Water quality - General requirements and guidance for microbiological examinations by culture (ISO 8199:2018)

This document specifies requirements and gives guidance for performing the manipulations common
to each culture technique for the microbiological examination of water, particularly the preparation of
samples, culture media, and general apparatus and glassware, unless otherwise required in the specific
standard. It also describes the various techniques available for detection and enumeration by culture
and the criteria for determining which technique is appropriate.
This document is mainly intended for examinations for bacteria, yeasts and moulds, but some aspects
are also applicable to bacteriophages, viruses and parasites. It excludes techniques not based on
culturing microorganisms, such as polymerase chain reaction (PCR) methods.

Wasserbeschaffenheit - Allgemeine Anforderungen und Anleitung für mikrobiologische Untersuchungen mittels Kulturverfahren (ISO 8199:2018)

Dieses Dokument enthält Anforderungen und eine Anleitung zu Arbeitsweisen, die, sofern in der spezifischen Norm nicht anderweitig gefordert, für jedes Kulturverfahren für die mikrobiologische Untersuchung von Wasser, besonders für die Aufbereitung von Proben, Nährmedien sowie allgemeinen Geräten und Glasgeräten üblich sind. Dieses Dokument beschreibt die unterschiedlichen verfügbaren Kulturverfahren zum Nachweis und zur Zählung sowie die Kriterien für die Auswahl des jeweilig geeigneten Verfahrens. Dieses Dokument ist hauptsächlich für Untersuchungen auf Bakterien, Hefen und Pilze bestimmt, aber einige Aspekte sind auch auf Bakteriophagen, Viren und Parasiten anwendbar. Verfahren, die nicht auf der Kultivierung von Mikroorganismen beruhen, z. B. Polymerase-Kettenreaktion (PCR), sind vom Anwendungsbereich ausgeschlossen.

Qualité de l'eau - Exigences et lignes directrices générales pour les examens microbiologiques sur milieu de culture (ISO 8199:2018)

Le présent document établit les exigences et fournit les lignes directrices sur les manipulations communes à chaque technique de culture pour l'examen microbiologique de l'eau, en particulier la préparation des échantillons, les milieux de culture ainsi que l'appareillage général et la verrerie, sauf autre exigence dans la norme spécifique. Il décrit également les diverses techniques disponibles pour la détection et le dénombrement sur milieu de culture et les critères permettant de déterminer la technique appropriée.
Le présent document concerne principalement les examens portant sur les bactéries, les levures et les moisissures, mais certains aspects peuvent aussi s'appliquer aux bactériophages, aux virus et aux parasites. Sont exclues les techniques ne reposant pas sur la culture de micro-organismes, telles que les méthodes de réaction en chaîne par polymérase (PCR).

Kakovost vode - Splošne zahteve in navodilo za mikrobiološke preiskave v kulturi (ISO 8199:2018)

Ta dokument določa zahteve in podaja navodilo za izvajanje manipulacij, ki so skupne vsaki tehniki kulture, za mikrobiološko preiskavo vode, zlasti za pripravo vzorcev, gojišča kultur ter splošnih aparatov in steklovine, razen če je v posebnem standardu zahtevano drugače. Opisuje tudi različne tehnike, ki so na voljo za ugotavljanje prisotnosti in števila v kulturi, ter merila za določitev ustrezne tehnike.
Ta dokument je namenjen predvsem za preiskave bakterij, plesni in kvasovk, nekateri vidiki pa se uporabljajo tudi za bakteriofage, viruse in parazite. V tem dokumentu niso zajete tehnike, ki ne temeljijo na gojenju mikroorganizmov, kot je verižna reakcija s polimerazo (PCR).

General Information

Status
Published
Public Enquiry End Date
04-Dec-2017
Publication Date
10-Apr-2019
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
04-Feb-2019
Due Date
11-Apr-2019
Completion Date
11-Apr-2019

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SLOVENSKI STANDARD
SIST EN ISO 8199:2019
01-maj-2019
1DGRPHãþD
SIST EN ISO 8199:2007
Kakovost vode - Splošne zahteve in navodilo za mikrobiološke preiskave v kulturi
(ISO 8199:2018)

Water quality - General requirements and guidance for microbiological examinations by

culture (ISO 8199:2018)

Wasserbeschaffenheit - Allgemeine Anforderungen und Anleitung für mikrobiologische

