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
ISO 15213-1:2023(E)

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© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Email: [email protected]
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Published in Switzerland

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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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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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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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ISO 15213-1:2023(E)

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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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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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://​w ww​.iso​.org/​obp
— IEC Electropedia: available at https://​w ww​.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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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.

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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 dealing
with the sampling of the product concerned, it is recommended that the parties concerned come to an
agreement on this subject.
Recommended sampling techniques are given in the following documents:
— ISO/TS 17728 for food and animal feed;
— ISO 707 for milk and milk products;
— ISO 6887-3 for raw molluscs, tunicates and echinoderms from primary production areas;
— ISO 13307 for primary production stage;
— ISO 17604 for carcasses;
— ISO 18593 for surfaces.
It is important that the laboratory receives a sample that is representative of the product under
consideration. The sample should not have been damaged or changed during transport or storage.

8 Preparation of test sample

Prepare the test sample from the laboratory sample in accordance with the specific International
Standard dealing with the product concerned. Follow the procedures as specified in the ISO 6887 series.
If there is no specific International Standard available, it is recommended that the parties concerned
come to an agreement on this subject.

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9 Procedure

9.1 General
A flow diagram of the procedure is given in Annex A.

9.2 Test portion, initial suspension and dilutions

Follow the procedures in accordance with the ISO 6887 series and the specific International Standard
dealing with the product concerned.
Prepare a single decimal dilution series from the test portion if the product is liquid, or from the initial
suspension in the case of other products.
A special protocol for preparing the initial suspension of feed samples is given in Annex D.

9.3 Heat treatment to select spores

If it is the intention to count only spores, heat the decimal dilution series to 80  °C in a water bath
(6.10) for 10 min ± 1 min. Heat treatment shall be given within 15 min after preparation of the initial
suspension to avoid germination of spores. If the tube is not placed in the water bath within 15 min, it
should be placed immediately in melting ice for a maximum of 2 h.
The temperature during heat treatment should be monitored by placing an appropriate thermometer in
a reference bottle of the same size as the sample bottle and containing the same volume of water at the
same initial temperature as the sample being treated (6.6). The tubes should not be hermetically sealed
during the heat treatment. The time taken to reach 80 °C shall not exceed 5 min and can be minimized
by ensuring the water level to be at least 4 cm above the level of the sample and equipping the water
bath with a circulating-water pump to maximize heat exchange.
Start the time of heating (10 min) when the temperature of the reference sample has reached 80 °C.
After heat treatment, the samples should be cooled immediately until 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).

9.4 Inoculation and incubation

9.4.1 Take two sterile Petri dishes with a diameter of approximately 90 mm (6.9). Transfer to each
dish, by means of a sterile pipette (6.7), 1 ml of the test sample if liquid, or 1 ml of the initial suspension
(10−1 dilution) in the case of other products. If plates from more than one dilution are prepared, this
may be reduced to one dish (see ISO 7218).

When, for certain products, it is necessary to estimate low numbers of sulfite-reducing Clostridium spp.,
the limit of enumeration may be lowered by a factor of 10 by examining 10 ml of the initial suspension
in three large (140 mm) Petri dishes (6.9).

9.4.2 Take one other sterile Petri dish (6.9). Use another sterile pipette (6.7) to dispense 1 ml of the
10−1 dilution (liquid product) or 1 ml of the 10−2 dilution (other products).

9.4.3 If necessary, repeat the procedure with further dilutions, using a new sterile pipette (6.7) for
each decimal dilution.

9.4.4 If appropriate and possible, select only the critical dilution steps (at least two consecutive
decimal dilutions) for the inoculation of the Petri dishes (6.9) 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).

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9.4.5 Pour about 12 ml to 15 ml for 90 mm Petri dishes or 45 ml to 50 ml for 140 mm Petri dishes of
the iron sulfite agar (ISA) medium (Clause B.2), molten and tempered at 44 °C to 47 °C (6.10), into each
Petri dish (6.9).

