Microbiology of the food chain - Horizontal method for the detection and enumeration of Enterobacteriaceae - Part 1: Detection of Enterobacteriaceae (ISO 21528-1:2017)

ISO 21528-1:2017 specifies a method, with enrichment, for the detection of Enterobacteriaceae. It is applicable to
-      products intended for human consumption and the feeding of animals, and
-      environmental samples in the area of primary production, food production and food handling.
This method is applicable
-      when the microorganisms sought are expected to need resuscitation by enrichment, and
-      when the number sought is expected to be below 100 per millilitre or per gram of test sample.
A limitation on the applicability of ISO 21528-1:2017 is imposed by the susceptibility of the method to a large degree of variability (see Clause 11).
NOTE       Enumeration can be carried out by calculation of the most probable number (MPN) after incubation in liquid medium. See Annex A.

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die Zählung von Enterobacteriaceae Teil 1: Nachweis von Enterobacteriaceae (ISO 21528-1:2017)

Microbiologie de la chaîne alimentaire - Méthode horizontale par la recherche et le dénombrement des Enterobacteriaceae - Partie 1: Recherche des Enterobacteriaceae (ISO 21528-1:2017)

L'ISO 21528-1:2017 spécifie une méthode pour la recherche des Enterobacteriaceae avec enrichissement. Elle est applicable:
-      aux produits destinés à l'alimentation humaine et animale, et
-      aux échantillons d'environnement pour la production au stade primaire, la production des aliments et la distribution des aliments.
Cette méthode est applicable:
-      lorsque les micro-organismes recherchés nécessitent une revivification par enrichissement, et
-      lorsque le nombre recherché est supposé être inférieur à 100 par millilitre ou par gramme d'échantillon pour essai.
Des limites sont imposées aux possibilités d'application de l'ISO 21528-1:2017 du fait que ces méthodes sont sujettes à de grandes variations (voir l'Article 11).
NOTE       Le dénombrement peut être effectué en calculant le nombre le plus probable (NPP) après incubation en milieu liquide. Voir l'Annexe A.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila enterobakterij - 1. del: Ugotavljanje prisotnosti enterobakterij (ISO 21528-1:2017)

Ta standard določa metodo najverjetnejšega števila (MPN) s predhodno obogatitvijo za ugotavljanje prisotnosti enterobakterij. Uporablja se za:
– izdelke, namenjene za prehrano ljudi in krmo živali; in
– okoljske vzorce na območju proizvodnje hrane in ravnanja s hrano; in
– vzorce iz primarne proizvodnje.
(referenčni dokument: ISO 21528-1)

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SLOVENSKI STANDARD
SIST EN ISO 21528-1:2017
01-september-2017
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje

prisotnosti in števila enterobakterij - 1. del: Ugotavljanje prisotnosti enterobakterij

(ISO 21528-1:2017)

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

Enterobacteriaceae - Part 1: Detection of Enterobacteriaceae (ISO 21528-1:2017)

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die

Zählung von Enterobacteriaceae Teil 1: Nachweis von Enterobacteriaceae (ISO 21528-

1:2017)

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

dénombrement des Enterobacteriaceae - Partie 1: Recherche des Enterobacteriaceae
(ISO 21528-1:2017)
Ta slovenski standard je istoveten z: EN ISO 21528-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 21528-1:2017 en

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

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SIST EN ISO 21528-1:2017
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SIST EN ISO 21528-1:2017
EN ISO 21528-1
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2017
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for the
detection and enumeration of Enterobacteriaceae - Part 1:
Detection of Enterobacteriaceae (ISO 21528-1:2017)

Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales

horizontale par la recherche et le dénombrement des Verfahren für den Nachweis und die Zählung von

Enterobacteriaceae - Partie 1: Recherche des Enterobacteriaceae Teil 1: Nachweis von

Enterobacteriaceae (ISO 21528-1:2017) Enterobacteriaceae (ISO 21528-1:2017)
This European Standard was approved by CEN on 27 April 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 21528-1:2017 E

worldwide for CEN national Members.
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SIST EN ISO 21528-1:2017
EN ISO 21528-1:2017 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 21528-1:2017
EN ISO 21528-1:2017 (E)
European foreword

