SIST EN ISO 6887-1:2017

Microbiology of the food chain - 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 6887-1:2017)

This document defines general rules for the aerobic preparation of the initial suspension and of dilutions
for microbiological examinations of products intended for human or animal consumption.
This document is applicable to the general case and other parts apply to specific groups of products
as mentioned in the foreword. Some aspects might also be applicable to molecular methods where
matrices can be associated with inhibition of the PCR steps and consequently affect the test result.
This document excludes preparation of samples for both enumeration and detection test methods
where preparation instructions are detailed in specific International Standards.

Mikrobiologie der Lebensmittelkette - Vorbereitung von Untersuchungsproben und Herstellung von Erstverdünnungen und von Dezimalverdünnungen für mikrobiologische Untersuchungen - Teil 1: Allgemeine Regeln für die Herstellung von Erstverdünnungen und Dezimalverdünnungen (ISO 6887-1:2017)

Dieser Teil von ISO 6887 legt allgemeinen Regeln für die aerobe Herstellung von Erstverdünnungen und Verdünnungen für mikrobiologische Untersuchungen von Produkten fest, die für den Verzehr durch Menschen oder Tiere vorgesehen sind.
Dieser Teil von ISO 6887 gilt für den allgemeinen Fall und andere Teile gelten für spezifische Produkt¬gruppen, wie im Vorwort dargelegt. Einige Aspekte sind möglicherweise auch auf molekulare Verfahren anwendbar, bei denen Matrices mit der Hemmung von PCR Schritten in Zusammenhang stehen und folglich das Untersuchungsergebnis beeinflussen können.
Dieser Teil von ISO 6887 schließt die Vorbereitung von Proben für Untersuchungsverfahren zur Zählung und zum Nachweis aus, bei denen die Anweisungen für die Vorbereitung in spezifischen Internationalen Normen ausführlich dargelegt sind.

Microbiologie des aliments - Préparation des échantillons, de la suspension mère et des dilutions décimales en vue de l'examen microbiologique - Partie 1: Règles générales pour la préparation de la suspension mère et des dilutions décimales (ISO 6887-1:2017)

ISO 6887-1:2017 définit des règles générales pour la préparation de la suspension mère et des dilutions réalisées en aérobiose, en vue des examens microbiologiques des produits destinés à la consommation humaine ou à l'alimentation animale.
ISO 6887-1:2017 est applicable aux cas généraux et les autres parties s'appliquent aux groupes de produits spécifiques indiqués dans l'Avant-propos. Certains aspects peuvent aussi être applicables aux méthodes moléculaires dans lesquelles les matrices peuvent être impliquées dans l'inhibition des étapes de PCR et, de ce fait, affectent le résultat d'essai.
ISO 6887-1:2017 exclut la préparation d'échantillons en vue des méthodes de dénombrement et de recherche dans lesquelles les instructions de préparation sont détaillées dans des Normes internationales spécifiques.

Mikrobiologija v prehranski verigi - Priprava preskusnih vzorcev, osnovne suspenzije in decimalnih razredčin za mikrobiološko preiskavo - 1. del: Splošna pravila za pripravo osnovne suspenzije in decimalnih razredčin (ISO 6887-1:2017)

Ta dokument določa splošna pravila za aerobno pripravo osnovne suspenzije in razredčin za mikrobiološke preiskave izdelkov, namenjenih za prehrano ljudi ali živali. Ta dokument se uporablja za splošne primere, drugi deli pa veljajo za posebne skupine izdelkov, kot je omenjeno v predgovoru. Nekateri vidiki se lahko uporabljajo tudi za molekularne metode, kjer je mogoče matrice povezati z zaviranjem verižne reakcije s polimerazo, kar vpliva na rezultate preskusa. Ta dokument ne zajema priprave vzorcev za preskusni metodi za ugotavljanje števila in odkrivanje, kadar so navodila glede priprave podrobneje opisana v določenih mednarodnih standardih.