Untersuchungen mittels Kulturverfahren (ISO 8199:2018)
Qualité de l'eau - Exigences et lignes directrices générales pour les examens
microbiologiques sur milieu de culture (ISO 8199:2018)
Ta slovenski standard je istoveten z: EN ISO 8199:2018
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.45 Preiskava vode na splošno Examination of water in
general
SIST EN ISO 8199:2019 en,fr,de

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

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SIST EN ISO 8199:2019
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SIST EN ISO 8199:2019
EN ISO 8199
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2018
EUROPÄISCHE NORM
ICS 07.100.20
English Version
Water quality - General requirements and guidance for
microbiological examinations by culture (ISO 8199:2018)

Qualité de l'eau - Exigences et lignes directrices Wasserbeschaffenheit - Allgemeine Anforderungen und

générales pour les examens microbiologiques sur Anleitung für mikrobiologische Untersuchungen

milieu de culture (ISO 8199:2018) mittels Kulturverfahren (ISO 8199:2018)
This European Standard was approved by CEN on 6 September 2018.

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: Rue de la Science 23, B-1040 Brussels

© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8199:2018 E

worldwide for CEN national Members.
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SIST EN ISO 8199:2019
EN ISO 8199:2018 (E)
Contents Page

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

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SIST EN ISO 8199:2019
EN ISO 8199:2018 (E)
European foreword

This document (EN ISO 8199:2018) has been prepared by Technical Committee ISO/TC 147 "Water

quality" in collaboration with Technical Committee CEN/TC 230 “Water analysis” 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 May 2019, and conflicting national standards shall be

withdrawn at the latest by May 2019.

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

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN ISO 8199:2007.

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 8199:2018 has been approved by CEN as EN ISO 8199:2018 without any modification.

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SIST EN ISO 8199:2019
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SIST EN ISO 8199:2019
INTERNATIONAL ISO
STANDARD 8199
Third edition
2018-10
Water quality — General requirements
and guidance for microbiological
examinations by culture
Qualité de l’eau — Exigences et lignes directrices générales pour les
examens microbiologiques sur milieu de culture
Reference number
ISO 8199:2018(E)
ISO 2018
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SIST EN ISO 8199:2019
ISO 8199:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST EN ISO 8199:2019
ISO 8199:2018(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ..............................................................................................................................................................................................................................vii

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

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

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

4 Principle ........................................................................................................................................................................................................................ 4

5 General measurement requirements ............................................................................................................................................. 4

5.1 Uniformity of temperatures ......................................................................................................................................................... 4

5.2 Incubation times .................................................................................................................................................................................... 4

5.3 Volumes and masses .......................................................................................................................................................................... 4

6 Diluents and culture media ....................................................................................................................................................................... 5

6.1 General ........................................................................................................................................................................................................... 5

6.2 Quality requirements of ingredients .................................................................................................................................... 5

6.3 Diluents ......................................................................................................................................................................................................... 5

7 Sterilization and decontamination .................................................................................................................................................... 5

7.1 Sterilization of apparatus and glassware ......................................................................................................................... 5

7.2 Sterilization of consumables ....................................................................................................................................................... 6

7.3 Decontamination of glassware and materials after use ...................................................................................... 6

7.4 Waste management ............................................................................................................................................................................. 6

8 Samples and sample handling ................................................................................................................................................................ 6

8.1 Sampling ....................................................................................................................................................................................................... 6

8.2 Sample preparation ............................................................................................................................................................................ 7

8.2.1 Waters and other aqueous matrices ............................................................................................................... 7

8.2.2 Swabs ......................................................................................................................................................................................... 7

9 Enumeration (quantitative) methods ............................................................................................................................................ 8

9.1 Inoculation of test portions in (or on) solid media ................................................................................................. 8

9.1.1 General...................................................................................................................................................................................... 8

9.1.2 Pour plate technique .................................................................................................................................................... 8

9.1.3 Spread plate technique ............................................................................................................................................... 9

9.1.4 Membrane filtration technique ........................................................................................................................10

9.1.5 Incubation ...........................................................................................................................................................................12

9.1.6 Counting and confirmation from solid media ......................................................................................12

9.1.7 General guidance for calculation of results .................. ..........................................................................13

9.1.8 Expression of results .................................................................................................................................................14

9.2 Enumeration using a liquid medium.................................................................................................................................24

9.2.1 General...................................................................................................................................................................................24

9.2.2 Procedure ............................................................................................................................................................................25

9.2.3 Choice of inoculation system ..............................................................................................................................25

9.2.4 Incubation ...........................................................................................................................................................................26

