemical confirmation tests are described in 9.6.
3.2
enumeration of sulfite-reducing Clostridium spp.
determination of the number of colony-forming units (cfu) of sulfite-reducing Clostridium spp. (3.1)
bacteria per gram, per millilitre, per square centimetre 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. Other plates are prepared under the same conditions using decimal dilutions
of the test sample. After solidification of the agar culture medium, an overlay is used to prevent the
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ISO 15213-1:2023(E)
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 needs to be performed before plating.
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 sulfite-reducing Clostridium spp. requires four successive stages as specified in
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
The plates are incubated under anaerobic conditions at 37 °C for 48 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
Typical colonies are picked for confirmation.
NOTE When no confirmation is performed, the results can be reported as “anaerobic sulfite-reducing
bacteria”.
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.
3
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SIST EN ISO 15213-1:2023
ISO 15213-1:2023(E)
6.2 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).
6.3 Incubator, capable of operating at 37 °C ± 1 °C.
6.4 pH-meter, having an accuracy of calibration of ± 0,1 pH unit at 25 °C.
6.5 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.6 Sterile bottles, flasks or tubes, of appropriate capacity. Bottles, flasks or tubes with non-toxic
metallic or plastic screwcaps may be used.
6.7 Sterile graduated pipettes or automatic pipettes, of nominal capacities of 10 ml and 1 ml.
6.8 Sterile loops, of approximately 1 µl volume, or inoculation needle or wire.
6.9 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
6.10 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 dea
...
SLOVENSKI STANDARD
oSIST prEN ISO 15213-1:2021
01-november-2021
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Clostridium spp. - 1. del: Preštevanje Clostridium spp., ki
reducirajo sulfit, s tehniko štetja kolonij (ISO/DIS 15213-1:2021)
Microbiology of the food chain - Horizontal method for the detection and enumeration of
Clostridium spp. - Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-
count technique (ISO/DIS 15213-1:2021)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Clostridium spp. - Teil 1: Zählung von Sulfit reduzierenden Clostridium spp.
durch Koloniezählverfahren (ISO/DIS 15213 1:2021)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Clostridium spp. - Partie 1: Dénombrement de Clostridium spp.
sulfito-réducteur par la technique par comptage des colonies (ISO/DIS 15213-1:2021)
Ta slovenski standard je istoveten z: prEN ISO 15213-1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 15213-1:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 15213-1:2021
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oSIST prEN ISO 15213-1:2021
DRAFT INTERNATIONAL STANDARD
ISO/DIS 15213-1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2021-09-13 2021-12-06
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Clostridium spp. —
Part 1:
Enumeration of sulfite-reducing Clostridium spp. by
colony-count technique
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche et le dénombrement de
Clostridium spp. —
Partie 1: Dénombrement de Clostridium spp. sulfito-réducteur par la technique par comptage des colonies
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 15213-1:2021(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2021
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oSIST prEN ISO 15213-1:2021
ISO/DIS 15213-1:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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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Email: copyright@iso.org
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Published in Switzerland
ii © ISO 2021 – All rights reserved
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oSIST prEN ISO 15213-1:2021
ISO/DIS 15213-1:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Preparation of dilutions . 3
4.3 Enumeration . 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. 4
9.1 General . 4
9.2 Test portion, initial suspension and dilutions . 4
9.3 Heat pre-treatment to select spores . 4
9.4 Inoculation and incubation . 5
9.5 Enumeration of typical colonies . 5
9.6 Confirmation of sulfite-reducing Clostridium spp . 6
10 Expression of results . 6
10.1 General . 6
10.2 Enumeration of sulfite-reducing Clostridium spp . 6
11 Performance characteristics of the method . 6
11.1 Interlaboratory study . 6
11.2 Repeatability limit . 7
11.3 Reproducibility limit . 7
12 Test report . 8
13 Quality assurance . 8
Annex A (normative) Flow diagram of the procedure . 9
Annex B (normative) Culture media and reagents .11
Annex C (informative) Method validation studies and performance characteristics .14
Annex D (informative) Special protocol for the enumeration of sulfite-reducing Clostridium
spp. in feed .18
Bibliography .22
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oSIST prEN ISO 15213-1:2021
ISO/DIS 15213-1:2021(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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
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.
