EN ISO 7937:2004

SLOVENSKI STANDARD
01-januar-2005
1DGRPHãþD
SIST EN 13401:1999
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje števila
Clostridium perfringens - Tehnika štetja kolonij (ISO 7937:2004)
Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of
Clostridium perfringens - Colony-count technique (ISO 7937:2004)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur Zählung
von Clostridium perfringens - Koloniezählverfahren (ISO 7937:2004)
Microbiologie des aliments - Méthode horizontale pour le dénombrement de Clostridium
perfringens - Technique par comptage des colonies (ISO 7937:2004)
Ta slovenski standard je istoveten z: EN ISO 7937:2004
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 7937
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2004
ICS 07.100.30 Supersedes EN 13401:1999
English version
Microbiology of food and animal feeding stuffs - Horizontal
method for the enumeration of Clostridium perfringens - Colony-
count technique (ISO 7937:2004)
Microbiologie des aliments - Méthode horizontale pour le Mikrobiologie von Lebensmitteln und Futtermitteln -
dénombrement de Clostridium perfringens - Technique par Horizontales Verfahren zur Zählung von Clostridium
comptage des colonies (ISO 7937:2004) perfringens - Koloniezählverfahren (ISO 7937:2004)
This European Standard was approved by CEN on 14 August 2004.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Central Secretariat or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2004 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 7937:2004: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 7937:2004) has been prepared by Technical Committee ISO/TC 34
"Agricultural food products" in collaboration with Technical Committee CEN/TC 275 "Food
analysis - Horizontal methods", the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of
an identical text or by endorsement, at the latest by February 2005, and conflicting national
standards shall be withdrawn at the latest by February 2005.

This document supersedes EN 13401:1999.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice
The text of ISO 7937:2004 has been approved by CEN as EN ISO 7937:2004 without any
modifications.
INTERNATIONAL ISO
STANDARD 7937
Third edition
2004-08-15
Microbiology of food and animal feeding
stuffs — Horizontal method for the
enumeration of Clostridium
perfringens — Colony-count technique
Microbiologie des aliments — Méthode horizontale pour le
dénombrement de Clostridium perfringens — Technique par comptage
des colonies
Reference number
ISO 7937:2004(E)
©
ISO 2004
ISO 7937:2004(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2004
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2004 – All rights reserved

ISO 7937:2004(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 2
4 Principle. 2
5 Diluent, culture media and reagents . 2
6 Apparatus and glassware. 7
7 Sampling. 7
8 Preparation of test sample. 7
9 Procedure. 7
10 Expression of results. 9
11 Test report. 11
Annex A (informative) Results of interlaboratory test . 12
Bibliography . 16

ISO 7937:2004(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 7937 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
This third edition cancels and replaces the second edition (ISO 7937:1997, EN 13401:1999), which has been
technically revised.
iv © ISO 2004 – All rights reserved

ISO 7937:2004(E)
Introduction
Because of the large variety of food and feed products, this horizontal method may not be appropriate in every
detail for certain products. In this case, different methods that are specific to these products may be used if
absolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this
horizontal method as far as possible.
When this International Standard is next reviewed, account will be taken of all information then available
regarding the extent to which this horizontal method has been followed and the reasons for deviations from
this method in the case of particular products.
The harmonization of test methods cannot be immediate and, for certain groups of products, International
Standards and/or national standards may already exist that do not comply with this horizontal method. It is
hoped that when such standards are reviewed they will be changed to comply with this international Standard
so that eventually the only remaining departures from this horizontal method will be those necessary for
well-established technical reasons.

