ISO/TS 22964:2006

TECHNICAL ISO/TS
SPECIFICATION 22964
IDF/RM
210
First edition
2006-02-01

Milk and milk products — Detection
of Enterobacter sakazakii
Lait et produits laitiers — Détection de l'Enterobacter sakazakii




Reference numbers
ISO/TS 22964:2006(E)
IDF/RM 210:2006(E)
©
ISO and IDF 2006

---------------------- Page: 1 ----------------------
ISO/TS 22964:2006(E)
IDF/RM 210:2006(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. Neither the ISO Central
Secretariat nor the IDF accepts any 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
and IDF national committees. In the unlikely event that a problem relating to it is found, please inform the ISO Central Secretariat at the
address given below.


©  ISO and IDF 2006
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 or IDF at the respective
address below.
ISO copyright office International Dairy Federation
Case postale 56 • CH-1211 Geneva 20 Diamant Building • Boulevard Auguste Reyers 80 • B-1030 Brussels
Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88
Fax + 41 22 749 09 47 Fax + 32 2 733 04 13
E-mail copyright@iso.org E-mail info@fil-idf.org
Web www.iso.org Web www.fil-idf.org
Published in Switzerland

ii © ISO and IDF 2006 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/TS 22964:2006(E)
IDF/RM 210:2006(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
4 Principle (see also annex A). 1
4.1 Pre-enrichment in non-selective liquid medium. 1
4.2 Enrichment in selective liquid medium . 1
4.3 Plating out and identification . 2
4.4 Confirmation. 2
5 Culture media and reagents . 2
5.1 General. 2
5.2 Culture media . 2
6 Apparatus and glassware. 7
7 Sampling. 8
8 Preparation of test sample. 8
9 Procedure (see the scheme in Annex A). 8
9.1 Test portion . 8
9.2 Pre-enrichment. 8
9.3 Selective enrichment. 8
9.4 Isolation of presumptive Enterobacter sakazakii . 8
9.5 Confirmation. 9
9.6 Interpretation of the results of the confirmation tests. 10
10 Control cultures . 11
11 Expression of results. 11
12 Test report. 11
Annex A (informative) Method flow scheme. 12
Bibliography . 13

© ISO and IDF 2006 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO/TS 22964:2006(E)
IDF/RM 210:2006(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.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of normative document:
⎯ an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members
of the parent committee casting a vote;
⎯ an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting
a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a
further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is
confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an
International Standard or be withdrawn.
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/TS 22964|IDF/RM 210 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO
and IDF.

iv © ISO and IDF 2006 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/TS 22964:2006(E)
IDF/RM 210:2006(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of
standard methods of analysis and sampling for milk and milk products.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting.
Publication as an International Standard requires approval by at least 50 % of IDF National Committees
casting a vote.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
Standing Committee may decide to publish another type of normative document which is called by IDF:
Reviewed method. Such a method represents an agreement between the members of a Standing Committee
and is accepted for publication if it is approved by at least 50 % of the committee members casting a vote. A
Reviewed method is equal to an ISO/PAS or ISO/TS and will, therefore, also be published jointly under ISO
conditions.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. IDF shall not be held responsible for identifying any or all such patent rights.
ISO/TS 22964|IDF/RM 210 was prepared by the International Dairy Federation (IDF) and Technical
Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published
jointly by IDF and ISO.
All work was carried out by the Joint ISO-IDF Action Team on Harmonization, of the Standing Committee on
Microbiological methods of analysis, under the aegis of its project leaders, Mr D.J.C. van den Berg (NL) and
Mr H. Joosten (CH).


© ISO and IDF 2006 – All rights reserved v

---------------------- Page: 5 ----------------------
ISO/TS 22964:2006(E)
TECHNICAL SPECIFICATION
IDF/RM 210:2006(E)

Milk and milk products — Detection of Enterobacter sakazakii
1 Scope
This Technical Specification specifies a method for the detection of Enterobacter sakazakii in milk powder and
powdered infant formula.
The method is also applicable to environmental samples collected from milk powder or infant formula factories.
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 8261|IDF 122, Milk and milk products — General guidance for the preparation of test samples, initial
suspensions and decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
presumptive Enterobacter sakazakii
microorganisms which form typical colonies on a chromogenic isolation agar, when tests are carried out in
accordance with this Technical Specification
3.2
Enterobacter sakazakii
microorganisms which form typical colonies on a chromogenic isolation agar, form yellow colonies on tryptone
soya agar and display biochemical characteristics as described, when tests are carried out in accordance with
this Technical Specification
4 Principle (see also annex A)
4.1 Pre-enrichment in non-selective liquid medium
The pre-enrichment medium is inoculated with the test portion and incubated at 37 °C ± 1 °C for 16 h to 20 h.
4.2 Enrichment in selective liquid medium
The selective enrichment medium is inoculated with the culture obtained in 4.1 and incubated at
44 °C ± 0,5 °C for 22 h to 26 h.
© ISO and IDF 2006 – All rights reserved 1