General Information

• Status: Published
• Public Enquiry End Date: 09-Mar-2014
• Publication Date: 16-May-2017
• ICS: 07.100.30 - Food microbiology
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 25-Apr-2017
• Due Date: 30-Jun-2017
• Completion Date: 17-May-2017
• Ref Project: EN ISO 6887-1:2017 - Microbiology of the food chain - 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 6887-1:2017)

Relations

• Replaces: SIST EN 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 (ISO 6887-1:1999)
• Effective Date: 01-Jun-2017

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 6887-1:2017
01-junij-2017
1DGRPHãþD
SIST EN ISO 6887-1:1999
0LNURELRORJLMDYSUHKUDQVNLYHULJL3ULSUDYDSUHVNXVQLKY]RUFHYRVQRYQH
VXVSHQ]LMHLQGHFLPDOQLKUD]UHGþLQ]DPLNURELRORãNRSUHLVNDYRGHO6SORãQD
SUDYLOD]DSULSUDYRRVQRYQHVXVSHQ]LMHLQGHFLPDOQLKUD]UHGþLQ,62
Microbiology of the food chain - 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 6887-1:2017)
Mikrobiologie der Lebensmittelkette - Vorbereitung von Untersuchungsproben und
Herstellung von Erstverdünnungen und von Dezimalverdünnungen für mikrobiologische
Untersuchungen - Teil 1: Allgemeine Regeln für die Herstellung von Erstverdünnungen
und Dezimalverdünnungen (ISO 6887-1:2017)
Microbiologie des aliments - Préparation des échantillons, de la suspension mère et des
dilutions décimales en vue de l'examen microbiologique - Partie 1: Règles générales
pour la préparation de la suspension mère et des dilutions décimales (ISO 6887-1:2017)
Ta slovenski standard je istoveten z: EN ISO 6887-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 6887-1:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 6887-1:2017

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SIST EN ISO 6887-1:2017


EN ISO 6887-1
EUROPEAN STANDARD

NORME EUROPÉENNE

April 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 6887-1:1999
English Version

Microbiology of the food chain - 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 6887-1:2017)
Microbiologie de la chaîne alimentaire - Préparation Mikrobiologie der Lebensmittelkette - Vorbereitung
des échantillons, de la suspension mère et des dilutions von Untersuchungsproben und Herstellung von
décimales en vue de l'examen microbiologique - Partie Erstverdünnungen und von Dezimalverdünnungen für
1: Règles générales pour la préparation de la mikrobiologische Untersuchungen - Teil 1: Allgemeine
suspension mère et des dilutions décimales (ISO 6887- Regeln für die Herstellung von Erstverdünnungen und
1:2017) Dezimalverdünnungen (ISO 6887-1:2017)
This European Standard was approved by CEN on 14 January 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 6887-1:2017 E
worldwide for CEN national Members.

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SIST EN ISO 6887-1:2017
EN ISO 6887-1:2017 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 6887-1:2017
EN ISO 6887-1:2017 (E)
European foreword
This document (EN ISO 6887-1:2017) 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 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 supersedes EN ISO 6887-1:1999.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, 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 6887-1:2017 has been approved by CEN as EN ISO 6887-1:2017 without any
modification.

3

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SIST EN ISO 6887-1:2017

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SIST EN ISO 6887-1:2017
INTERNATIONAL ISO
STANDARD 6887-1
Second edition
2017-03
Microbiology of the food chain —
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
Microbiologie de la chaîne alimentaire — Préparation des
échantillons, de la suspension mère et des dilutions décimales en vue
de l’examen microbiologique —
Partie 1: Règles générales pour la préparation de la suspension mère
et des dilutions décimales
Reference number
ISO 6887-1:2017(E)
©
ISO 2017

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SIST EN ISO 6887-1:2017
ISO 6887-1:2017(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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2017 – All rights reserved

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SIST EN ISO 6887-1:2017
ISO 6887-1:2017(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Diluents . 3
5.1 Basic materials . 3
5.2 Diluents for general use . 3
5.2.1 Peptone salt solution . 3
5.2.2 Buffered peptone water . 4
5.2.3 Double-strength buffered peptone water . 4
5.3 Diluents for special purposes . 4
5.4 Distribution and sterilization of the diluent . 4
5.5 Performance testing for diluents . 5
6 Apparatus . 5
7 Sampling . 6
8 Preparation of samples. 6
8.1 General . 6
8.2 Frozen products . 7
8.2.1 General. 7
8.2.2 Small samples defrosted before testing . 7
8.2.3 Large pieces or blocks sampled while frozen . 7
8.3 Hard and dry products . 8
8.4 Dehydrated and other low-moisture products. 8
8.5 Liquid and non-viscous products. 8
8.6 Acidic products . 8
8.7 High-fat (over 20 %) foods . 9
8.8 Multi-component products. 9
8.9 Packaged products . 9
8.10 Surface samples (swabs and other devices) .10
9 Specific procedures.10
9.1 Test portion and initial suspension (primary dilution) .10
9.2 Duration of the procedure .11
9.3 Pooling and compositing procedures for qualitative tests.11
10 Further dilutions .11
10.1 Decimal dilution series .11
10.2 Other dilution series .12
Annex A (informative) Illustrations of pooling and compositing procedures.13
Annex B (informative) Method for sampling frozen test pieces or blocks .18
Annex C (informative) Data showing reliability of test results according to size of test portions .20
Annex D (informative) Verification protocol for pooling samples for qualitative tests .23
Bibliography .26
© ISO 2017 – All rights reserved iii

