SIST EN ISO 22964:2017
(Main)Microbiology in the food chain - Horizontal method for the detection of Cronobacter spp. (ISO 22964:2017)
Microbiology in the food chain - Horizontal method for the detection of Cronobacter spp. (ISO 22964:2017)
This standard describes the detection of Enterobacter sakazakii (Reference document: ISO/TS 22964)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis von Cronobacter spp. (ISO 22964:2017)
Dieses Dokument legt ein horizontales Verfahren zum Nachweis von Cronobacter spp. fest.
Vorbehaltlich der in der Einleitung erörterten Einschränkungen gilt dieses Dokument für
- Lebensmittelprodukte und Zutaten, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind, und
- Umgebungsproben im Bereich der Herstellung von Lebensmitteln und des Umgangs mit Lebensmitteln.
Microbiologie de la chaine alimentaire - Méthode horizontale pour la détection de Cronobacter spp. (ISO 22964:2017)
ISO 22964:2017 spécifie une méthode horizontale pour la recherche de Cronobacter spp.
Sous réserve des restrictions exposées dans l'introduction, le présent document est applicable aux:
- produits et ingrédients alimentaires destinés à la consommation humaine et à l'alimentation animale;
- échantillons environnementaux prélevés dans les secteurs de la production et de la manutention des aliments.
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti Cronobacter spp. (ISO 22964:2017)
Ta standard opisuje način zaznavanja organizmov Enterobacter sakazakii (referenčni dokument: ISO/TS 22964)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 22964:2017
01-julij-2017
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti Cronobacter spp. (ISO 22964:2017)
Microbiology in the food chain - Horizontal method for the detection of Cronobacter spp.
(ISO 22964:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis von
Cronobacter spp. (ISO 22964:2017)
Microbiologie de la chaine alimentaire - Méthode horizontale pour la détection de
Cronobacter spp. (ISO 22964:2017)
Ta slovenski standard je istoveten z: EN ISO 22964:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 22964:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 22964:2017
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SIST EN ISO 22964:2017
EN ISO 22964
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2017
EUROPÄISCHE NORM
ICS 67.100.01; 07.100.30
English Version
Microbiology of the food chain - Horizontal method for the
detection of Cronobacter spp. (ISO 22964:2017)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche de Cronobacter spp. (ISO Verfahren zum Nachweis von Cronobacter spp. (ISO
22964:2017) 22964:2017)
This European Standard was approved by CEN on 13 March 2017.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22964:2017 E
worldwide for CEN national Members.
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SIST EN ISO 22964:2017
EN ISO 22964:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 22964:2017
EN ISO 22964:2017 (E)
European foreword
This document (EN ISO 22964:2017) has been prepared by Technical Committee ISO/TC 34 "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 October 2017, and conflicting national standards shall
be withdrawn at the latest by October 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 22964:2017 has been approved by CEN as EN ISO 22964:2017 without any modification.
3
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SIST EN ISO 22964:2017
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SIST EN ISO 22964:2017
INTERNATIONAL ISO
STANDARD 22964
First edition
2017-04
Microbiology of the food chain —
Horizontal method for the detection
of Cronobacter spp.
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche de Cronobacter spp.
Reference number
ISO 22964:2017(E)
©
ISO 2017
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SIST EN ISO 22964:2017
ISO 22964:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 22964:2017
ISO 22964:2017(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Principle . 2