9.4.6 Carefully mix the inoculum with the medium by rotating the Petri dishes (6.9) and allow the
mixture to solidify by leaving the Petri dishes standing on a cool horizontal surface.

9.4.7 After complete solidification, pour about 5  ml of the ISA medium (Clause B.2) for 90 mm
Petri dishes (6.9) or 10 ml for 140 mm Petri dishes (6.9) as overlay, to prevent the development of
spreading colonies on the surface of the medium. Allow to solidify as specified in 9.4.6.

9.4.8 Invert the plates obtained in 9.4.7 and incubate the plates at 37 °C (6.3) in an anaerobic
atmosphere (6.1).

9.5 Enumeration of typical colonies

9.5.1 After 48 h ± 2 h of incubation, examine the plates (see 9.4.8) for presumptive sulfite-reducing
Clostridium spp.

Typical colonies, which show black or grey to yellow-brown staining on the ISA medium, are counted.
Upon removal of the plates from the anaerobic atmosphere, plates shall be counted within 30 min as
the colour of the colonies can rapidly fade and disappear upon exposure to oxygen. If anaerobic jars are
used, the plates should be checked jar by jar or in small portions if the incubation was performed in an
anaerobic incubator (6.1, 6.3).
NOTE Diffuse, unspecific blackening of the medium can occur. The growth of anaerobic bacteria, which
produce hydrogen (not H2S), can also reduce the sulfite present and lead to a general blackening of the medium,
which makes enumeration of typical colonies difficult.

9.5.2 Select the plates (see 9.5.1) containing less than 150 presumptive colonies (for 90 mm
Petri dishes) or less than 360 colonies (for 140 mm Petri dishes). Count these colonies and record their
number as presumptive colonies per dish. Then choose at random five such colonies for subculturing
for the confirmation tests (see 9.6).

For enumeration of plates with low or high numbers of presumptive colonies, see ISO 7218.

9.6 Confirmation of sulfite-reducing Clostridium spp.

9.6.1 For confirmation, take five presumptive colonies from each dish retained for enumeration (see
9.5.2). If more than one morphology is present among the colonies, select one of each morphology for
subculture and confirmation.

9.6.2 Streak each of the selected colonies with a sterile loop (6.8) onto two non-selective blood agar
plates, e.g. Columbia blood agar (Clause B.3). If blood is not available, Columbia agar base or another
nutrient-rich medium (e.g. tryptone soya agar or brain heart infusion agar) can be used.

Allow the plates to equilibrate at room temperature if they were stored at a lower temperature. If
necessary, dry the surface of the plates before use (see ISO 11133).
Several isolates can be streaked onto identified sectors of each of the two non-selective agar plates.
Streaks should obtain well-isolated colonies.
From each pair of plates, one is incubated in an aerobic atmosphere and the other in an anaerobic
atmosphere (6.1) at 37 °C for 20 h ± 2 h (6.3). After incubation, the plates can be refrigerated at 5 °C
(6.5) for a maximum of 48 h before reading. For plates which were incubated anaerobically, maintain
the anaerobic atmosphere.

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9.6.3 Typical colonies are confirmed as follows:

— If growth from one typical colony occurs on the anaerobically incubated (blood) agar plate and
not on the aerobically incubated (blood) agar plate, the colony belongs to the genus Clostridia. This
colony and other colonies with the same morphology on ISA medium are counted as sulfite-reducing
Clostridium spp.
— If growth occurs from one typical colony on both the anaerobically and aerobically incubated
blood agar plates, the colony does not belong to the genus Clostridia. Therefore, this colony and
other colonies with the same morphology on ISA medium cannot be counted as sulfite-reducing
Clostridium spp.
NOTE Alternative procedures (see ISO  7218) can be used to confirm the isolate as sulfite-reducing
Clostridium spp., provided that the suitability of the alternative procedure has been validated (see ISO 16140-4 or
ISO 16140-6).