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SIST EN ISO 6887-1:2017
ISO 6887-1:2017(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 Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
This second edition cancels and replaces the first edition (ISO 6887-1:1999), which has been technically
revised.
A list of parts in the ISO 6887 series can be found on the ISO website.
iv © ISO 2017 – All rights reserved

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SIST EN ISO 6887-1:2017
ISO 6887-1:2017(E)

Introduction
Because of the large variety of food and animal feed products, this horizontal method might not be
appropriate in every detail for certain products. In this case, different methods which are specific to
these products can be used if absolutely necessary for justified technical reasons.
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.
This document defines the general rules for the preparation of samples, initial suspensions and
subsequent dilutions for microbiological examination. The remaining parts of ISO 6887 give specific
rules for the preparation of samples and initial suspensions, each covering the variety of food and feed
products and environmental samples to which ISO 6887 applies.
For a number of products, it is necessary to take special precautions, especially when preparing the
initial suspension, because of the physical state of the product (such as dry products, highly viscous
products) or the presence of inhibitory substances (such as spices, high salt content) or the acidity, etc.
These are covered in general terms in this document.
Any special diluents or practices required for particular products or microorganisms in specific
standard methods take priority over the general rules listed in the ISO 6887 series. These can include
the following:
— specific rehydration procedures for foods of low water activity to minimize osmotic shock;
— the use of adequate temperatures to aid suspension of cocoa, gelatine, milk powder, etc.;
— resuscitation procedures for the improved recovery of stressed microorganisms resulting from
food processing and storage;
— homogenization procedures and duration specific to certain products (e.g. cereals) and/or to certain
determinations (e.g. yeasts and moulds).
© ISO 2017 – All rights reserved v

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SIST EN ISO 6887-1:2017

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SIST EN ISO 6887-1:2017
INTERNATIONAL STANDARD ISO 6887-1:2017(E)
Microbiology of the food chain — 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
WARNING — The use of this document may involve hazardous materials, operations and
equipment. It is the responsibility of the user of this document to establish appropriate safety
and health practices and to determine the applicability of regulatory limitations before use.
1 Scope
This document defines general rules for the aerobic preparation of the initial suspension and of dilutions
for microbiological examinations of products intended for human or animal consumption.
This document is applicable to the general case and other parts apply to specific groups of products
as mentioned in the foreword. Some aspects might also be applicable to molecular methods where
matrices can be associated with inhibition of the PCR steps and consequently affect the test result.
This document excludes preparation of samples for both enumeration and detection test methods
where preparation instructions are detailed in specific International Standards.
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 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 .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
laboratory sample
sample prepared for sending to the laboratory and intended for inspection or testing
[SOURCE: ISO 7002:1986, A.19]
© ISO 2017 – All rights reserved 1

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SIST EN ISO 6887-1:2017
ISO 6887-1:2017(E)

3.2
composite sample
mixed sample of a number of the same items of food, animal feed, animals or environment, from which
a test portion is taken for examination in the laboratory
Note 1 to entry: See illustration of a composite sample in Annex A.
3.3
pooled sample
mixed sample of a number of the same items of food, animal feed, animals or environment, where the
complete mixture is the test portion and is taken as a whole for examination in the laboratory
Note 1 to entry: See illustration of a pooled sample in Annex A.
3.4
test sample
sample prepared from the laboratory sample (3.1) according to the procedure specified in the method of
test and from which test portions (3.5) are taken
Note 1 to entry: Preparation of the laboratory sample before the test portion is taken is infrequently used in
microbiological examinations.
[SOURCE: ISO 7002:1986, A.47]
3.5
test portion
measured (volume or mass) representative sample taken from the laboratory sample (3.1) for use in the
preparation of the initial suspension (3.6)
Note 1 to entry: Sometimes preparation of the laboratory sample (3.1) is required before the test portion is taken,
but this is infrequently used in microbiological examinations.
3.6
initial suspension
primary dilution
suspension, solution or emulsion obtained after a weighed or measured quantity of the product under
examination (or of a test sample prepared from the product) has been mixed with, normally, a nine-fold
quantity of diluent, allowing large particles, if present, to settle
Note 1 to entry: Nine-fold quantities of diluent are normally used to produce a decimal dilution series, but other
ratios may be required for specific purposes, such as to enumerate low numbers.
3.7
further dilution
suspension or solution obtained by mixing a measured volume of the initial suspension (3.6) with an
x-fold volume of diluent and by repeating this operation with further dilutions until a dilution series,
suitable for the inoculation of culture media, is obtained
Note 1 to entry: Ten-fold dilutions are normally used to produce a decimal dilution series, but other ratios may be
required for specific purposes.
3.8
pooled test portions
mixture of test portions from a number of the same items of food, animal feed, animals or environment,
where the complete mixture is the test portion examined
Note 1 to entry: See illustration of pooled test portions in Annex A.
2 © ISO 2017 – All rights reserved