5.1 Non-selective pre-enrichment in BPW . 2
5.2 Enrichment in a selective medium (CSB) . 2
5.3 Plating out and identification on chromogenic agar (CCI agar) . 2
5.4 Confirmation . 2
6 Culture media and reagents . 2
7 Equipment and consumables . 2
8 Sampling . 3
9 Preparation of test sample . 3
10 Procedure (as shown in Annex A) . 3
10.1 Test portion . 3
10.2 Pre-enrichment . 4
10.3 Enrichment . 4
10.4 Isolation of presumptive Cronobacter spp. 4
10.5 Confirmation . 4
10.5.1 General. 4
10.5.2 Purification of colonies . . 5
10.5.3 Biochemical confirmation . 5
10.6 Interpretation of biochemical results. 6
11 Expression of results . 6
12 Performance characteristics of the method . 7
12.1 Interlaboratory study . 7
12.2 Sensitivity . 7
12.3 Specificity . 7
12.4 LOD .
50 7
13 Test report . 7
Annex A (normative) Diagram of the test procedure . 8
Annex B (normative) Composition, preparation and performance testing of culture media
and reagents . 9
Annex C (informative) Distinction of Cronobacter spp. from other genera .17
Annex D (informative) Method validation studies and performance characteristics .19
Bibliography .22
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SIST EN ISO 22964:2017
ISO 22964:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation 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: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/275, Food Analysis — Horizontal methods, in collaboration with ISO Technical
Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the
agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This first edition cancels and replaces ISO/TS 22964:2006, which has been technically revised with the
following changes:
— the scope has been extended to Cronobacter spp. detection in food products for humans and feeding
animals and environmental samples and the title changed accordingly;
— the enrichment broth, modified lauryl sulfate tryptose broth (mLST), has been replaced by
Cronobacter selective broth (CSB);
— the isolation agar, Enterobacter sakazakii isolation agar (ESIA) has been replaced by chromogenic
Cronobacter isolation (CCI) agar;
— several confirmation tests have been replaced by other tests according to Table 1 of this document.
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SIST EN ISO 22964:2017
ISO 22964:2017(E)
Introduction
This document describes a horizontal method for the detection of Cronobacter spp. in food, in animal
feed and in environmental samples. The main changes, listed in the foreword, introduced in this
[2]
document compared to ISO/TS 22964:2006 are considered as major (see ISO 17468 ).
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, which 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 document 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 document so that eventually the only remaining departures from this horizontal method will
be those necessary for well-established technical reasons.
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SIST EN ISO 22964:2017
INTERNATIONAL STANDARD ISO 22964:2017(E)
Microbiology of the food chain — Horizontal method for
the detection of Cronobacter spp.
1 Scope
This document specifies a horizontal method for the detection of Cronobacter spp.
Subject to the limitations discussed in the introduction, this document is applicable to
— food products and ingredients 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 documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www.e lectropedia. org/
— ISO Online browsing platform: available at http:// www. iso. org/o bp
3.1
Cronobacter spp.
[10]
microorganisms which form typical colonies on chromogenic Cronobacter isolation (CCI) agar and
which display the biochemical characteristics described, when tests are carried out in accordance with
this document
3.2
detection of Cronobacter spp.
determination of Cronobacter spp. (3.1) in a particular mass or volume of product or surface area when
tests are carried out in accordance with this document
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SIST EN ISO 22964:2017
ISO 22964:2017(E)
4 Abbreviated terms
For the purposes of this document, the following abbreviations apply.
BPW buffered peptone water
CCI chromogenic Cronobacter isolation
CSB Cronobacter selective broth
TSA tryptone soya agar
5 Principle
5.1 Non-selective pre-enrichment in BPW
A test portion is inoculated into BPW, then incubated between 34 °C and 38 °C for 18 h ± 2 h.
NOTE Cronobacter spp. can be present in small numbers accompanied by other Enterobacteriaceae such as
E. cloacae that could interfere with their detection.
5.2 Enrichment in a selective medium (CSB)
The selective enrichment medium is inoculated with the culture obtained in 5.1 and incubated at
41,5 °C ± 1 °C for 24 h ± 2 h.
5.3 Plating out and identification on chromogenic agar (CCI agar)
The chromogenic agar is streaked for isolation with the enrichment culture obtained in 5.2 and
incubated at 41,5 °C ± 1 °C for 24 h ± 2 h.