INTERNATIONAL STANDARD ISO 7937:2004(E)

Microbiology of food and animal feeding stuffs — Horizontal
method for the enumeration of Clostridium perfringens —
Colony-count technique
1 Scope
This International Standard describes a horizontal method for the enumeration of viable Clostridium
perfringens. It is applicable to
 products intended for human consumption and the feeding of animals, and
 environmental samples in the area of food production and food handling.
2 Normative references
The following referenced documents are indispensable for the application 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 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions
ISO 6887-2, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 2: Specific rules for the preparation of meat and
meat products
ISO 6887-3, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 3: Specific rules for the preparation of fish and
fishery products
ISO 6887-4, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 4: Specific rules for the preparation of products
other than milk and milk products meat and meat products, and fish and fishery products
ISO 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations
ISO 8261, Milk and milk products — General guidance for the preparation of test samples, initial suspensions
and decimal dilutions for microbiological examination
ISO/TS 11133-1, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production
of culture media — Part 1 : General guidelines on quality assurance for the preparation of culture media in the
laboratory
ISO/TS 11133-2:2003, Microbiology of food and animal feeding stuffs — Guidelines on preparation and
production of culture media — Part 2: Practical guidelines on performance testing of culture media
ISO 7937:2004(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Clostridium perfringens
C. perfringens
bacteria that form characteristic colonies (black precipitate, caused by the reduction of sulfite to sulfide, which
colours the colonies black) in the specified selective medium, and which give positive confirmatory reactions
when the test is carried out by either of two techniques specified in this International Standard
3.2
enumeration of C. perfringens
determination of the number of culturable and confirmed Clostridium perfringens bacteria per millilitre or per
gram of sample when the test is carried out by the method specified in this International Standard
4 Principle
4.1 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.
Further 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 an overlay of the same medium.
4.2 The plates are incubated anaerobically at 37 °C for 20 h ± 2 h.
4.3 The characteristic colonies are enumerated.
4.4 The numbers of characteristic colonies are confirmed and the number of C. perfringens per millilitre or
per gram of sample is calculated.
5 Diluent, culture media and reagents
See ISO 7218, ISO/TS 11133-1 and ISO/TS 11133-2 for the preparation, production and performance testing
of culture media.
5.1 Diluent
See the relevant part of ISO 6887 or ISO 8261.
5.2 Sulfite-cycloserine agar (SC)
NOTE This was originally designated “egg-yolk-free TSC” (see [1]).
2 © ISO 2004 – All rights reserved

ISO 7937:2004(E)
5.2.1 Base
5.2.1.1 Composition
Enzymatic digest of protein 15,0 g
Enzymatic digest of soya 5,0 g
Yeast extract 5,0 g
Disodium disulfite (Na S O ), anhydrous 1,0 g
2 2 5
a
Ammonium iron(III) citrate 1,0 g
b
Agar 9,0 g to 18,0 g
Water 1 000 ml
a
This reagent should contain at least 15 % (mass fraction) of iron.
b
Depending on the gel strength of the agar.

5.2.1.2 Preparation
Dissolve the components in the water by boiling. Adjust the pH so that after sterilization it will be 7,6 ± 0,2 at
25 °C. Dispense the base into flasks or bottles of appropriate capacity. Sterilize for 15 min at 121 °C. Store in
a refrigerator at 5 °C ± 3 °C. Discard unused medium 2 weeks after preparation.
In some cases (see 9.4.3.1), it may be necessary to prepare dishes of SC agar base medium for confirmation
with the nitrate motility medium (5.5) and the lactose-gelatin medium (5.8). For this purpose, transfer portions
of about 15 ml of the base [melted and cooled to approximately 44 °C to 47 °C using a water bath (6.10)] into
Petri dishes and allow to solidify. Immediately before use, dry the plates (see ISO 7218).
5.2.2 D-Cycloserine solution
5.2.2.1 Composition
a
D-Cycloserine 4,0 g
Water 100 ml
a
Use white crystalline powder only.

5.2.2.2 Preparation
Dissolve the D-cycloserine in the water and sterilize the solution by filtration.
Store in a refrigerator at 3 °C ± 2 °C.
Discard unused solution 4 weeks after preparation.
5.2.3 Complete medium
Immediately before use in the pour-plate method (see 9.2), to each 100 ml of sterile molten base (5.2.1)
cooled to 44 °C to 47 °C, add 1 ml of D-cycloserine solution (5.2.2).
5.2.4 Performance testing for the quality assurance of SC medium
For the definition of selectivity and productivity, refer to ISO/TS 11133-1. To check the performance, refer to
ISO/TS 11133-2:2003, Table B.1 [see TS(C)].
ISO 7937:2004(E)
5.3 Fluid thioglycollate medium
5.3.1 Composition
Enzymatic digest of casein 15,0 g
L-Cystine 0,5 g
D-Glucose 5,5 g
Yeast extract 5,0 g
Sodium chloride 2,5 g
Sodium thioglycollate (mercaptoacetate) 0,5 g
a
Agar
0,5 g to 2,0 g
Resazurin 0,001 g
Water 1 000 ml
a
Depending on the gel strength of the agar.