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SIST EN ISO 6887-1:2017
ISO 6887-1:2017(E)

3.9
pooled (pre-)enriched test portions
individually (pre-)enriched test portions from a number of the same items of food, animal feed, animals
or environment, from which specified volumes are combined for further examination
Note 1 to entry: See illustration of pooled (pre-)enriched test portions in Annex A.
3.10
specific standard
International Standard or guidance document describing the examination of a specific product (or group
of products) for the detection or enumeration of a specific microorganism (or group of microorganisms)
4 Principle
Preparation of the initial suspension (3.6) in such a way as to obtain as uniform a distribution as
possible of the microorganisms contained in the test portion (3.5).
Preparation, if necessary, of further dilutions (3.7) in order to reduce the number of microorganisms
per unit volume to allow, after incubation, observation of their growth or not (in the case of tubes or
bottles) or colony counting (in the case of plates), as stated in each specific standard.
NOTE In order to restrict the range of enumeration to a given optimum interval, or if high numbers of
microorganisms are foreseen, it is possible to inoculate only the necessary (decimal) dilutions needed to achieve
the enumeration according to the calculations described in ISO 7218.
5 Diluents
5.1 Basic materials
To improve the reproducibility of test results, it is recommended that either ready-made diluents or
dehydrated basic components or a dehydrated complete preparation should be used. In all cases, the
manufacturer’s instructions shall be followed rigorously.
Chemical products shall be of recognized analytical quality and suitable for microbiological
examinations.
The water used shall be distilled water or of equivalent quality (see ISO 7218 or ISO 11133).
For more detailed rules on preparation and performance testing of culture media, see ISO 11133.
5.2 Diluents for general use
5.2.1 Peptone salt solution
5.2.1.1 Composition
Enzymatic digest of casein 1,0 g
Sodium chloride 8,5 g
Water 1 000 ml
5.2.1.2 Preparation
Dissolve the components in the water in flasks, bottles or test tubes (6.4) by heating, if necessary.
Adjust the pH if necessary so that, after sterilization, it is 7,0 ± 0,2 at 25 °C.
© ISO 2017 – All rights reserved 3

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SIST EN ISO 6887-1:2017
ISO 6887-1:2017(E)

5.2.2 Buffered peptone water
5.2.2.1 Composition
a
Peptone 10,0 g
Sodium chloride 5,0 g
Disodium hydrogen phosphate dodecahydrate 9,0 g
b
(Na HPO ·12H O) ‡
2 4 2
Potassium dihydrogen phosphate (KH PO ) ‡ 1,5 g
2 4
Water 1 000 ml
a
  For example, enzymatic digest of casein.
b
  If disodium hydrogen phosphate with a different water content is
used, amend the mass of the ingredient accordingly. For example, in
case of anhydrous disodium hydrogen phosphate (Na HPO ), use 3,57 g.
2 4
‡  Buffer ingredients, see 5.2.3.
5.2.2.2 Preparation
Dissolve the components in the water in flasks, bottles or test tubes (6.4), by heating if necessary.
Adjust the pH, if necessary, so that after sterilization, it is 7,0 ± 0,2 at 25 °C.
5.2.3 Double-strength buffered peptone water
This diluent may be necessary for high acid samples (see 8.6) and is prepared by dissolving double the
quantities of a complete dehydrated medium in 1 000 ml of water and processing in the same manner.
If the diluent is prepared from individual ingredients, only double the quantities of the two buffer
ingredients (marked ‡) are required.
5.3 Diluents for special purposes
See the specific standard or part of ISO 6887 appropriate to the product concerned.
5.4 Distribution and sterilization of the diluent
Dispense the diluent in volumes as necessary for the preparation of the initial suspensions into vessels
(6.4) of appropriate capacity.
Dispense further diluent in volumes as necessary for the preparation of the (decimal or other ratio)
dilutions into vessels (6.4) of appropriate capacity.
The tolerance allowable on final diluent volumes, after sterilization, shall not exceed ±2 %.
In order to enumerate several groups of microorganisms using different culture media, it may be
necessary to distribute all the diluents (or some of them) in quantities greater than 9,0 ml into vessels
(6.4) of appropriate size.
Stopper the vessels loosely to allow for expansion on heating.
S
...

