Iso 21528 1 2004

INTERNATIONAL ISO
STANDARD 21528-1
First edition
2004-08-15
Microbiology of food and animal feeding

stuffs — Horizontal methods for the
detection and enumeration of
Enterobacteriaceae —
Part 1:
Detection and enumeration by MPN
iTeh STANDARD PREVIEW
technique with pre-enrichment
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Microbiologie des aliments — Méthodes horizontales pour la recherche
et le dénombrement des Enterobacteriaceae —
ISO 21528-1:2004
Partie 1: Recherche et dénombrement à l'aide de la technique NPP
https://standards.iteh.ai/catalog/standards/sist/2f5ef1a0-d39a-489b-ac2d-
avec préenrichissement
970987355d9a/iso-21528-1-2004
Reference number
ISO 21528-1:2004(E)
© ISO 2004
ISO 21528-1:2004(E)
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ISO 21528-1:2004
970987355d9a/iso-21528-1-2004
© 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,
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ii © ISO 2004 – All rights reserved

ISO 21528-1:2004(E)
Contents Page
Foreword ............................................................................................................................................................ iv
Introduction ........................................................................................................................................................ v
1 Scope...................................................................................................................................................... 1
2 Normative references ........................................................................................................................... 1
3 Terms and definitions........................................................................................................................... 2
4 Principle ................................................................................................................................................. 2
4.1 Detection of Enterobacteriaceae (see Annex A) ................................................................................. 2
4.2 Enumeration by the MPN technique (see Annex B) ........................................................................... 3
5 Diluent, culture media and reagent ..................................................................................................... 3
6 Apparatus and glassware..................................................................................................................... 7
7 Sampling ................................................................................................................................................ 7
8 Preparation of test sample ................................................................................................................... 7
9 Procedure............................................................................................................................................... 7
9.1 iTeh STANDARD PREVIEW
General ................................................................................................................................................... 7
9.2 Detection method .................................................................................................................................. 8
9.3 (standards.iteh.ai)
Enumeration method (MPN method)................................................................................................... 8
9.4 Isolation and selection for confirmation............................................................................................. 9
9.5 Subculturing selected colonies ...........................................................................................................
ISO 21528-1:2004 9
9.6 Biochemicalhttps://standards.iteh.ai/catalog/standards/sist/2f5ef1a0-d39a-489b-ac2d-
confirmation tests........................................................................................................... 9
10 970987355d9a/iso-21528-1-2004
Expression of results............................................................................................................................ 9
10.1 Confirmed positive tubes ..................................................................................................................... 9
10.2 Detection method ................................................................................................................................ 10
10.3 Enumeration method: Calculation of the most probable number (MPN)...................................... 10
11 Precision .............................................................................................................................................. 10
12 Test report............................................................................................................................................ 10
Annex A (normative) Diagram of procedure of the detection method........................................................ 11
Annex B (normative) Diagram of procedure for MPN technique................................................................. 12
© ISO 2004 – All rights reserved iii

ISO 21528-1:2004(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.
This first edition of ISO 21528-1, together with ISO 21528-2, cancels and replaces the following standards:
 ISO 5552:1997, Meat and meat products — Detection and enumeration of Enterobacteriaceae without
resuscitation — MPN technique and colony-count technique;

(standards.iteh.ai)
ISO 7402:1993, Microbiology — General guidance for the enumeration of Enterobacteriaceae without
resuscitation — MPN technique and colony-count technique;
ISO 21528-1:2004
 ISO 8523:1991, Microbiology — General guidance for the detection of Enterobacteriaceae with pre-
enrichment.
970987355d9a/iso-21528-1-2004
ISO 21528-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
ISO 21528 consists of the following parts, under the general title Microbiology of food and animal feeding
stuffs — Horizontal methods for the detection and enumeration of Enterobacteriaceae:
 Part 1: Detection and enumeration by MPN technique with pre-enrichment
 Part 2: Colony-count method
iv © ISO 2004 – All rights reserved

ISO 21528-1:2004(E)
Introduction
This part of ISO 21528 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.
When this part of ISO 21528 is next reviewed, account will be taken of all information then available regarding
the extent to which the guidelines have been followed and the reasons for deviation from them 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 part of ISO 21528 so
that eventually the only remaining departures from this horizontal method will be those necessary for well-
established technical reasons.

