kSIST FprEN ISO 15213-1:2022

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/FDIS 15213-1:2022)

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/FDIS 15213-1:2022)

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/FDIS 15213-1:2022)

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/FDIS 15213-1:2022)

General Information

• Status: Not Published
• Public Enquiry End Date: 29-Nov-2021
• ICS: 07.100.30 - Food microbiology
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 5020 - Formal vote (FV) (Adopted Project)
• Start Date: 29-Nov-2022
• Due Date: 17-Jan-2023
• Completion Date: 21-Dec-2022
• Directive: 2017/625 - Regulation (EU) 2017/625 of the European Parliament and of the Council of 15 March 2017 on official controls and other official activities performed to ensure the application of food and feed law, rules on animal health and welfare, plant health and plant protection products
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: FprEN ISO 15213-1 - 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/FDIS 15213-1:2022)

Standards Content (Sample)

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

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Published in Switzerland
ii © ISO 2021 – All rights reserved
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oSIST prEN ISO 15213-1:2021
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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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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.

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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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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).
4 © ISO 2021 – All rights reserved
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oSIST prEN ISO 15213-1:2021
ISO/DIS 15213-1:2021(E)
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

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

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,

whi
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