SLOVENSKI STANDARD
oSIST prEN ISO 6887-1:2014
01-februar-2014
0LNURELRORJLMDYSUHKUDQVNLYHULJL3ULSUDYDY]RUFHY]DSUHVNXãDQMHWHURVQRYQHLQ
GHFLPDOQLKUD]UHGþLQ]DPLNURELRORãNRSUHLVNDYRGHO6SORãQDSUDYLOD]D
SULSUDYRRVQRYQHLQGHFLPDOQLKUD]UHGþLQ,62',6
Microbiology of the food chain - 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/DIS 6887-1:2013)
Mikrobiologie der Lebensmittelkette - Vorbereitung von Untersuchungsproben und
Herstellung von Erstverdünnungen und von Dezimalverdünnungen für mikrobiologische
Untersuchungen - Teil 1: Allgemeine Regeln für die Herstellung von Erstverdünnungen
und Dezimalverdünnungen (ISO/DIS 6887-1:2013)
Microbiologie des aliments - Préparation des échantillons, de la suspension mère et des
dilutions décimales en vue de l'examen microbiologique - Partie 1: Règles générales
pour la préparation de la suspension mère et des dilutions décimales (ISO/DIS 6887-
1:2013)
Ta slovenski standard je istoveten z: prEN ISO 6887-1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 6887-1:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 6887-1:2014

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oSIST prEN ISO 6887-1:2014
DRAFT INTERNATIONAL STANDARD
ISO/DIS 6887-1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2013-11-28 2014-04-28
Microbiology of the food chain — 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
Microbiologie de la chaîne alimentaire — Préparation des échantillons, de la suspension mère et des
dilutions décimales en vue de l’examen microbiologique —
Partie 1: Règles générales pour la préparation de la suspension mère et des dilutions décimales
[Revision of first edition (ISO 6887-1:1999)]
ICS: 07.100.30
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ISO/DIS 6887-1:2013(E)
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Copyright notice
This ISO document is a Draft International Standard and is copyright-protected by ISO. Except as
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Contents Page
Foreword . v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle. 3
5 Diluents. 3
5.1 Basic materials . 3
5.2 Diluents for general use . 3
5.3 Diluents for special purposes . 4
5.4 Distribution and sterilization of the diluent . 4
6 Apparatus . 5
7 Sampling. 6
8 Preparation of samples . 6
8.1 Frozen products . 6
8.2 Hard and dry products . 7
8.3 Dehydrated and other low moisture products. 7
8.4 Liquid and non-viscous products . 8
8.5 Acidic products . 8
8.6 High (over 20%) fat foods . 8
8.7 Multi-component products . 8
8.8 Packaged products . 9
8.9 Surface samples (swabs and other devices) . 9
9 Specific procedures . 9
9.1 Test portion and initial suspension (primary dilution) . 9
9.2  Duration of the procedure
9.3 Pooling and compositing procedures for qualitative tests . 11
10 Further dilutions
10.1 Decimal dilution series
10.2 Other dilution series
Annex A (informative) Illustrations of pooling and compositing procedures . 13
A.1 Composited samples
A.2 Pooled samples
A.3 Pooled test portions
A.4 Pooled (pre-) enriched test portions
Annex B (normative) Method for sampling frozen test pieces or blocks. 17
B.1 Non-homogeneous blocks . 17
B.2 Homogeneous test pieces . 17
Annex C (informative) Reliability of test results according to size of test portions
Annex D (informative) Verification protocol for pooling samples for qualitative tests
D.1 Pooling tests
D.2 Confirmation of findings
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oSIST prEN ISO 6887-1:2014
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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 6887-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
This document cancels and replaces ISO 6887-1:1999.
ISO 6887 consists of the following parts, under the general title Microbiology of the food chain — Preparation
of test samples, initial suspension and decimal dilutions for microbiological examination:
 Part 1: General rules for the preparation of the initial suspension and dilutions
 Part 2: Specific rules for the preparation of meat and meat products
 Part 3: Specific rules for the preparation of fish and fishery products
 Part 4: Specific rules for the preparation of miscellaneous products
[This part includes sample preparation for a variety of products not covered in the other parts, as follows:
very acidic products; very hard products; cereals and cereal products; animal feeds; gelatine; margarines
and non-dairy spreads; dehydrated and low a products; eggs and egg products; fermented products;
w
bakery goods; and beverages.]
 Part 5: Specific rules for the preparation of milk and milk products
 Part 6: Specific rules for the preparation of samples taken at the primary production stage.
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Introduction
Because of the large variety of food and animal 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 part of ISO 6887 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 part of ISO 6887 so
that eventually the only remaining departures from this horizontal method will be those necessary for
well-established technical reasons.
This part of ISO 6887 defines the general rules for the preparation of samples, initial suspensions and
subsequent dilutions for microbiological examination. The remaining parts of ISO 6887 give specific rules for
the preparation of samples and initial suspensions, each covering the variety of food and feed products and
environmental samples to which ISO 6887 applies.
For a number of products, it is necessary to take special precautions, especially when preparing the initial
suspension, because of the physical state of the product (such as dry products, highly viscous products) or
the presence of inhibitory substances (such as spices, high salt content) or the acidity, etc. These are covered
in general terms in this part of ISO 6887.
Any special diluents or practices required for particular products or microorganisms in specific standard
methods should still be used in preference to the general rules listed in this series of standards. These may
include:
 specific rehydration procedures for foods of low water activity to minimize osmotic shock;
 the use of adequate temperatures to aid suspension of cocoa, gelatine, milk powder, etc.;
 resuscitation procedures for the improved recovery of stressed microorganisms resulting from food
processing and storage;
 homogenization procedures and duration specific to certain products (e.g. cereals) and/or to certain
determinations (e.g. yeasts and moulds).
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DRAFT INTERNATIONAL STANDARD ISO/DIS 6887-1