This document (EN ISO 21528-1:2017) 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 January 2018, and conflicting national standards shall

be withdrawn at the latest by January 2018.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN 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 21528-1:2017 has been approved by CEN as EN ISO 21528-1:2017 without any

modification.
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SIST EN ISO 21528-1:2017
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SIST EN ISO 21528-1:2017
INTERNATIONAL ISO
STANDARD 21528-1
Second edition
2017-06
Microbiology of the food chain —
Horizontal method for the detection
and enumeration of
Enterobacteriaceae —
Part 1:
Detection of Enterobacteriaceae
Microbiologie de la chaîne alimentaire — Méthode horizontale par
la recherche et le dénombrement des Enterobacteriaceae —
Partie 1: Recherche des Enterobacteriaceae
Reference number
ISO 21528-1:2017(E)
ISO 2017
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SIST EN ISO 21528-1:2017
ISO 21528-1:2017(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

the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
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 21528-1:2017
ISO 21528-1:2017(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

4.1 Enrichment in non-selective medium ................................................................................................................................. 2

4.2 Isolation and selection for confirmation .......................................................................................................................... 2

4.3 Confirmation ............................................................................................................................................................................................. 2

5 Diluent, culture media and reagent .................................................................................................................................................. 2

6 Equipment and consumables .................................................................................................................................................................. 2

7 Sampling ........................................................................................................................................................................................................................ 3

8 Preparation of test sample ......................................................................................................................................................................... 3

9 Procedure..................................................................................................................................................................................................................... 3

9.1 General ........................................................................................................................................................................................................... 3

9.2 Test portion and initial suspension ....................................................................................................................................... 4

9.3 Enrichment ................................................................................................................................................................................................. 4

9.4 Isolation and selection for confirmation .......................................................................................................................... 4

9.4.1 Isolation ................................................................................................................................................................................... 4

9.4.2 Selection of colonies for confirmation .......................................................................................................... 4

9.5 Subculturing selected colonies.................................................................................................................................................. 4

9.6 Biochemical confirmation tests ................................................................................................................................................ 4

9.6.1 Oxidase reaction ............................................................................................................................................................... 4

9.6.2 Fermentation test ............................................................................................................................................................ 5

10 Expression of results ........................................................................................................................................................................................ 5

11 Precision ....................................................................................................................................................................................................................... 5

11.1 Interlaboratory study ........................................................................................................................................................................ 5

11.2 Sensitivity .................................................................................................................................................................................................... 5

11.3 Specificity .................................................................................................................................................................................................... 5

12 Test report ................................................................................................................................................................................................................... 5

13 Quality assurance ................................................................................................................................................................................................ 6

Annex A (informative) Enumeration by MPN technique .................................................................................................................. 7

Annex B (normative) Culture media and reagents .............................................................................................................................10

Annex C (informative) Method validation studies and performance characteristics .....................................15

Bibliography .............................................................................................................................................................................................................................17

© ISO 2017 – All rights reserved iii
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SIST EN ISO 21528-1:2017
ISO 21528-1:2017(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 meaning of ISO specific terms and expressions related to conformity assessment,

as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the

Technical Barriers to Trade (TBT) see the following URL: www . i so .org/ iso/ foreword .html

This document was prepared by the European Committee for Standardization (CEN) Technical

Committee CEN/TC 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 second edition cancels and replaces the first edition (ISO 21528-1:2004), which has been technically

revised with the following main changes:
— the MPN method has become an informative Annex A;

— the pre-enrichment step in BPW followed by enrichment in EE broth has been changed to enrichment

[7]

in BPW and confirmation now takes place in Glucose OF medium instead of using glucose agar;

— performance testing for the quality assurance of the culture media has been added;

— performance characteristics for this method have been added to Annex C.
A list of all the parts in the ISO 21528 series can be found on the ISO website.
iv © ISO 2017 – All rights reserved
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SIST EN ISO 21528-1:2017
ISO 21528-1:2017(E)
Introduction

This document is intended to provide general guidance for the examination of products not dealt

with by existing International Standards and to be taken into account by organizations preparing

microbiological test methods for application to foods or animal feeding stuffs. Because of the large

variety of products within this field of application, these guidelines may not be appropriate in every

detail for certain products, and for some other products it may be necessary to use different methods.