10 Expression of results
For calculation of the results, follow the procedure(s) in accordance with ISO 7218. Calculate and report
the results as the number of sulfite-reducing Clostridium  spp. in cfu per gram, per millilitre or per
square centimetre. When the sampled area is not known, report as per sampling device, such as a cloth,
sponge swab or stick.
If heat treatment for the selection of spores (9.3) was used, the result is reported as number of sulfite-
reducing Clostridium spp. spores in cfu per gram, per millilitre, per square centimetre or per sampling
device.
In cases where no typical colonies of the target organism have been detected, follow ISO 7218 for the
expression of results for special cases.

11 Validation of the method

11.1 Interlaboratory study

Results of the interlaboratory study (step 6 in ISO 17468) to determine the performance characteristics
of the method are described in 11.2.
NOTE In the interlaboratory study, the repeatability and reproducibility were only determined at a low level,
as the enumeration of sulfite-reducing Clostridium spp. is performed as a hygiene indicator and at low levels (e.g.
the maximum acceptable levels for infant formula are between 10 cfu/g and 30 cfu/g).

11.2 Performance characteristics

The performance characteristics of the method (repeatability and reproducibility standard
deviations) were determined in an interlaboratory study. It is possible that the values derived from the
interlaboratory study are not applicable to concentration ranges and (food) categories other than those
used in the study. All data are given in Annex C.
A summary of different values of the interlaboratory repeatability standard deviation (sr) is given in
Table 1.

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ISO 15213-1:2023(E)

Table 1 — Summary of sr values from the interlaboratory study

sr values of low
(Food) category (Food) item
inoculation level
Ready-to-eat, ready-to-reheat meat products Corned beef 0,091
Multi-component foods or meal components Instant soup 0,049
infant formula and infant cereals Powdered infant formula 0,040
Eggs and egg products (derivates) Egg powder 0,11
Processed fruits and vegetables Canned pineapple 0,060
Environmental samples (food or feed production) Environmental swab 0,054
Pet food and animal feed Feed silage 0,048

A summary of different values of the interlaboratory reproducibility standard deviation (sR) is given in
Table 2.

Table 2 — Summary of sR values from the interlaboratory study

sR values of low
(Food) category (Food) item
inoculation level
Ready-to-eat, ready-to-reheat meat products Corned beef 0,93
Multi-component foods or meal components Instant soup 0,41
infant formula and infant cereals Powdered infant formula 0,12
Eggs and egg products (derivates) Egg powder 0,61
Processed fruits and vegetables Canned pineapple 0,35
Environmental samples (food or feed production) Environmental swab 0,20
Pet food and animal feed Feed silage 0,26

12 Test report
The test report shall specify at least the following:
— the test method used, with reference to this document, i.e. ISO 15213-1;
— the sampling method used, if known;
— all operating conditions not specified in this document, or regarded as optional or informative
(including informative annexes), together with details of any incidents which can have influenced
the test result(s);
— any deviations from this document;
— all information necessary for the complete identification of the sample;
— the test result(s) obtained;
— the date of the test;
— when necessary or if requested by the client, an estimate of the measurement uncertainty of
quantitative test results, in accordance with ISO 19036:2019, Clause 9.

13 Quality assurance
The laboratory should have a quality control system to ensure that the equipment, reagents and
techniques are suitable for the method. The use of positive controls, negative controls and blanks are
part of the method. Performance testing of culture media is specified in Annex  B and described in
ISO 11133.

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 ISO 15213-1:2023(E)

Annex A
(normative)

Flow diagram of the procedure

Figure A.1 shows the flow diagram of procedure for the enumeration of sulfite-reducing Clostridium spp.
by colony-count technique in food, animal feed, environmental and primary production stage samples.