Microbiology of the food chain — 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
WARNING — The use of this standard may involve hazardous materials, operations and equipment. It
is the responsibility of the user of this standard to establish appropriate safety and health practices
and to determine the applicability of regulatory limitations before use.

1 Scope
This part of ISO 6887 defines general rules for the aerobic preparation of the initial suspension and of dilutions
for microbiological examinations of products intended for human or animal consumption.
This part of ISO 6887 is applicable to the general case and other parts apply to specific groups of products as
detailed in the Foreword. Some aspects may also be applicable to molecular methods where matrices may be
associated with inhibition of the PCR steps and consequently affect the test result.
This part of ISO 6887 excludes preparation of samples for both enumeration and detection test methods
where preparation instructions are detailed in specific International Standards.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations.
3 Terms and definitions
For the purposes of all parts of ISO 6887, the following definitions apply.
3.1
laboratory sample
sample prepared for sending to the laboratory and intended for inspection or testing
[ISO 7002]

3.2
composite sample
mixed sample of a number of items of the same type of food, animal feed, animals or environment, from which
a test portion is taken for examination in the laboratory.

NOTE See illustration of a composite sample in Annex A.
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3.3
pooled sample
mixed sample of a number of items of the same type of food, animal feed, animals or environment, where the
complete mixture is the test portion and is taken as a whole for examination in the laboratory

NOTE See illustration of a pooled sample in Annex A.

3.4
test sample
sample prepared from the laboratory sample according to the procedure specified in the method of test and
from which test portions are taken
[ISO 7002]
NOTE Preparation of the laboratory sample before the test portion is taken is infrequently used in
microbiological examinations.
3.5
test portion
measured (volume or mass) representative sample taken from the laboratory sample for use in the
preparation of the initial suspension
NOTE Sometimes preparation of the laboratory sample (3.4) is required before the test portion is taken but
this is infrequently used in microbiological examinations.

3.6
initial suspension
primary dilution
suspension, solution or emulsion obtained after a weighed or measured quantity of the product under
examination (or of a test sample prepared from the product) has been mixed with, normally, a nine-fold
quantity of diluent, allowing large particles, if present, to settle
NOTE 1 Nine-fold dilutions are normally used to produce a decimal dilution series, but other ratios may be required for
specific purposes.
3.7
further dilutions
suspensions or solutions obtained by mixing a measured volume of the initial suspension (3.6) with an x-fold
volume of diluent and by repeating this operation with further dilutions until a dilution series, suitable for the
inoculation of culture media, is obtained
NOTE 1 Nine-fold dilutions are normally used to produce a decimal dilution series, but other ratios may be required for
specific purposes.
3.8
pooled test portions
mixture of test portions from a number of items of the same type of food, animal feed, animals or environment,
where the complete mixture is the test portion examined