9.2.5 Interpretation of results .........................................................................................................................................26

9.2.6 Uncertainty of test results ....................................................................................................................................27

9.2.7 Determination of MPN values ............................................................................................................................27

10 Detection (qualitative) methods .......................................................................................................................................................30

10.1 General ........................................................................................................................................................................................................30

10.2 Procedure .................................................................................................................................................................................................31

10.3 Uncertainty of test results ..........................................................................................................................................................31

11 Performance characteristics of methods .................................................................................................................................31

12 Analytical quality control .........................................................................................................................................................................32

12.1 General ........................................................................................................................................................................................................32

© ISO 2018 – All rights reserved iii
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ISO 8199:2018(E)

12.2 Internal quality control .................................................................................................................................................................32

12.2.1 General...................................................................................................................................................................................32

12.2.2 Process controls ............................................................................................................................................................32

12.3 External quality assessment.....................................................................................................................................................33

Annex A (informative) Criteria for the choice of technique.......................................................................................................35

Annex B (informative) Confidence intervals for colony count technique and choice of

method of calculation in special cases ........................................................................................................................................40

Annex C (normative) Counting and calculations with two Petri dishes per dilution ......................................44

Annex D (normative) Composition, preparation and performance testing of diluents ...............................51

Bibliography .............................................................................................................................................................................................................................55

iv © ISO 2018 – All rights reserved
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SIST EN ISO 8199:2019
ISO 8199:2018(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 of the voluntary nature of standards, 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 www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.

This third edition cancels and replaces the second edition (ISO 8199:2005), which has been technically

revised. The main changes compared with the previous edition are as follows.

— Clauses have been added for terms and definitions, detection (qualitative) methods, performance

characteristics and analytical quality control (AQC).

— The clauses referencing culture media and diluent preparation and QC have been revised to align

with ISO 11133 and have been included in a new Annex D.

— The subclause on general guidance for the calculation of results for solid media techniques has

[9]

been updated to reflect the changes in ISO 7218:2007/Amd.1:2013 on which the relevant clauses

and subclauses in the second edition were based. Modifications have been made, however, to take

account of water microbiology techniques (e.g. membrane filtration) and to allow for dilutions other

than ten-fold dilutions.

— Annex B has been added to give guidance on confidence intervals when calculating special cases,

relating to the update of the subclause on general guidance for the calculation of results for solid

media techniques.

— Annex C has been added to describe calculations when using duplicate dishes per dilution, relating

to the update of the subclause on general guidance for the calculation of results for solid media

techniques.

— The subclause relating to enumeration using liquid media had been expanded and includes

additional guidance on the use of MPN calculators. The former Annex B containing MPN tables has

been removed.
— The title of this document has been amended to reflect these changes.
© ISO 2018 – All rights reserved v
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SIST EN ISO 8199:2019
ISO 8199:2018(E)

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
vi © ISO 2018 – All rights reserved
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SIST EN ISO 8199:2019
ISO 8199:2018(E)
Introduction

Techniques for the detection and enumeration of microorganisms based on their ability to grow on or

in specified culture media are an important and widely used means of assessing the microbiological

quality of water. The purpose of this document is to gather in a single document the information

common to the various techniques. This reduces repetition of technical details in individual standards

and facilitates choice of the technique most suitable for a particular situation. Other guidance has been

included on general topics of relevance to these techniques, such as analytical quality control, method

performance characteristics and uncertainty of test results.
© ISO 2018 – All rights reserved vii
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SIST EN ISO 8199:2019
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SIST EN ISO 8199:2019
INTERNATIONAL STANDARD ISO 8199:2018(E)
Water quality — General requirements and guidance for
microbiological examinations by culture

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all of the safety problems, if any, associated with its

use. It is the responsibility of the user to establish appropriate safety and health practices.

IMPORTANT — It is absolutely essential that tests conducted according to this document be

carried out by suitably trained staff.
1 Scope

This document specifies requirements and gives guidance for performing the manipulations common

to each culture technique for the microbiological examination of water, particularly the preparation of

samples, culture media, and general apparatus and glassware, unless otherwise required in the specific

standard. It also describes the various techniques available for detection and enumeration by culture

and the criteria for determining which technique is appropriate.