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ISO 15213-1:2023(E)

Figure A.1 — Flow diagram of procedure for the enumeration of sulfite-reducing

Clostridium spp. by colony-count technique

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 ISO 15213-1:2023(E)

Annex B
(normative)

Culture media and reagents

B.1 General
The general specifications of ISO 11133 are applicable to the preparation and performance testing of
the culture media described in this annex. If culture media or reagents are prepared from dehydrated
complete media/reagents or if ready-to-use media/reagents are used, follow the manufacturer’s
instructions regarding preparation, storage conditions, expiry date and use.
The shelf life of the media indicated in this annex has been determined in some studies. The user shall
verify this under its own storage conditions (in accordance with ISO 11133).
Performance testing of culture media is described in Clause B.4.

B.2 Iron sulfite agar (ISA)

B.2.1 Composition

Peptonea   15,0 g
Enzymatic digest of soya   5,0 g
Yeast extract   5,0 g
Sodium disulfite (sodium metabisulfite), (CAS Registry Number®d 0,5 g
anhydrous (Na2S2O5) 7681-57-4)
Iron(III) ammonium citrate (C6H8FeNO4)b (CAS No. 1185-57-5) 1,0 g
Agarc   9,0 to 18,0 g
Water   1 000 ml
a For example, enzymatic digest of casein.

b This reagent should contain at least 150 g/kg of iron.

c Depending on the gel strength of the agar.
d Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical
Society (ACS). This information is given in the convenience of users of this document and does not
constitute an endorsement by ISO of the product named. Equivalent products may be used if they
can be shown to lead to the same results.

B.2.2 Preparation
Dissolve the ingredients in water, by heating if necessary.
Adjust the pH, if necessary, so that after sterilization it is 7,6 ± 0,2 at 25 °C (6.4).
Sterilize for 15 min in the autoclave (6.2) set at 121 °C.
Store the medium, at 5 °C (6.5) for up to four weeks in closed containers or tubes (6.6). Prior to use, the
stored medium is melted completely and cooled down to 44 °C to 47 °C (6.10) before use.

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ISO 15213-1:2023(E)

B.3 Columbia blood agar (CBA)

B.3.1 Columbia blood agar base

B.3.1.1 Composition

Enzymatic digest of animal tissue   23,0 g

Starch soluble (C12H22O11) (CAS No. 9005-84-9) 1,0 g
Sodium chloride (NaCl) (CAS No. 7647-14-5) 5,0 g
Agar a   8,0 to 18,0 g
Water   1 000 ml
a Depending on the gel strength of the agar.

B.3.1.2 Preparation

Dissolve the ingredients in water, by heating if necessary.

Adjust the pH, if necessary, so that after sterilization it is 7,3 ± 0,2 at 25 °C (6.4).
Dispense the medium in flasks (6.6) of suitable capacity to obtain portions appropriate for the test.
Sterilize for 15 min in the autoclave (6.2) set at 121  °C. Store the medium, at 5  °C (6.5) for up to
four weeks in closed containers or tubes (6.6).

B.3.2 Defibrinated blood (horse or sheep blood)

B.3.3 Complete base

B.3.3.1 Composition

Base (B.3.1) 100 ml

Defibrinated blood (B.3.2) 5 ml

B.3.3.2 Preparation

Add the blood to the base previously cooled to 44 °C to 47 °C (6.10). Mix well.

B.3.3.3 Preparation of sheep blood agar plates

Dispense the medium (B.3.3.1) into sterile Petri dishes (6.9) in portions appropriate for the test. Allow
to solidify.
Immediately before use, dry the agar plates following the procedure as given by ISO 11133. Store the
poured plates, protected for drying, at 5 °C (6.5) for up to four weeks.

B.4 Performance testing

For the definition of selectivity and productivity refer to ISO 11133. In general, follow the procedures
for performance testing described in ISO 11133.
The performance testing for the quality assurance of the culture media is given in Table B.1.