NOTE See illustration of pooled test portions in Annex A.
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3.9
pooled (pre-) enriched test portions
individually (pre-) enriched test portions from a number of items of the same type of food, animal feed, animals
or environment, from which specified volumes are combined for further examination

NOTE See illustration of pooled (pre-) enriched test portions in Annex A.
3.10
specific standard
an International Standard or guidance document describing the examination of a specific product (or group of
products) for the detection or enumeration of a specific microorganism (or group of microorganisms)
4 Principle
Preparation of the initial suspension (3.6) in such a way as to obtain as uniform a distribution as possible of
the microorganisms contained in the test portion (3.5).
Preparation, if necessary, of further dilutions (3.7) in order to reduce the number of microorganisms per unit
volume to allow, after incubation, observation of their growth or not (in the case of tubes or bottles) or colony
counting (in the case of plates), as stated in each specific standard.
NOTE In order to restrict the range of enumeration to a given optimum interval, or if high numbers of microorganisms
are foreseen, it is possible to inoculate only the necessary (decimal) dilutions (at least two successive dilutions) needed to
achieve the enumeration according to the calculations described in ISO 7218.
5 Diluents
5.1 Basic materials
To improve the reproducibility of test results, it is recommended that either ready-made diluents or dehydrated
basic components or a dehydrated complete preparation should be used. In all cases, the manufacturer's
instructions shall be followed rigorously.
Chemical products shall be of recognized analytical quality and suitable for microbiological examinations.
The water used shall be distilled water or of equivalent quality (see ISO 7218 or ISO 11133 [2]).
For more detailed rules on preparation of culture media see ISO 11133 [2].
All diluents shall be tested for acceptable performance in accordance with ISO 11133 [2].
5.2 Diluents for general use
5.2.1 Peptone salt solution
5.2.1.1 Composition
Enzymatic digest of casein 1,0 g
Sodium chloride 8,5 g
Water 1 000 ml
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5.2.1.2 Preparation
Dissolve the components in the water in flasks, bottles or test tubes (6.4), by heating if necessary.
Adjust the pH if necessary so that, after sterilization, it is 7,0  0,2 at 25 °C.
5.2.2 Buffered peptone water
5.2.2.1 Composition
1
Peptone 10,0 g
Sodium chloride 5,0 g
Disodium hydrogen phosphate dodecahydrate 9,0 g
(Na HPO ,12H O)
2 4 2
Potassium dihydrogen phosphate (KH PO ) 1,5 g
2 4
Water 1 000 ml
1
: For example enzymatic digest of casein
5.2.2.2 Preparation
Dissolve the components in the water in flasks, bottles or test tubes (6.4), by heating if necessary.
Adjust the pH, if necessary, so that after sterilization it is 7,0  0,2 at 25 °C.
5.2.3 Double-strength buffered peptone water
This diluent may be necessary for high acid samples (see 8.5) and is prepared by dissolving double the
quantities of a complete dehydrated medium or the dry ingredients given at 5.2.2.1 in 1 000 ml of water and
processing in the same manner. Alternatively only double the quantities of buffer ingredients may be used if
the diluent is prepared from individual ingredients.
5.3 Diluents for special purposes
See the specific standard or part of ISO 6887 appropriate to the product concerned.
5.4 Distribution and sterilization of the diluent
Dispense the diluent in volumes as necessary for the preparation of the initial suspensions into vessels (6.4)
of appropriate capacity.
Dispense further diluent in volumes as necessary for the preparation of the (decimal or other ratio) dilutions
into vessels (6.4) of appropriate capacity in quantities such that, after sterilization, each vessel contains
9,0 ml  0.2 ml. The tolerance allowable on this final volume, after sterilization, shall not exceed  2 %.
NOTE In order to enumerate several groups of microorganisms using different culture media, it may be necessary to
distribute all the diluents (or some of them) in quantities greater than 9,0 ml into vessels (6.4) of appropriate size.
Stopper the vessels loosely to allow for expansion on heating.
Sterilize in the autoclave at 121 °C  3 °C for 15 min (see ISO 7218).
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After autoclaving, check the volumes from a proportion of the batch of diluent prepared are within the
permitted tolerance of  2 %. This may be achieved either destructively by emptying the contents of the
vessels into a tared container after autoclaving, or non-destructively by marking and weighing vessels
positioned through the autoclave both before and after autoclaving.
An alternative method to ensure diluent volumes meet the permitted tolerance is autoclaving bulk volumes