5.4 Confirmation
Typical colonies are selected from the chromogenic agar, purified on a non-selective agar such as TSA
and biochemically characterized.
6 Culture media and reagents
For current laboratory practice, see ISO 7218 and ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
For performance testing of culture media see ISO 11133 and/or Annex B.
7 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
7.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).
As specified in ISO 7218.
7.2 Incubators, capable of operating in the range 34 °C to 38 °C, 37 °C ± 1 °C and 41,5 °C ± 1 °C.
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SIST EN ISO 22964:2017
ISO 22964:2017(E)
7.3 Sterile loops, of approximate diameter 3 mm (10 μl volume), and of 1 µl volume, and inoculation
needle or wire.
7.4 pH meter, having an accuracy of calibration of ± 0,1 pH unit at 25 °C.
7.5 Flasks and bottles, with closures, of suitable capacities for use in the preparation of enrichment
broths and agars and their storage.
7.6 Sterile graduated pipettes or automatic pipettes, of nominal capacities 10 ml, 1 ml and 0,1 ml.
7.7 Tubes (plugged or with caps) or culture bottles, of appropriate capacity, with non-toxic metallic
caps with liners or plastic disposable caps (see ISO 7218).
7.8 Petri dishes, of diameter approximately 90 mm.
7.9 Spectrophotometer, capable of measuring absorption of light with a wavelength of 405 nm.
7.10 Pestle and mortar.
7.11 Refrigerators, capable of operating at 5 °C ± 3 °C
7.12 Water baths, capable of operating between 47 °C and 50 °C and at 37 °C ± 1 °C.
7.13 Drying cabinet (or oven ventilated by convection), capable of being maintained between 25 °C
and 50 °C.
8 Sampling
Sampling is not part of the method specified in this document. See the specific International Standard
dealing with the product concerned. If there is no specific International Standard dealing with sampling
of the product concerned, it is recommended that the interested parties come to an agreement on this
subject.
[3] [4]
A recommended sampling method is given in ISO/TS 17728 for food and animal feed, and ISO 18593
for sampling of surfaces.
It is important that the laboratory receives a sample which is truly representative and which has not
been damaged or changed during transport or storage (see ISO 7218).
9 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International
Standard dealing with the product concerned: see ISO 6887 (all parts). If there is no specific
International Standard, it is recommended that the parties concerned come to an agreement on this
subject.
10 Procedure (as shown in Annex A)
10.1 Test portion
In general, to prepare the primary dilution, add 10 g or 10 ml of the test sample (Clause 9) to 90 ml of pre-
enrichment medium (B.1) (BPW), to yield a tenfold dilution. Pre-warm the BPW to room temperature
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ISO 22964:2017(E)
before use. For specific products, follow the procedures specified in ISO 6887 (all parts). For dry milk,
follow ISO 6887-5.
This document has been validated for test portions of 10 g. A smaller size of the test portion may be
used without the need of additional validation/verification providing that the same ratio between pre-
enrichment broth and test portion is maintained. A larger test portion than that initially validated may
be used, if a validation/verification study has shown that there are no negative effects on the detection
of Cronobacter spp.
NOTE 1 Validation can be conducted in accordance with the appropriate documents of ISO 16140 (all parts).
Verification for pooling samples can be conducted in accordance with the protocol described in ISO 6887-1:2017,
Annex D (verification protocol for pooling samples for qualitative tests).
NOTE 2 Large sample sizes can compromise the recovery of stressed Cronobacter spp. when interfering
[5[,[6]
microflora are present, such as probiotics.
For preparing quantities larger than 10 g, BPW should be pre-warmed between 34 °C and 38 °C (7.2)
before inoculated with the test portion.
10.2 Pre-enrichment
Incubate the inoculated pre-enrichment medium prepared in accordance with 10.1 between 34 °C and
38 °C (7.2) for 18 h ± 2 h.