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ISO 21528-1:2004
970987355d9a/iso-21528-1-2004
© ISO 2004 – All rights reserved v

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ISO 21528-1:2004
970987355d9a/iso-21528-1-2004
INTERNATIONAL STANDARD ISO 21528-1:2004(E)
Microbiology of food and animal feeding stuffs — Horizontal

methods for the detection and enumeration of
Enterobacteriaceae —
Part 1:
Detection and enumeration by MPN technique with
pre-enrichment
1 Scope
This part of ISO 21528 specifies a method, with pre-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 food production and food handling.
(standards.iteh.ai)
Enumeration is carried out by calculation of the most probable number (MPN) after incubation at 37 °C
1)
(or 30 °C) in liquid medium. ISO 21528-1:2004
This method is applicable 970987355d9a/iso-21528-1-2004
 when the microorganisms sought are expected to need resuscitation before enrichment, and
 when the number sought is expected to be in the range 1 to 100 per millilitre or per gram of test sample.
A limitation on the applicability of this part of ISO 21528 is imposed by the susceptibility of the method to a
large degree of variability (see Clause 11).
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 6887-1:1999, 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
1) The temperature of 37 °C is generally used when the enumeration of Enterobacteriaceae is for a hygienic indicator.
Alternatively, a temperature of 30 °C can be chosen when the enumeration of Enterobacteriaceae is conducted for
technological purposes and includes psychrotrophic Enterobacteriaceae.
© ISO 2004 – All rights reserved 1

ISO 21528-1:2004(E)
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
and decimal dilutions for microbiological examination — Part 3: Specific rules for the preparation of fish and
fishery products
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 7218:1996, 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
3 Terms and definitions iTeh STANDARD PREVIEW

(standards.iteh.ai)
For the purposes of this document, the following terms and definitions apply.
3.1 ISO 21528-1:2004

Enterobacteriaceae https://standards.iteh.ai/catalog/standards/sist/2f5ef1a0-d39a-489b-ac2d-
microorganisms that form characteristic colonies on violet red bile glucose agar and that ferment glucose and
970987355d9a/iso-21528-1-2004
show a negative oxidase reaction when the tests are carried out in accordance with the methods specified in
this part of ISO 21528
3.2
detection of Enterobacteriaceae
determination of the presence or absence of these bacteria, in a particular quantity of product, when tests are
carried out in accordance with this part of ISO 21528
3.3
enumeration of Enterobacteriaceae
most probable number of Enterobacteriaceae found per millilitre or per gram of the test sample when the test
is carried out according to the method specified in this part of ISO 21528
4 Principle
4.1 Detection of Enterobacteriaceae (see Annex A)
4.1.1 Pre-enrichment in non-selective medium
Buffered peptone water (BPW) is inoculated with the test portion, then incubated at 37 °C (or 30 °C)1) for
18 h ± 2 h.
2 © ISO 2004 – All rights reserved