This first edition of ISO 15213-1, together with ISO 15213-2 and ISO 15213-3, cancels and replaces
ISO 15213:2003 and ISO 7937:2004, which have been technically revised. The main changes in this first
edition, compared to ISO 15213:2003, are the following:
— the scope is enlarged to samples from the primary production stage;
— the scope of the method is changed from bacteria to Clostridium spp.; therefore, typical colonies on
the iron sulfite agar plates are confirmed;
— the concentration of sulfite in the Iron Sulfite Agar (ISA) is reduced from 1,0 g/l into 0,5 g/l;
— the heat treatment of 10 min at 80 °C is optional, in case of high background flora, or for the
enumeration of only spores of sulfite-reducing Clostridium spp. present in the sample;
— the option for using tubes is removed;
— the option for incubating the samples at 50 °C for the enumeration of thermophilic sulfite-reducing
bacteria is removed;
— a description is given how the confirmation of typical colonies has to be performed;
— in Annex C the performance characteristics are given;
— Annex D provides a special protocol for the enumeration of sulfite-reducing Clostridium spp. in feed.
iv © ISO 2021 – All rights reserved
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oSIST prEN ISO 15213-1:2021
ISO/DIS 15213-1:2021(E)
Introduction
Sulfite-reducing Clostridium spp. are anaerobic, Gram-positive, spore-forming, rod-shaped bacteria
which belong to the family of the Bacillaceae. The most important species which belong to this group
are Clostridium (C.) perfringens, C. bifermentans, C. sporogenes and C. botulinum. Some species can cause
foodborne illness. As ubiquitous bacteria they are predominantly found in nature.
Sulfite-reducing Clostridium spp., including C. perfringens, are widely used as microbial indicators of
clostridial contamination in the manufacturing of foods (e.g. meat production). These have the capacity
to produce heat resistant spores. Outside the dairy industry, the use of sulfite-reducing Clostridium
spp. as a microbial indicator is limited to a relatively small number of foods. Its current application
in non-dairy foods is either an indication of faecal contamination (especially C. perfringens, see also
ISO 15213-2 and ISO 15213-3) and/or as an indicator of sanitation/process control related to potential
growth and survival of anaerobic spore-forming bacteria.
This part of ISO 15213 describes the horizontal method for the enumeration of sulfite-reducing
Clostridium spp. in food, feed, environmental samples, and samples from the primary production stage.
The method for the enumeration of C. perfringens is described in part 2. Part 3 describes the method
for the detection of C. perfringens. These three parts are published into one 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 may
be similar.
The main technical changes listed in the Foreword, introduced in this document compared to
ISO 15213:2003 are considered as major (see ISO 17468).
These changes have a major impact on the performance characteristics of the method.
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oSIST prEN ISO 15213-1:2021
DRAFT INTERNATIONAL STANDARD ISO/DIS 15213-1:2021(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Clostridium spp. —
Part 1:
Enumeration of sulfite-reducing Clostridium spp. by
colony-count technique
1 Scope
This document specifies the enumeration of sulfite-reducing anaerobes and sulfite-reducing Clostridium
spp. by the colony-count technique. This part of ISO 15213 is applicable to
— products intended for human consumption,
— products intended for animal feeding,
— environmental samples in the area of food and feed production, handling, and
— 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.
This technique is suitable for, but not limited to, use for the enumeration of microorganisms in test
samples and is based on a minimum of 10 colonies counted in 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.
NOTE This method has been validated in an interlaboratory study for the following food categories:
1) Ready-to-eat, ready-to-reheat meat products;
2) Eggs and egg products (derivates);
3) Processed fruits and vegetables;
4) Infant formula and infant cereals;
5) Multi-component foods or meal components;
and 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 foods. Since the method is not commonly used for samples in the primary production stage,
this category was not included in the validation study. Therefore, no performance characteristics were
obtained for this category. For detailed information on the validation see Clause 11 and Annex C.
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ISO/DIS 15213-1:2021(E)
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 food and animal feeding stuffs — 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
terminological 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
sulfite-reducing Clostridium spp
genus of microorganisms of the family of Clostridiaceae, usually capable of growth in/on Iron Sulfite
Agar (ISA) under anaerobic conditions, forming typical or less typical colonies, and displaying certain
characteristics with biochemical confirmation tests
Note 1 to entry: The biochemical confirmation tests are described in 9.6.