Nevertheless, it is hoped that in all cases, every attempt will be made to apply the guidelines provided

as far as possible and that deviations from them will only be made if absolutely necessary for technical

reasons.

The main changes, listed in the foreword, introduced in this document compared to ISO 21528-1:2004

are considered as major (see ISO 17468).

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.
© ISO 2017 – All rights reserved v
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SIST EN ISO 21528-1:2017
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SIST EN ISO 21528-1:2017
INTERNATIONAL STANDARD ISO 21528-1:2017(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Enterobacteriaceae —
Part 1:
Detection of Enterobacteriaceae

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

detecting Enterobacteriaceae 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 a method, with enrichment, for the detection of Enterobacteriaceae. It is

applicable to
— products intended for human consumption and the feeding of animals, and

— environmental samples in the area of primary production, food production and food handling.

This method is applicable

— when the microorganisms sought are expected to need resuscitation by enrichment, and

— when the number sought is expected to be below 100 per millilitre or per gram of test sample.

A limitation on the applicability of this document is imposed by the susceptibility of the method to a

large degree of variability (see Clause 11).

NOTE Enumeration can be carried out by calculation of the most probable number (MPN) after incubation in

liquid medium. See Annex A.
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 2017 – All rights reserved 1
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SIST EN ISO 21528-1:2017
ISO 21528-1:2017(E)

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
Enterobacteriaceae

microorganism that forms characteristic colonies on violet red bile glucose agar and that ferment

glucose and show a negative oxidase reaction when the tests are carried out in accordance with the

methods specified in this document
3.2
detection of Enterobacteriaceae

determination of Enterobacteriaceae (3.1), in a particular mass or volume of product or surface area,

when tests are carried out in accordance with this document
4 Principle
4.1 Enrichment in non-selective medium

Buffered peptone water (BPW) is inoculated with the test portion, and then incubated at 37 °C (or

30 °C) for 18 h.

NOTE The incubation temperature of 37 °C for enrichment and isolation/enumeration on plating medium is

generally used when the detection and enumeration of Enterobacteriaceae is for a hygiene indicator. Alternatively,

a temperature of 30 °C can be chosen when the detection or enumeration of Enterobacteriaceae is conducted for

technological purposes and includes psychrotrophic Enterobacteriaceae. In this document, 37 °C will be used

throughout the text.
4.2 Isolation and selection for confirmation

Violet red bile glucose (VRBG) agar is inoculated with the culture obtained after enrichment in

BPW, then incubated at 37 °C. It is examined after 24 h to detect the presence of typical colonies of

presumptive Enterobacteriaceae.
4.3 Confirmation

Typical colonies of presumptive Enterobacteriaceae are subcultured onto non-selective medium, and

confirmed by means of tests for the fermentation of glucose and the presence of oxidase.

5 Diluent, culture media and reagent
For current laboratory practice, see ISO 7218.

Composition of culture media and reagents and their preparation are specified in Annex B.

For performance testing of culture media, see ISO 11133 and Annex B.
6 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.

6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), as specified in ISO 7218.

6.2 Incubator, capable of operating at 37 °C ± 1 °C (or 30 °C ± 1 °C).
2 © ISO 2017 – All rights reserved
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SIST EN ISO 21528-1:2017
ISO 21528-1:2017(E)

6.3 Drying cabinet (ventilated by convection) or incubator, capable of operating between 25 °C

and 50 °C.

6.4 Water bath, or similar apparatus, capable of being maintained between 47 °C to 50 °C.

6.5 Containers (e.g. bottles, tubes, flasks), suitable for the sterilization and storage of culture media.

6.6 Test tubes or flasks of appropriate capacity.
6.7 Petri dishes, made of glass or plastics, of diameter 90 mm to 100 mm.

6.8 Loops (of diameter approximately 3 mm) and wires, made of platinum/iridium or

nickel/chromium, or glass rods, or equivalent sterile disposable loops or inoculating needles.

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

6.10 pH-meter, accurate to within ±0,1 pH unit at 25 °C.
6.11 Homogenizer, as specified in ISO 7218.
7 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 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:
— ISO/TS 17728 for food and animal feed;
— ISO 13307 for primary production stage;
— ISO 17604 for carcasses;
— ISO 18593 for environmental samples.