5.3.2 Preparation
Dissolve the components in the water by boiling. Adjust the pH so that after sterilization it will be 7,1 ± 0,2 at
25 °C.
Dispense 10 ml portions into tubes and sterilize at 121 °C for 15 min.
Before use, this medium shall be de-aerated.
5.3.3 Performance testing for the quality assurance of thioglycollate broth
For the definition of selectivity and productivity, refer to ISO/TS 11133-1. To check the performance, refer to
ISO/TS 11133-2:2003, Table B.4.
5.4 Lactose sulfite medium (LS) (optional)
5.4.1 Base medium
5.4.1.1 Composition
Enzymatic digest of casein 5,0 g
Yeast extract 2,5 g
Sodium chloride 2,5 g
Lactose 10 g
L-Cysteine hydrochloride 0,3 g
Water 1 000 ml
5.4.1.2 Preparation
Dissolve the components in the water by boiling (if necessary). Adjust the pH so that after sterilization it will be
7,1 ± 0,2 at 25 °C.
Dispense 8 ml portions into test tubes with inverted Durham tubes (6.7) and sterilize at 121 °C for 15 min.
The medium may be stored at 3 °C ± 2 °C for up to 4 weeks.
4 © ISO 2004 – All rights reserved

ISO 7937:2004(E)
5.4.2 Disodium disulfite solution
5.4.2.1 Composition
Disodium disulfite (Na S O ), anhydrous 1,2 g
2 2 5
Water 100 ml
5.4.2.2 Preparation
Dissolve the disodium disulfite in the water and sterilize the solution by filtration.
Use the solution within a day.
5.4.3 Ammonium iron(III) citrate solution
5.4.3.1 Composition
Ammonium iron(III) citrate 1 g
Water 100 ml
5.4.3.2 Preparation
Dissolve the ammonium iron(III) citrate in the water and sterilize the solution by filtration.
Use the solution within a day.
5.4.4 Complete medium
If the medium is not used on the day of preparation, just prior to completion de-aerate the medium by heating
and then cool rapidly. If the medium is in screw-cap bottles, loosen the caps before heating and tighten them
before cooling.
Then add 0,5 ml of the disodium disulfite solution (5.4.2) and 0,5 ml of the ammonium iron(III) citrate solution
(5.4.3) to each 8 ml of base (5.4.1).
Use the complete medium within a day.
5.5 Nitrate motility medium (optional)
5.5.1 Composition
Enzymatic digest of casein 5,0 g
Meat extract 3,0 g
Galactose 5,0 g
Glycerol 5,0 g
Potassium nitrate (KNO) 1,0 g
Disodium hydrogen orthophosphate (Na HPO) 2,5 g
2 4
a
Agar 1,0 g to 5,0 g
Water 1 000 ml
a
Depending on the gel strength of the agar.

ISO 7937:2004(E)
5.5.2 Preparation
Dissolve the components in the water by boiling. Adjust the pH so that it will be 7,3 ± 0,2 at 25 °C after
sterilization.
Transfer the medium to culture tubes in 10 ml quantities and sterilize at 121 °C for 15 min. If not used the
same day, store in a refrigerator at 5 °C ± 3 °C. Just prior to use, heat in boiling water or steam for 15 min and
then cool rapidly to the incubation temperature.
Discard unused medium 4 weeks after preparation.
5.6 Nitrite detection reagent (optional)
5.6.1 5-Amino-2-naphthalenesulfonic acid (5-2-ANSA) solution
Dissolve 0,1 g of 5-2-ANSA in 100 ml of 15 % (v
...