and dispensing the required amounts into sterile vessel aseptically.
6 Apparatus
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following:
6.1 Apparatus for dry sterilization (oven) and wet sterilization (autoclave)
See ISO 7218.
6.2 Homogenizer
6.2.1 Rotary homogenizer (blender)
See ISO 7218. If a large sample is to be homogenized, the equipment should include a sterile 1 litre bowl.
6.2.2 Peristaltic homogenizer
See ISO 7218. With sterile bags or filter bags to retain particulate material where necessary.
6.3 Mechanical stirrer
See ISO 7218.
6.4 Flasks, test tubes or screw-cap bottles, of appropriate capacities.
6.5 Total-delivery graduated pipettes, of nominal capacities 1 ml and 10 ml, graduated in 0,1 ml and
0,5 ml divisions respectively. Mechanical pipettors of suitable accuracy may also be used (see ISO 7218).
6.6 pH-meter, capable of being read to the nearest 0,01 pH unit at 25 °C, enabling measurements to be
made which are accurate to  0,1 pH unit (see ISO 7218).
6.7 Balance, capable of weighing to the nearest 0,01 g (see ISO 7218).
6.8 Sterile sample trays, of appropriate dimensions.
6.9 Sterile scissors, forceps or tongs, straight scalpels or knives, and spatulas.
6.10 Special opening equipment such as bottle and can openers.
6.11 Equipment for collecting test portions from frozen laboratory samples
6.11.1 Variable speed electric drill, with maximum speed in use of 900 r/min, or hand drill.
6.11.2 Sterile wood bit for electric drill, of 14 mm or 16 mm diameter.
6.11.3 Sterile wood chisel, of 20 mm width, and hammer or plastic mallet.
6.11.4 Other apparatus that does not cause overheating or contamination of the sample.
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6.11.5 Equipment for cauterization of sample surfaces (e.g. portable gas blowtorch).
6.11.6 Template for surface sampling, metallic frame of appropriate dimensions to delineate the surface to
be sampled, sterilized by flaming after immersion in 70% (volume fraction) alcohol.
6.12 Water bath, capable of being maintained at 44 °C to 47 °C or as stated for specific purposes.
6.13 Sterile wide-necked bowls, containers or plastic bags of 500 ml capacity.
6.14 Sterile glass beads or balls to disperse samples such as swabs.
7 Sampling
Carry out sampling in accordance with the specific standard appropriate to the product concerned or see ISO
17728 [3]. If specific sampling instructions are not available, it is recommended that agreement be reached on
this subject by the parties concerned.
Some guidance is given in other parts of ISO 6887 on sampling specific to certain products (see, for example,
ISO 6887-3).
8 Preparation of samples
Requirements for the different general categories of products subjected to microbiological examination are
given in this clause. For specific requirements on certain product types, see the appropriate part of ISO 6887
for further information.
In other cases, see the specific standard appropriate to the product concerned. If such a specific standard is
not available, it is recommended that agreement be reached on this subject by the parties concerned.
8.1 Frozen products
8.1.1 General
Frozen products are considered under two headings:
 small laboratory samples that may be defrosted before testing;
 large pieces or blocks from which laboratory samples or test portions are taken without defrosting.
8.1.2 Small samples defrosted before testing
These samples include packaged retail products of all types (generally under 2 kg), including cuts and
portions of meat and fish, vegetables, desserts and prepared multi-component products.
All such products stored and submitted to the laboratory frozen should be brought to a consistency that allows
sampling in the original packaging. This can be achieved by standing at 18 °C to 27 °C (laboratory ambient
temperature) for a maximum of 3 h, or at 5 °C  3 °C for a maximum of 24 h. Store thawing samples on
separate trays (6.8) to prevent cross-contamination from any ‘drip’ (thaw liquid) leaking through the packaging.
Samples shall be tested as quickly as possible after this, even if the product is still partially frozen when taking
the test portion as addition of the diluent at ambient laboratory temperature will facilitate full defrosting.
Defrosting in a temperature-controlled water bath or under running cold water is not recommended as this
may result in contamination of the sample if the packaging is not completely watertight.
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8.1.3 Large pieces or blocks sampled while frozen
8.1.3.1 General
These samples include large pieces or blocks of frozen products (generally over 2 kg), including carcases and
joints of meat, and fish that has been block frozen in bulk.
Separate the sample from any packaging using scissors or a scalpel (6.9), and place it on a tray (6.8), with a
flat side facing upwards.
Three options for sampling exist dep
...