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 ISO 15213-1:2023(E)

Table B.1 — Performance testing for the quality assurance of the culture media
Control WDCM Reference Method of
Medium Function Incubation Criteriac,e
strains numbersa medium control
P R ≥ 0,5
A suitable
00007b (P R ≥ 0,7 when
Clostridium non-selective
Productivity Quantitative compared to ISA
(48 ± 2) h/ perfringens 00080 medium for
batch already
(37 ± 1) °C anaerobes
ISA validated)
anaerobic
atmosphere Growth (1 to 2)
Escherichia 00013 or
Specificity — Qualitative No blackening of
colid 00012
colonies
(20 ± 2) h / Good growth (2)
(37 ± 1) °C Clostridium
CBA Confirmation 00007b — Qualitative Colonies with
anaerobic perfringens
atmosphere beta-haemolysis
a Refer to the reference strain catalogue on http://​w ww​.wfcc​.info for information on culture collection strain numbers and contact
details; WDCM: World Data Centre for Microorganisms.
b Strain to be used as a minimum.
c P R = productivity ratio.
d Strain free of choice; one of the strains has to be used as a minimum.
e Growth is categorized as 0: no growth; 1: weak growth (partial inhibition); 2: good growth (see ISO 11133).

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ISO 15213-1:2023(E)

Annex C
(informative)

Performance characteristics of the method

An interlaboratory study involving 20 laboratories in 7 countries was carried out. The following (food)
items [representing the (food) categories as indicated] were included in the study: canned corned beef,
instant soup, powdered infant formula, egg powder, canned pineapple, environmental swabs and feed
silage. The (food) samples were each tested at a low level of contamination. The study was organized
in 2019 by Merck KGaA, Darmstadt, Germany and FrieslandCampina as part of the development of this
document.
The values of the performance characteristics, for each (food) item and category, derived from this
interlaboratory study are shown in Tables C.1 to C.7, and were calculated in accordance with ISO 17468.

Table C.1 — Results of data analysis obtained with canned corned beef

(category: ready-to-eat, ready-to-reheat meat products)
Parameter Low level
Number of participating collaborators 20
Number of collaborators retained after evaluation of the data 13
Number of samples 26
Number of sample results retained after evaluation of the data 26
Mean value Σa (log10 cfu/g) 0,61
Interlaboratory repeatability standard deviation, sr (log10 cfu/g) 0,091
Interlaboratory reproducibility standard deviation, sR (log10 cfu/g) 0,931
NOTE 1 Strain used for inoculation: C. perfringens (WDCM 00201).
NOTE 2 The found sR is high, due to big variances found by the different participants for
canned corned beef.

Table C.2 — Results of data analysis obtained with instant soup

(category: multi-component foods or meal components)
Parameter Low level
Number of participating collaborators 20
Number of collaborators retained after evaluation of the data 13
Number of samples 26
Number of sample results retained after evaluation of the data 26
Mean value Σa (log10 cfu/g) 0,58
Interlaboratory repeatability standard deviation, sr (log10 cfu/g) 0,049
Interlaboratory reproducibility standard deviation, sR (log10 cfu/g) 0,41
NOTE Strain used for inoculation: C. perfringens (WDCM 00201).

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Table C.3 — Results of data analysis obtained with powdered infant formula

(category: infant formula and infant cereals)
Parameter Low level
Number of participating collaborators 20
Number of collaborators retained after evaluation of the data 8
Number of samples 16
Number of sample results retained after evaluation of the data 16
Mean value Σa (log10 cfu/g) 0,48
Interlaboratory repeatability standard deviation, sr (log10 cfu/g) 0,040
Interlaboratory reproducibility standard deviation, sR (log10 cfu/g) 0,12
NOTE Strain used for inoculation: C. sporogenes (WDCM 00008).

