prEN ISO 15213-2

Microbiology of the food chain - Horizontal method for the detection and enumeration of Clostridium spp. - Part 2: Enumeration of Clostridium perfringens by colony-count technique (ISO/DIS 15213-2:2022)

This document specifies the enumeration of Clostridium (C.) perfringens by 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 technique is intended to be used when the number of colonies sought is expected to be more than 10 per ml or per g of the test sample.

Mikrobiologie der Nahrungskette - Horizontales Verfahren zum Nachweis und zur Aufzählung von Clostridium spp. - Teil 2: Zählung von Clostridium perfringens durch Koloniezählverfahren (ISO/DIS 15213‑2:2022)

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement de Clostridium spp. - Partie 2: Dénombrement de Clostridium perfringens par la technique de comptage des colonies (ISO/DIS 15213-2:2022)

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila Clostridium spp. - 2. del: Preštevanje Clostridium perfringens s tehniko štetja kolonij (ISO/DIS 15213-2:2022)

General Information

• Status: Not Published
• Publication Date: 12-Nov-2023
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463 - Microbiology of the food chain
• Current Stage: 4599 - Dispatch of FV draft to CMC - Finalization for Vote
• Start Date: 27-Apr-2023
• Completion Date: 27-Apr-2023
• Directive: 2017/625 - Regulation (EU) 2017/625 of the European Parliament and of the Council of 15 March 2017 on official controls and other official activities performed to ensure the application of food and feed law, rules on animal health and welfare, plant health and plant protection products
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: oSIST prEN ISO 15213-2:2022 - Microbiology of the food chain - Horizontal method for the detection and enumeration of Clostridium spp. - Part 2: Enumeration of Clostridium perfringens by colony-count technique (ISO/DIS 15213-2:2022)

Relations

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

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN ISO 15213-2:2022
01-november-2022
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje

prisotnosti in števila Clostridium spp. - 2. del: Preštevanje Clostridium perfringens

s tehniko štetja kolonij (ISO/DIS 15213-2:2022)

Microbiology of the food chain - Horizontal method for the detection and enumeration of

Clostridium spp. - Part 2: Enumeration of Clostridium perfringens by colony-count

technique (ISO/DIS 15213-2:2022)
Mikrobiologie der Nahrungskette - Horizontales Verfahren zum Nachweis und zur

Aufzählung von Clostridium spp. - Teil 2: Zählung von Clostridium perfringens durch

Koloniezählverfahren (ISO/DIS 15213-2:2022)

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le

dénombrement de Clostridium spp. - Partie 2: Dénombrement de Clostridium perfringens

par la technique de comptage des colonies (ISO/DIS 15213-2:2022)
Ta slovenski standard je istoveten z: prEN ISO 15213-2
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 15213-2:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 15213-2:2022
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oSIST prEN ISO 15213-2:2022
DRAFT INTERNATIONAL STANDARD
ISO/DIS 15213-2
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2022-09-13 2022-12-06
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Clostridium spp. —
Part 2:
Enumeration of Clostridium perfringens by colony-count
technique

Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche et le dénombrement de

Clostridium spp. —

Partie 2: Dénombrement de Clostridium perfringens par la technique par comptage des colonies

ICS: 07.100.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
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oSIST prEN ISO 15213-2:2022
ISO/DIS 15213-2:2022(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 15213-2
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Clostridium spp. —
Part 2:
Enumeration of Clostridium perfringens by colony-count
technique

Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche et le dénombrement de

Clostridium spp. —

Partie 2: Dénombrement de Clostridium perfringens par la technique par comptage des colonies

ICS: 07.100.30
This document is circulated as received from the committee secretariat.
COPYRIGHT PROTECTED DOCUMENT
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© ISO 2022
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NOT BE REFERRED TO AS AN INTERNATIONAL

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RECIPIENTS OF THIS DRAFT ARE INVITED
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oSIST prEN ISO 15213-2:2022
ISO/DIS 15213-2:2022(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 ............................................................................................................ 5

9.3 Heat pre-treatment to select spores .................................................................................................................................... 5

9.4 Inoculation and incubation.......................................................................................................................................................... 5

9.5 Enumeration of typical colonies ............................................................................................................................................. 6

9.6 Confirmation of C. perfringens .................................................................................................................................................. 6

9.6.1 Selection of colonies for confirmation ............................................................................................................ 6

9.6.2 Acid phosphatase test .................................................................................................................................................... 6