10.3 Enrichment
After incubation of the inoculated pre-enrichment medium, mix well and transfer 0,1 ml of the obtained
culture 10.2 into 10 ml of CSB (B.2) and mix well. Incubate at 41,5 °C (7.2) for 24 h ± 2 h.
10.4 Isolation of presumptive Cronobacter spp.
Allow the CCI (B.3) plates to equilibrate at room temperature if they are stored at a lower temperature.
If necessary, dry the surface of the plates (7.13) following the procedure given in ISO 11133.
From enrichment culture, mix well and inoculate, by means of a 10 µl loop (7.3), the surface of the CCI
agar (B.3) to obtain well-separated colonies. Incubate the plate at 41,5 °C (7.2) for 24 h ± 2 h.
After incubation, examine the chromogenic plate for the presence of typical colonies of presumptive
Cronobacter.
Typical Cronobacter colonies on CCI are small to medium-sized (1 mm to 3 mm) and blue to blue-green
in colour. Non-Cronobacter colonies are often white or white with a green centre, grey or black. Some
naturally pigmented colonies of non-Cronobacter can appear yellow or red.
10.5 Confirmation
10.5.1 General
For confirmation, sub-culture from the selective medium CCI (see 10.4) five marked typical or suspect
colonies. In the case that colonies are not well separated, it might be necessary to streak a typical colony
first on the selective agar (B.3) again.
If on the dish there are fewer than five typical or suspect colonies, take all the marked colonies for
confirmation.
Use pure cultures for biochemical confirmation.
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ISO 22964:2017(E)
10.5.2 Purification of colonies
Streak the selected colonies onto the surface of a non-selective agar such as TSA (B.4) so as to gain well-
isolated colonies.
Incubate the plates inverted between 34 °C and 38 °C (7.2) for 21 h ± 3 h.
If the cultures on the non-selective agar are mixed, sub-culture the suspect colony onto a further plate
of the non-selective agar and incubate between 34 °C and 38 °C (7.2) for 21 h ± 3 h to obtain a pure
culture.
It is possible to first test the most characteristic colony from the selective agar plate. If positive, it is not
necessary to test other colonies. If negative, progress through the other selected colonies until either all
are negative or a positive is found.
Strains can be kept on the non-selective agar at 5 °C (7.11), but cannot be stored for more than seven
days. Fresh subcultures of the colonies should be obtained before performing confirmation tests.
10.5.3 Biochemical confirmation
10.5.3.1 General
Carry out the confirmation tests listed in Table 1.
Table 1 — Confirmation tests for Cronobacter spp.
Oxidase Acid from:
Hydrolysis of 4-Nitrophenyl α-D-glucopyranoside substrate D-Arabitol
L-Lysine decarboxylase D-Sorbitol
L-Ornithine decarboxylase D-Sucrose
Methyl Red (optional) α-Methyl-D-glucoside (optional)
Voges-Proskauer (optional)
NOTE 1 If shown to be reliable, miniaturized galleries for the biochemical identification of Cronobacter spp.,
can be used (see ISO 7218).
NOTE 2 Other alternative procedures can be used to confirm the isolate as Cronobacter spp., provided that the
suitability of the alternative procedure has been verified (see also ISO 7218).
10.5.3.2 Oxidase
Using a platinum-iridium or plastic loop (7.3), take a portion of a well-isolated colony from each
individual plate (10.5.2) and streak it on to a filter paper moistened with the oxidase reagent (B.5.1);
the appearance of a mauve, violet or deep blue colour within 10 s indicates a positive reaction. If a
commercially available oxidase test kit is used, follow the manufacturer’s instructions.
10.5.3.3 Hydrolysis of 4 Nitrophenyl (PNP) α-D-glucopyranoside substrate
Using a loop or wire (7.3), suspend an individual colony grown on the non-selective agar such as TSA
(10.5.2) in 2 ml of physiological salt solution, 0,85 % NaCl (B.5.2.4). Add 2 ml of the α-G
...
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