Table C.4 — Results of data analysis obtained with egg powder

(category: eggs and egg products (derivates))
Parameter Low level
Number of participating collaborators 20
Number of collaborators retained after evaluation of the data 7
Number of samples 14
Number of sample results retained after evaluation of the data 14
Mean value Σa (log10 cfu/g) 0,52
Interlaboratory repeatability standard deviation, sr (log10 cfu/g) 0,11
Interlaboratory reproducibility standard deviation, sR (log10 cfu/g) 0,61
NOTE Strain used for inoculation: C. sporogenes (WDCM 00008).

Table C.5 — Results of data analysis obtained with canned pineapple

(category: processed fruits and vegetables)
Parameter Low level
Number of participating collaborators 20
Number of collaborators retained after evaluation of the data 13
Number of samples 26
Number of sample results retained after evaluation of the data 26
Mean value Σa (log10 cfu/g) 0,60
Interlaboratory repeatability standard deviation, sr (log10 cfu/g) 0,060
Interlaboratory reproducibility standard deviation, sR (log10 cfu/g) 0,35
NOTE Strain used for inoculation: C. bifermentans (WDCM 00079).

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ISO 15213-1:2023(E)

Table C.6 — Results of data analysis obtained with environmental swab

(category: environmental samples (food or feed production))
Parameter Low level
Number of participating collaborators 20
Number of collaborators retained after evaluation of the data 14
Number of samples 28
Number of sample results retained after evaluation of the data 28
Mean value Σa (log10 cfu/g) 0,60
Interlaboratory repeatability standard deviation, sr (log10 cfu/g) 0,054
Interlaboratory reproducibility standard deviation, sR (log10 cfu/g) 0,20
NOTE Strain used for inoculation: C. bifermentans (WDCM 00079).

Table C.7 — Results of data analysis obtained with feed silage

(category: pet food and animal feed)
Parameter Low level
Number of participating collaborators 20
Number of collaborators retained after evaluation of the data 13
Number of samples 26
Number of sample results retained after evaluation of the data 26
Mean value Σa (log10 cfu/g) 0,61
Interlaboratory repeatability standard deviation, sr (log10 cfu/g) 0,048
Interlaboratory reproducibility standard deviation, sR (log10 cfu/g) 0,26
NOTE Strain used for inoculation: C. perfringens (WDCM 00201).

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 ISO 15213-1:2023(E)

Annex D
(informative)

Special protocol for the enumeration of sulfite-reducing

Clostridium spp. in feed

D.1 General
This annex describes a special protocol[11] for preparation of the initial suspension of feed for the
enumeration of sulfite-reducing Clostridium spp. Feed can contain additives such as copper that can
influence the enumeration results. This problem can be solved using a special diluent and different
ratios for preparation of the initial suspension.
The performance testing of the diluents is described in Clause D.6.

D.2 Principle
A homogeneous initial suspension is prepared from the sample with a buffer solution containing
polyoxyethylene-80-sorbitan monolaurate (PSM) and peptone. In cases where there are critical
amounts of copper in the sample, a chelating agent has to be added during preparation of the initial
suspension.

D.3 Culture media and reagents

The general specifications of ISO 11133 are applicable to the preparation and performance testing of
the culture media described in this annex. If culture media or reagents are prepared from dehydrated
complete media/reagents or if ready-to-use media/reagents are used, follow the manufacturer’s
instructions regarding preparation, storage conditions, expiry date and use.
The shelf life of the media indicated in this annex has been determined in some studies. The user shall
verify these under their own storage conditions (as specified in accordance with ISO 11133).

D.3.1 Initial feed diluent solution

D.3.1.1 Composition

Enzymatic digest of animal tissues   1,0 g

Sodiumdihydrogenphosphate-dihydrate (NaH2PO4 · 2 H2O) (CAS No. 13472-35-0) 0,58 g
Di-Sodiumhydrogenphosphate-dihydrate (Na2HPO4 · 2 H2O) (CAS No. 7558-79-4) 2,5 g
Sodium chloride (NaCl)   4,0 g
Polyoxyethylene-80-sorbitan monolaurate (C46H52N5O8P) (CAS No. 9005-65-6) 0,3 g
Water   1 000 ml

D.3.1.2 Preparation

Dissolve the components in water, by heating if necessary.

