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)

ISO 6887-1:2017 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.
ISO 6887-1:2017 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.
ISO 6887-1:2017 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)

Dieses Dokument 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.
Dieses Dokument gilt für den allgemeinen Fall und andere Teile gelten für spezifische Produkt¬gruppen, wie im Vorwort dargelegt. Einige Aspekte könnten auch auf molekulare Verfahren anwendbar sein, bei denen Matrices mit der Hemmung von PCR Schritten in Zusammenhang stehen und folglich das Untersuchungsergebnis beeinflussen können.
Dieses Dokument 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 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 (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
Publication Date
04-Apr-2017
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
05-Apr-2017
Completion Date
05-Apr-2017

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SLOVENSKI STANDARD
SIST EN ISO 6887-1:2017
01-junij-2017
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SIST EN ISO 6887-1:1999
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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

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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.
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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. +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 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
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
For example, enzymatic digest of casein.
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.
...

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