ISO 21528-1:2004(E)
4.1.2 Enrichment in selective liquid medium
The enrichment broth [buffered brilliant green bile glucose broth (EE broth)] is inoculated with the culture
obtained after pre-enrichment, then incubated at 37 °C (or 30 °C)1) for 24 h ± 2 h.
4.1.3 Isolation and selection for confirmation
A selective solid medium (violet red bile glucose agar) is inoculated with the culture obtained after enrichment
in EE broth, then incubated at 37 °C (or 30 °C)1). It is examined after 24 h ± 2 h to check for the presence of
colonies presumed by their characteristics to be Enterobacteriaceae.
4.1.4 Confirmation
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.
4.2 Enumeration by the MPN technique (see Annex B)
4.2.1 Pre-enrichment in non-selective medium
A test portion of x g is added to 9 x ml of buffered peptone water (BPW) and homogenized. One or more 10-
fold dilutions (according to the expected level of contamination) are prepared in BPW. Aliquots (10 ml) of this
initial dilution are transferred to three tubes. Then 3 × 1 ml of the initial dilution are added to 9 ml of BPW and
3 × 1 ml of each further dilution are added to 9 ml of BPW. These tubes are incubated 37 °C (or 30 °C)1) for
18 h ± 2 h. iTeh STANDARD PREVIEW
4.2.2 (standards.iteh.ai)
Enrichment in selective liquid medium
Tubes of liquid enrichment broth (EE broth) are inoculated with each tube of culture obtained after pre-
ISO 21528-1:2004
× 3). The tubes are incubated at 37 °C (or 30 °C)1) for 24 h ± 2 h.
enrichment (at least 3https://standards.iteh.ai/catalog/standards/sist/2f5ef1a0-d39a-489b-ac2d-
970987355d9a/iso-21528-1-2004
4.2.3 Isolation and selection for confirmation
A selective solid medium (violet red bile glucose agar) is inoculated with a loop from each of the incubated
cultures obtained after enrichment in EE broth, then incubated at 37 °C (or 30 °C)1). It is examined after
24 h ± 2 h to check for the presence of colonies presumed by their characteristics to be Enterobacteriaceae.
4.2.4 Confirmation
Colonies of presumptive Enterobacteriaceae are subcultured on non-selective medium, then confirmed by

means of tests for the fermentation of glucose and the presence of oxidase.
4.2.5 Calculation
The most probable number of Enterobacteriaceae per millilitre or per gram of the test sample is calculated
from the number of confirmed positive tubes using the MPN table (see ISO 7218).
5 Diluent, culture media and reagent

For current laboratory practice, see ISO 7218 and ISO/TS 11133-1 and ISO/TS 11133-2.
5.1 Diluent: buffered peptone water (BPW)
See ISO 6887-1:1999, 5.2.2.
BPW is used as the non-selective pre-enrichment medium for the enumeration method.

ISO 21528-1:2004(E)
5.2 Culture media
5.2.1 Enrichment medium: Buffered brilliant green bile glucose broth (EE broth)
5.2.1.1 Composition
Enzymatic digest of animal tissues 10,0 g

Glucose 5,0 g
Disodium hydrogen phosphate (Na2HPO4) 6,45 g
Potassium dihydrogen phosphate (KH2PO4) 2,0 g
Beef bile for bacteriological use 20,0 g
Brilliant green (bacteriological quality) 0,0135 g
Water 1 000 ml
5.2.1.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by boiling. Do not heat the medium
for longer than 30 min. Cool the medium rapidly.
Adjust the pH, if necessary, so that after boiling it is 7,2 ± 0,2 at 25 °C.
Dispense the medium in 10 ml amounts into sterile tubes of appropriate capacity (6.5).
Do not sterilize the medium.

(standards.iteh.ai)
ISO 21528-1:2004
The medium may be stored for up to 1 month at 5 °C ± 3 °C.
5.2.1.3
970987355d9a/iso-21528-1-2004
Performance testing for the quality assurance of the culture medium
For the definition of selectivity and productivity, refer to ISO/TS 11133-1. For the performance criteria, refer to
ISO/TS 11133-2:2003, Table B.3.
5.2.2 Violet red bile glucose (VRBG) agar
5.2.2.1 Components

Yeast extract 3,0 g
Bile salts No. 3 1,5 g
Glucose 10,0 g
Sodium chloride 5,0 g
Neutral red 0,03 g
Crystal violet 0,002 g
Agar 9 g to 18 ga)
Water 1 000 ml
a) Depending on the gel strength of the agar.
4 © ISO 2004 – All rights reserved