Adjust the pH, if necessary, so that after sterilization it is 7,0 ± 0,2 at 25 °C (6.4).
Sterilize for 15 min in the autoclave (6.2) set at 121 °C.

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ISO 15213-1:2023(E)

Store the medium in closed containers (6.6) at 5 °C (6.5) for up to four weeks.

D.3.2 Feed diluent solution

D.3.2.1 Composition

Sodiumdihydrogenphosphate-dihydrate (NaH2PO4 · 2 H2O) (CAS No. 13472-35-0) 0,58 g

Di-Sodiumhydrogenphosphate-dihydrate (Na2HPO4 · 2 H2O) (CAS No. 7558-79-4) 2,5 g
Sodium chloride (NaCl)   4,0 g
Water   1 000 ml

D.3.2.2 Preparation

Dissolve the components in water, by heating if necessary.

Adjust the pH, if necessary, so that after sterilization it is 7,0 ± 0,2 at 25 °C (6.4).
Sterilize for 15 min in the autoclave (6.2) set at 121 °C.
Store the medium in closed containers (6.6) at 5 °C (6.5) for up to four weeks.

D.3.3 Polyoxyethylene-80-sorbitan monolaurate (PSM) (CAS No. 9005-65-6)

D.4 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.

D.4.1 Sterile storage containers for initial feed diluent solution (D.3.1) and feed diluent solution
(D.3.2), e.g. flasks or storage bottles with a nominal volume of 500  ml, 1  000  ml or 2  500  ml and
matching caps or stoppers.

D.4.2 Sterile laboratory bottles with nominal volumes of 500 ml and 1 000 ml with iso-thread or
caps.

D.4.3 Sterile bottles or flasks with a capacity of at least 50 ml and matching caps or stoppers.

D.4.4 Shaking device, preferably a horizontal shaker that can be set at 120 r/min to 180 r/min.

D.4.5 Paddle blender with sterile bags (minimal capacity is 400 ml).

D.4.6 Sterile graduated serological pipettes (5 ml) for complete discharge with wide tips.

D.5 Procedure
D.5.1 Test portion and preparation of initial suspension
Recommended amounts of sample and corresponding volumes of initial feed diluent (D.3.1) are
described in Table D.1, Part A. Treat the initial suspension with the recommended procedure shown in
Table D.1, Part B.

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Table D.1 — Recommended amounts of sample, corresponding initial feed

diluent solution volumes and procedure
Part A Part B
Recommended quantities of sample and volumes of ready-to- Resulting dilution factor and
use initial feed diluent solution for the preparation of initial recommended treatment of initial
sample suspensions for various sample types suspensions
Sample type Critical copper Amount of Initial feed diluent Dilution factor of Treatment of the
content sample (D.3.1) the initial initial suspension
mg/kg g or ml ml suspension
Additives Not applicable 4 196 1:50
Pre-mixtures,
≥ 400 380
mineral feed
20 1:20
Strongly
≥ 400 380
swelling feed
Shaking device
Hay, straw and (20 min) or paddle
Not applicable 20 380 1:20
silage blender (5 min)
Single feed Not applicable
Milk replacer Not applicable 20 180 1:10
Compound feed ≥ 200
Liquid feed Not applicable 20 180 1:10
Paste-like and Paddle blender
≥ 400 5 90 + 5 g PSM 1:20
oleaginous feed (5 min)

D.5.2 Shaking procedure

Weigh the recommended quantity of sample (see Table D.1, Part A) into a bottle (D.4.2). Add the initial
feed diluent solution (D.3.1) and shake the mixture immediately vigorously by hand for about 1 min.
Treat the initial suspension for 20 min on a shaking device (D.4.4).