9.6.3 SIM agar test ........................................................................................................................................... ................................ 7

9.6.4 Differentiation between human pathogenic and non-pathogenic

C. perfringens strains (optional) ........................................................................................................................... 7

9.6.5 Interpretation ....................................................................................................................................................................... 7

10 Expression of results ....................................................................................................................................................................................... 7

11 Performance characteristics of the method ........................................................................................................................... 7

11.1 Interlaboratory study ....................................................................................................................................................................... 7

11.2 Repeatability limit ............................................................................................................................................................................... 8

11.3 Reproducibility limit ......................................................................................................................................................................... 8

12 Test report .................................................................................................................................................................................................................. 9

13 13 Quality assurance ....................................................................................................................................................................................... 9

Annex A (normative) Flow diagram of the procedure ...................................................................................................................10

Annex B (normative) Culture media and reagents ............................................................................................................................11

Annex C (informative) Method validation studies and performance characteristics .................................16

Annex D (informative) Molecular differentiation between pathogenic and non-pathogenic

C. perfringens ........................................................................................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................41

iii
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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 on 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 the following URL: www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,

in collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/

TC 463, Microbiology of the food chain, in accordance with the Agreement on technical cooperation

between ISO and CEN (Vienna Agreement).

This first edition of ISO 15213-2 cancels and replaces ISO 7937:2004. ISO 15213-1 cancels and replaces

ISO 15213:2003. Both ISO 15213:2003 and ISO 7937:2004 have been technically revised. ISO 15213-3 is

a new developed standard.
The main changes compared with the previous edition are as follows:
— the Scope is enlarged to samples from the primary production stage;

— the heat treatment of 10 min at 80 °C has been made optional, in case of high background flora, or

for the enumeration of only spores of Clostridium (C.) perfringens present in the sample;

— the selective medium has been re-named from sulfite-cycloserine agar (SC) to tryptose sulfite

cycloserine agar (TSC) without changes in the formulation;
— the confirmation methods described are modified according to ISO 14189;
— a flow diagram in Annex A gives a short description of the procedure;

— in Annex D (informative) two molecular methods are described for differentiation between

pathogenic and non-pathogenic C. perfringens and one molecular method for the differentiation of

C. perfringens type A strains carrying a chromosomally encoded cpe gene or a plasmid encoded cpe

gene.
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

Clostridium (C.) perfringens is a Gram-positive, anaerobic, spore-forming bacterium. As a ubiquitous

bacterium, C. perfringens is predominantly found in soil, but also in the intestinal tract of humans and

animals. Therefore, the presence of C. perfringens in high numbers can be an indication of inadequate

preparation or handling of food.

High numbers of C. perfringens in ready-to-eat-food can cause human illness, mainly diarrhoea. The

strains are classified into toxin types, depending on the ability to produce different so called “major”

and “minor” toxins. Food poisonings are caused by C. perfringens isolates with the ability to produce

C. perfringens enterotoxin (CPE).

A characteristic feature is the heat resistance of the spores; they have the ability to germinate and

multiply in ready-to-eat food after the cooking process. Ingestion of contaminated food is followed

by gastrointestinal disease, when enzyme-resistant C. perfringens enterotoxins are set free during

sporulation in the small intestine. The strains are classified into different types.

This document describes the horizontal method for the enumeration of Clostridium (C.) perfringens in

food, feed, environmental samples, and samples from the primary production stage. The method for

the enumeration of sulfite-reducing Clostridium spp. is described in ISO 15213-1. The method for the

detection of C. perfringens is described in ISO 15213-3. These three parts are published as a series

of International Standards because the methods are closely linked to each other. These methods are

often conducted in association with each other in a laboratory and the media and their performance

characteristics can be similar.

The main technical changes listed in the Foreword, introduced in this document compared with

ISO 7937:2004 are considered as major (see ISO 17468).

These changes have a major impact on the performance characteristics of the method.

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oSIST prEN ISO 15213-2:2022
DRAFT INTERNATIONAL STANDARD ISO/DIS 15213-2:2022(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Clostridium spp. —
Part 2:
Enumeration of Clostridium perfringens by colony-count
technique

WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests

for enumeration Clostridium perfringens are only undertaken in properly equipped laboratories,

under the control of a skilled microbiologist, and that great care is taken in the disposal of all

incubated materials. Persons using this document should be familiar with normal laboratory

practice. This document does not purport to address all of the safety aspects, if any, associated

with its use. It is the responsibility of the user to establish appropriate safety and health

practices.
1 Scope

This document specifies the enumeration of Clostridium (C.) perfringens by colony-count technique.