ISO 21528-1:2004(E)
5.2.2.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by boiling.
Adjust the pH, if necessary, so that after boiling it is 7,4 ± 0,2 at 25 °C.
Dispense the culture medium into sterile tubes or flasks (6.5) of appropriate capacity.
Do not sterilize the medium.
Use the molten medium within 4 h of its preparation.
5.2.2.3 Preparation of agar plates
Immediately transfer approximately 15 ml of the culture medium, cooled to between 44 °C and 47 °C (6.4), to
Petri dishes (6.7) and allow to solidify.
Just before use, dry the plates, preferably with the lids off and the agar surface downwards, in a drying cabinet
(6.3) until the agar is dry.
If prepared in advance, the undried plates may be stored in conditions that do not change their composition for
up to 2 weeks at 5 °C ± 3 °C.
5.2.2.4 Performance testing for the quality assurance of the culture medium

For the definition of selectivity and productivity, refer to ISO/TS 11133-1. For the performance criteria, refer to
ISO/TS 11133-2:2003, Table B.1.
(standards.iteh.ai)
5.2.3 Nutrient agar
ISO 21528-1:2004
5.2.3.1 Composition
970987355d9a/iso-21528-1-2004
Meat extract 3,0 g
Sodium chloride 5,0 g
Agar 9 g to 18 g a)
Water 1 000 ml
a) Depending on the gel strength of the agar.
5.2.3.2 Preparation
Dissolve the components or the dehydrated complete medium in the water, by heating if necessary.
Adjust the pH, if necessary, so that after sterilization it is 7,3 ± 0,2 at 25 °C.
Dispense the culture medium into sterile tubes or flasks (6.5) of appropriate capacity.
Sterilize for 15 min in an autoclave (6.1) set at 121 °C.
5.2.3.3 Preparation of agar plates
Transfer portions of about 15 ml of the culture medium, melted and cooled to approximately 47 °C, to Petri
dishes (6.7) and allow to solidify.

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INTERNATIONAL ISO

Microbiology of food and animal feeding

iTeh STANDARD PREVIEW

ii © ISO 2004 – All rights reserved

© ISO 2004 – All rights reserved iii

 Part 1: Detection and enumeration by MPN technique with pre-enrichment

 Part 2: Colony-count method

iv © ISO 2004 – All rights reserved

iTeh STANDARD PREVIEW

© ISO 2004 – All rights reserved v

Microbiology of food and animal feeding stuffs — Horizontal

© ISO 2004 – All rights reserved 1

3 Terms and definitions iTeh STANDARD PREVIEW

3.1 ISO 21528-1:2004

4.1 Detection of Enterobacteriaceae (see Annex A)

4.1.1 Pre-enrichment in non-selective medium

2 © ISO 2004 – All rights reserved

4.1.2 Enrichment in selective liquid medium

4.1.3 Isolation and selection for confirmation

4.2 Enumeration by the MPN technique (see Annex B)

4.2.1 Pre-enrichment in non-selective medium

Colonies of presumptive Enterobacteriaceae are subcultured on non-selective medium, then confirmed by

5 Diluent, culture media and reagent

5.1 Diluent: buffered peptone water (BPW)

See ISO 6887-1:1999, 5.2.2.

© ISO 2004 – All rights reserved 3

5.2 Culture media

Enzymatic digest of animal tissues 10,0 g

Do not sterilize the medium.

5.2.2 Violet red bile glucose (VRBG) agar

Enzymatic digest of animal tissues 7,0 g

4 © ISO 2004 – All rights reserved

Do not sterilize the medium.

Use the molten medium within 4 h of its preparation.

5.2.2.3 Preparation of agar plates

iTeh STANDARD PREVIEW

Sterilize for 15 min in an autoclave (6.1) set at 121 °C.

5.2.3.3 Preparation of agar plates

© ISO 2004 – All rights reserved 5

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