D.5.3 Paddle blender method

Weigh the recommended quantity of sample (see Table D.1, Part  A) and, if appropriate, PSM into a
paddle blender bag (D.4.5). Add the initial feed diluent solution (D.3.1) and homogenize the mixture in
the paddle blender (D.4.5) for 5 min.

D.5.4 First dilution

Prepare a first dilution (resulting in a dilution factor of 1:100) using feed diluent solution (D.3.2)
according to Table D.2 from the treated initial suspension (D.5.2 or D.5.3). Keep the initial suspension
homogenous for pipetting. Transfer the necessary volume of initial suspension (D.5.2 or D.5.3) into
containers (D.4.2) using a pipette (D.4.6).

Table D.2 — Recommended volumes for the preparation of first dilutions

Premixtures, mineral feed, strongly
Other solid
Sample type Additives swelling, oleaginous or paste-like feed,
and liquid feed
silage, hay, straw
Treated initial suspension D.5.2 or D.5.3
Dilution factor of initial suspension 1:50 1:20 1:10
Volume of initial suspension
5 ml 5 ml 5 ml
(D.5.2 or D.5.3)
+ Feed diluent solution (D.3.2) + 5 ml + 20 ml + 45 ml
Dilution factor of first dilution
1:100 (10 to 2)
(D.5.2 or D.5.3)

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ISO 15213-1:2023(E)

From the first dilution, further serial dilutions are done according to D.5.4 using sterile bottles or flasks
(D.4.3). The following steps for enumeration of sulfite-reducing Clostridium spp. are done according to
the procedure described in the main text of this document (starting at 9.3).

D.6 Performance testing

For the definition of selectivity and productivity, refer to ISO 11133. In general, follow the procedures
for performance testing described in ISO 11133.
The performance testing for the quality assurance of the feed diluent is given in Table D.3.

Table D.3 — Performance testing for the quality assurance of the feed diluents
Control WDCM Method of
Medium Function Incubation Criteria
strains numbersa control
±30 % colonies / to (±30 % of
Initial feed diluent
45 min to 1 h/ Clostridium original count) on a suitable
solution, feed Diluent 00007b Quantitative
20 °C to 25 °C perfringens non-selective medium for
diluent solution
anaerobes
a Refer to the reference strain catalogue on http://​w ww​.wfcc​.info for information on culture collection strain numbers and contact
details; WDCM: World Data Centre for Microorganisms.
b Strain to be used as a minimum.

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Bibliography

[1] ISO 707, Milk and milk products — Guidance on sampling

[2] ISO  13307, Microbiology of food and animal feed — Primary production stage — Sampling
techniques
[3] ISO  15213-2,1)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
[4] ISO/TS  15213-3,2)Microbiology of the food chain — Horizontal method for the detection and
enumeration of Clostridium spp. — Part 3: Detection of Clostridium perfringens
[5] ISO 16140-4, Microbiology of the food chain — Method validation — Part 4: Protocol for method
validation in a single laboratory
[6] ISO  16140-6, Microbiology of the food chain — Method validation — Part 6: Protocol for the
validation of alternative (proprietary) methods for microbiological confirmation and typing
procedures
[7] ISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment
or revision of a standardized reference method
[8] ISO 17604, Microbiology of the food chain — Carcass sampling for microbiological analysis
[9] ISO/TS 17728, Microbiology of the food chain — Sampling techniques for microbiological analysis
of food and feed samples
[10] ISO 18593, Microbiology of the food chain — Horizontal methods for surface sampling
[11] VDLUFA 28.3.2, Futtermitteluntersuchung – Bestimmung von Sulfitreduzierenden Clostridien

1) Under preparation. Stage at the time of publication: ISO/DIS 15213-2:2022.

2) Under preparation. Stage at the time of publication: ISO/CD TS 15213-3:2018.

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ISO 15213-1:2023(E)

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