---------------------- Page: 6 ----------------------
ISO/TS 22964:2006(E)
IDF/RM 210:2006(E)
4.3 Plating out and identification
A chromogenic agar is inoculated with the enrichment culture obtained in 4.2 and incubated at 44 °C ± 1 °C
for 22 h to 26 h.
4.4 Confirmation
Typical colonies are selected from the chromogenic agar, and isolates producing a yellow pigment on tryptone
soya agar are biochemically characterized.
5 Culture media and reagents
5.1 General
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized
water or water of equivalent purity. The water shall be free from substances that might inhibit the growth of
microorganisms under the test conditions specified in this Technical Specification. See also ISO 6887-1 and
ISO 8261|IDF 122.
In order to improve the reproducibility of the results, it is recommended that, for the preparation of culture
media, dehydrated basic components or dehydrated complete media be used. In that case, follow the
manufacturer's instructions rigorously. See also ISO 6887-1.
The pH values given refer to a temperature of 25 °C. Adjustments, if necessary, are made by adding either
hydrochloric acid [c(HCl) = 1 mol/l] or sodium hydroxide solution [c(NaOH) = 1 mol/l].
If not used immediately, store the prepared culture media and reagents under conditions that do not produce
any change in their composition, in the dark at a temperature between 0 °C and 5 °C, for no longer than
1 month, unless otherwise stated.
5.2 Culture media
5.2.1 Buffered peptone water (BPW)
5.2.1.1 Composition
Enzymatic digest of casein 10,0 g
Sodium chloride (NaCl) 5,0 g
Disodium hydrogen phosphate dodecahydrate (Na HPO ·12 HO) 9,0 g
2 4 2
Potassium dihydrogen phosphate (KH PO) 1,5 g
2 4
Water 1 000 ml
5.2.1.2 Preparation
Dissolve each of the components in the water, by heating if necessary. Adjust the pH, if necessary, to
7,0 ± 0,2 at 25 °C. Distribute the BPW in flasks or tubes according to the analytical needs. Sterilize at 121 °C
for 15 min.
2 © ISO and IDF 2006 – All rights reserved

---------------------- Page: 7 ----------------------
ISO/TS 22964:2006(E)
IDF/RM 210:2006(E)
5.2.2 Modified lauryl sulfate tryptose broth (mLST)/vancomycin medium
5.2.2.1 Modified lauryl sulfate tryptose broth (mLST)
5.2.2.1.1 Composition
Sodium chloride (NaCl) 34,0 g
Enzymatic digest of animal and plant tissue 20,0 g
Lactose (C H O) 5,0 g
12 22 11
Potassium dihydrogen phosphate (KH PO) 2,75 g
2 4
Dipotassium hydrogen phosphate (K HPO) 2,75 g
2 4
Sodium lauryl sulfate (C H NaOS) 0,1 g
12 25 5
Water 1 000 ml
5.2.2.1.2 Preparation
Dissolve each of the components in the water, by heating if necessary.
Adjust the pH, if necessary, to 6,8 ± 0,2 at 25 °C. Dispense 10 ml of mLST into tubes of dimensions
18 mm × 160 mm.
Sterilize the tubes at 121 °C for 15 min.
5.2.2.2 Vancomycin solution
5.2.2.2.1 Composition
Vancomycin 10 mg
Water 10 ml
5.2.2.2.2 Preparation
Dissolve the vancomycin in the distilled water. Mix and sterilize by filtration.
The vancomycin solution may be kept at 0 °C to 5 °C for 15 days.
5.2.2.3 mLST/vancomycin medium
Add 0,1 ml of vancomycin solution (5.2.2.2.2) to 10 ml of mLST solution (5.2.2.1.2) so as to obtain a final
vancomycin concentration of 10 µg per millilitre of mLST.
The complete mLST/vancomycin medium may be kept at 0 °C to 5 °C for 1 day.
© ISO and IDF 2006 – All rights reserved 3

---------------------- Page: 8 ----------------------
ISO/TS 22964:2006(E)
IDF/RM 210:2006(E)
TM
1)
5.2.3 Enterobacter sakazakii isolation agar (ESIA )
5.2.3.1 Composition
Pancreatic peptone of casein 7,0 g
Yeast ex
...