This document is applicable to:
— products intended for human consumption;
— products for feeding animals;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.

NOTE This method has been validated in an interlaboratory study for the following food categories:

— ready-to-eat, ready-to-reheat meat products;
— eggs and egg products (derivates);
— processed fruits and vegetables;
— infant formula and infant cereals;
— multi-component foods or meal components.
It has also been validated for the following other categories:
— pet food and animal feed;
— environmental samples (food or feed production).

As this method has been validated for at least five food categories, this method is applicable for a broad

range of food. For detailed information on the validation see Clause 11 and Annex C. Since the method is

not commonly used for samples in the primary production stage, this category was not included in the

interlaboratory study. Therefore, no performance characteristics were obtained for this category.

This horizontal method was originally developed for the examination of all samples belonging to the

food chain. Based on the information available at the time of publication of this document, this method

is considered to be fully suited to the examination of all samples belonging to the food chain. However,

because of the large variety of products in the food chain, it is possible that this horizontal method is not

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oSIST prEN ISO 15213-2:2022
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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 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 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

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
confirmed Clostridium (C.) perfringens

bacteria that produce characteristic colonies in the specified selective medium under obligate anaerobic

conditions and possess the enzyme acid phosphatase
3.2
human pathogenic Clostridium (C.) perfringens

confirmed C. perfringens 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 plasmids. These isolates are able to

produce CPE in the small intestine on sporulation and cause human illness.
3.3
presumptive Clostridium (C.) perfringens

spore-forming bacteria forming countable typical colonies in a specific selective medium under obligate

anaerobic conditions

Note 1 to entry: Presumptive C. perfringens are spore-forming bacteria that are able to produce typical colonies

under the conditions specified in this document.
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

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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, a heat treatment of 10 min at 80 °C needs

to be performed before plating. Additionally, a method for molecular differentiation between human

pathogenic and non-pathogenic C. perfringens strains is described in Annex D.

When the number of cfu is expected to be at or near the limit of determination, the use of duplicate

plates is preferable. If duplicate plates are used the minimum for the sum of colonies 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 with overlay is especially suited for the enumeration of products expected to

contain spreading colonies that can obscure colonies of the target microorganisms.

The enumeration of C. perfringens requires four successive stages as specified in Annex A.

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

Petri dishes are inoculated with a specified quantity of the test sample if the initial product is liquid,

or a specified quantity of the initial suspension, in the case of other products. Additional Petri dishes

are inoculated, under the same conditions, using decimal dilutions of the test sample or of the initial

suspension. A selective medium is added (poured-plate technique) and then overlaid with the same

medium.

The plates are incubated under anaerobic conditions at 37 °C. for 20 h. The number of colonies presumed

to be C. perfringens, because of their characteristic appearance, is recorded.
4.4 Confirmation

Confirmatory tests are carried out. The result is calculated as the colony count of confirmed

C. perfringens per sample volume. Additionally, the method described in Annex D may be used for

molecular differentiation between human pathogenic and non-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/or 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.
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.

6.4 Freezer, capable of operating at -20 °C ± 2 °C and at -70 °C ± 3 °C.
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6.5 Incubator(s), capable of operating at 37 °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 or plastic screw-caps may be used.

6.9 Sterile graduated pipettes or automatic pipettes, of nominal capacities 10 ml, 1 ml and 0,1 ml.

6.10 Sterile loops, of approximate diameter 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 and (optional) large size (diameter

approximately 140 mm).

6.12 Thermostatically controlled water bath, capable of operating at 44 °C to 47 °C and 80 °C ± 2 °C.

7 Sampling

Sampling is not part of the method specified in this document. Follow the specific International

Standard dealing with the product concerned. If there is no specific International Standard dealing

with the sampling of the product concerned, it is recommended that the parties concerned come to an

agreement on this subject.
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 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 flow diagram of the procedure is given in Annex A.
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9.2 Test portion, initial suspension and dilutions

Prepare the test sample in accordance with the specific International Standard dealing with the product

concerned. If there is no specific International Standard, it is recommended that the parties concerned

come to an agreement on this subject. Prepare a single decimal dilution series from the test portion if

the product is liquid, or from the in
...