3.2
enumeration of sulfite-reducing Clostridium spp
determination of the number of colony-forming units (cfu) of sulfite-reducing Clostridium spp. (3.1)
bacteria per ml or per g or sample when the specified test is conducted
Note 1 to entry: The specified test is described 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. Other plates are prepared under the same conditions using decimal dilutions of
the test sample. If it is the intention to count only spores, heat treatment of 10 min at 80 °C needs to be
performed before plating.
When the number of cfu is expected to be at or near the limit of determination of the method, the use of
duplicate plates is preferable. If duplicate plates are used the minimum for the sum of colonies should
be 10. 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 is especially suited for the enumeration of products expected to contain
spreading colonies that can obscure colonies of the target microorganisms.
The enumeration of sulfite-reducing Clostridium spp. requires four successive stages as specified in
Annex A.
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4.2 Preparation of dilutions
For the preparation of decimal dilutions from the test portion, follow the procedure as specified in
ISO 6887 (all parts).
4.3 Enumeration
The plates are incubated under anaerobic conditions at 37 °C for 48 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 commences due to the formation of iron(II)sulphide as a result of
the reaction between sulphide ions and trivalent iron [Fe(III)] present in the medium.
4.4 Confirmation
Typical colonies are picked for confirmation.
NOTE When no confirmation is performed, the results can be reported as ‘anaerobic sulfite-reducing
bacteria’.
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/or Annex B.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following:
6.1 Appropriate apparatus for achieving an anaerobic atmosphere.
6.2 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).
6.3 Drying cabinet or oven, ventilated by convection, capable of operating between 25 °C and 50 °C.
6.4 Incubator, capable of operating at 37 °C ± 1 °C.
6.5 pH-meter, having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C.
6.6 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.7 Sterile flasks, bottles or tubes, of appropriate capacity. Bottles, flasks or tubes with non-toxic
metallic of plastic screw-caps may be used.
6.8 Sterile graduated pipettes or automatic pipettes, of nominal 10 ml and 1 ml.
6.9 Sterile loops, of approximately 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 Water bath, capable of operating at 44 °C to 47 °C and 80 °C ± 2 °C.
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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:
— ISO/TS 17728 for food and animal feed;
— ISO 707 for milk and milk products;
— ISO 6887-3 for fish and fishery products;
— 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. 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 ISO 6887 (all parts).
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
A diagram of the procedure is given in Annex A.
9.2 Test portion, initial suspension and dilutions
Refer to ISO 6887 (all parts) and the specific International Standard dealing with the product concerned.
A proposal for preparing the initial suspension of feed samples is given in Annex D.
9.3 Heat pre-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 for
10 min ± 1 min. Heat treatment shall be given within 15 min after preparation of the initial suspension
to avoid germination of spores. The temperature 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. The time taken to reach 80 °C
shall not exceed 15 min and can be minimised by ensuring the water level to be at least 4 cm above the
level of the sample and that water in the water bath is circulated 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, feed silage).
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9.4 Inoculation and incubation
9.4.1 Take two sterile Petri dishes (6.10). Transfer to each dish, by means of a sterile pipette (6.8), 1 ml
−1
of the test sample if liquid, or 1 ml of the initial suspension (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).
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 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 that will give colony counts of between 10 and
150 colonies per plate (on 90 mm Petri dishes) or between 10 and 360 colonies per plate (on 140 mm
Petri dishes).
9.4.5 Pour about 12 ml to 15 ml of the iron sulfite agar medium (see B.2), molten and tempered at
44 °C to 47 °C (6.11), into each Petri dish. The time elapsed between the end of the preparation of the
−1
initial suspension (or of the 10 dilution if the product is liquid) and the moment the inoculum comes
into contact with the culture medium (see B.2) should ideally be less than 20 min and shall not exceed 45
min unless specific conditions for example soaking of dehydrated samples to reduce osmotic shock, are
given in the relevant International Standard.
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 medium (see B.2) 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 and place them in anaerobic jars (6.1) and incubate (6.4) the plates at 37 °C for
48 h ± 2 h in an anaerobic atmosphere.
9.5 Enumeration of typical colonies
9.5.1 After 48 h ± 2 h of incubation, examine the plates (9.4.7) for presumptive sulfite-reducing
Clostridium spp.
Typical colonies, which show black or grey to yellow-brown staining on the iron sulfite agar medium,
are counted.
Since the colour of the colonies rapidly fades and finally disappear, the plates have to be counted within
30 min after completion of the anaerobic incubation. If more anaerobic jars are used, the plates should
be checked jar by jar or in small lots 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,
2
whi
...
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