It is important that the laboratory receives a sample that is representative and the sample should not

have been damaged or changed during transport or storage.
8 Preparation of test sample

Prepare the test sample in accordance with the specific International Standard appropriate to the

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

parties concerned come to an agreement on this subject.
9 Procedure
9.1 General
See ISO 7218.
© ISO 2017 – All rights reserved 3
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SIST EN ISO 21528-1:2017
ISO 21528-1:2017(E)
9.2 Test portion and initial suspension

In general, an amount of test portion (mass or volume) is added to a quantity of BPW (mass or volume)

to yield a 10-fold dilution. For example, a 10 g test portion is mixed with 90 ml of BPW.

This document has been validated for test portions of 10 g or ml. A smaller test portion may be

used, without the need for additional validation/verification, providing that the same ratio between

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 adverse effects on the detection

of Enterobacteriaceae.

NOTE 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.
9.3 Enrichment
Incubate the initial suspension (9.2) at 37 °C for 18 h ± 2 h.

Continue the procedure with isolation and selection of colonies for confirmation (9.4).

9.4 Isolation and selection for confirmation
9.4.1 Isolation

Using a loop (6.8), streak from the incubated enrichment medium (see 9.3) the surface of a plate

containing the selective medium (B.2) and incubate the plate at 37 °C (see note in Clause 4) for 24 h ± 2 h.

9.4.2 Selection of colonies for confirmation

Characteristic colonies are pink to red or purple (with or without precipitation haloes).

Mark suspect colonies from the incubated plates (see 9.4.1). Select at least one typical or suspect colony

for subculture (see 9.5) and biochemical confirmation tests (see 9.6). If this is negative, select up to four

more suspect colonies.

If more than one morphology is present among the colonies, select one colony of each morphology for

subculture.

Certain Enterobacteriaceae may cause decolouration of their colonies or of the medium. Therefore,

when no characteristic colonies are present, choose whitish colonies for confirmation.

9.5 Subculturing selected colonies

Streak onto non-selective medium (e.g. nutrient agar plates) (B.3) each of the colonies selected for

confirmation (see 9.4.2).
Incubate these plates at 37 °C for 24 h ± 2 h.

Select a well-isolated colony from each of the incubated plates for the biochemical confirmation tests

(see 9.6).
9.6 Biochemical confirmation tests
9.6.1 Oxidase reaction

Using a platinum/iridium loop, wire or glass rod (6.8), take a portion of each well-isolated colony (see

9.5) and streak onto a filter paper moistened with the oxidase reagent (B.5) or onto a commercially

available disc or stick. A nickel/chromium loop or wire shall not be used.
4 © ISO 2017 – All rights reserved
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SIST EN ISO 21528-1:2017
ISO 21528-1:2017(E)

Consider the test to be negative if the colour of the filter paper does not turn dark blue purple within 10 s.

Consult the manufacturer’s instructions for ready-to-use discs or sticks.
9.6.2 Fermentation test

Using a wire (6.8), stab the same colonies selected in 9.6 that gave a negative oxidase test into tubes

containing Glucose OF medium (B.4). Overlay the surface of the medium with minimal 1 cm of sterile

mineral oil (B.6).
Incubate these tubes at 37 °C for 24 h ± 2 h.

If a yellow colour develops throughout the content of the tube, regard the reaction as being positive.

10 Expression of results

If any of the selected typical colonies (see 9.4.2) from a subculture (see 9.4.1) is oxidase-negative and

glucose-positive, the sample from which the subculture was derived shall be regarded as being positive

for Enterobacteriaceae. In accordance with the interpretation of the results, indicate Enterobacteriaceae

detected or not detected in a test portion of x g or x ml of product, or on the surface area swabbed, or in

entire objects (e.g. boot socks).
11 Precision
11.1 Interlaboratory study

The performance characteristics of the method were determined in an interlaboratory study to

determine the specificity, sensitivity and the LOD of the method. The data are summarized in

Annex C. The values derived from the interlaboratory study may not be applicable to food types other

than those given in Annex C.

NOTE In this document, the word “type” is combined with “food” to improve the readability of this document.

However, the word “food” is interchangeable with “feed” and the other areas of the food cha

...

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