This document is mainly intended for examinations for bacteria, yeasts and moulds, but some aspects

are also applicable to bacteriophages, viruses and parasites. It excludes techniques not based on

culturing microorganisms, such as polymerase chain reaction (PCR) methods.
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 7704, Water quality — Evaluation of membrane filters used for microbiological analyses

ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and

performance testing of culture media
ISO 19458, Water quality — Sampling for microbiological analysis
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:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
accuracy
closeness of agreement between a test result and the accepted reference value
[SOURCE: ISO 6107-8:1993, 1, modified — The note has been deleted.]
© ISO 2018 – All rights reserved 1
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SIST EN ISO 8199:2019
ISO 8199:2018(E)
3.2
bias

difference between the expectation of the test results and an accepted reference value

[SOURCE: ISO 5725-1:1994, 3.8, modified — Note 1 to entry has been deleted.]
3.3
confirmed count

count (3.4) corrected for not confirmed presumptive counts (3.9) by further testing of the

presumptive objects
3.4
count

observed number of objects such as colonies or cells determined by direct counting, or

most probable number (MPN) estimation based on statistical calculation using the number of positive

units or presumptive positive units in a dilution series of a test sample (3.16)

[SOURCE: ISO 6107-6:2004, 22, modified — “or presumptive positive units” has been added.]

3.5
detection level

minimum concentration of organisms that produce evidence of growth with a probability of p = 0,95

when inoculated into a specified culture medium and incubated under defined conditions

Note 1 to entry: The theoretical level that conforms to this definition is an average of three viable cells in an

inoculum volume.
[SOURCE: ISO 13843:2017, 3.10]
3.6
intralaboratory reproducibility
intermediate precision

closeness of agreement between test results obtained with the same method on the same or similar test

materials in the same laboratory with different operators using different equipment

3.7
limit of determination

lowest analyte concentration per analytical portion where the expected relative standard uncertainty

equals a specified value
[SOURCE: ISO 13843:2017, 3.17]
3.8
precision

closeness of agreement between independent test results obtained under stipulated conditions

[SOURCE: ISO 5725-1:1994, 3.12, modified — Notes 1 to 3 to entry have been deleted.]

3.9
presumptive count

colony count (3.4) or most probable number (MPN) estimate based on the number of colonies or reaction

vessels that have an outward appearance that is interpreted as typical of a target organism

[SOURCE: ISO 6107-6:2004, 62, modified — “fermentation tubes” has been replaced with “reaction

vessels”.]
2 © ISO 2018 – All rights reserved
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SIST EN ISO 8199:2019
ISO 8199:2018(E)
3.10
relative standard deviation
rel

estimate of the standard deviation of a population from a sample of n results divided by the mean of

that sample
[SOURCE: ISO 13843:2017, 3.30]
3.11
repeatability
measurement repeatability

measurement precision (3.8) under a set of repeatability conditions (3.12) of measurement

[SOURCE: ISO 13843:2017, 3.32]
3.12
repeatability conditions

condition of measurement, out of a set of conditions that includes the same measurement procedure,

same operators, same measuring system, same operating conditions and same location, and replicate

measurements on the same or similar objects over a short period of time
[SOURCE: ISO 13843:2017, 3.33]
3.13
reproducibility
measurement reproducibility

measurement precision (3.8) under reproducibility conditions (3.14) of measurement

Note 1 to entry: Relevant statistical terms are given in ISO 5725-1 and ISO 5725-2.

[SOURCE: ISO 13843:2017, 3.34]
3.14
reproducibility conditions

condition of measurement, out of a set of conditions that includes different locations, operators,

measuring systems, and replicate measurements on the same or similar objects
[SOURCE: ISO 13843:2017, 3.35]
3.15
test portion
specified quantity of the sample that is taken for analysis
EXAMPLE 0,1 ml, 1 ml, 100 ml of sample.
3.16
test sample

undiluted, diluted or otherwise prepared test portion (3.15) of a sample to be tested, after completion

of all preparation steps such as centrifugation, filtration, homogenization, pH adjustment and

determination of ionic strength
[SOURCE: ISO 6107-6:2004, 92, modified — Note 1 to entry has been deleted.]
3.17
trueness

closeness of agreement between the average value obtained from a large series of test results and an

accepted reference value

Note 1 to entry: The measure of trueness is usually expressed in terms of bias (3.2).

[SOURCE: ISO 6107-8:1993, 63]
© ISO 2018 – All rights reserved 3
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ISO 8199:2018(E)
3.18
uncertainty of counting

relative standard deviation (3.10) of results of repeated counting of the colonies or particles of the same

plate(s) or field(s) under stipulated conditions (same person, different persons in one laboratory, or

different laboratories)
[SOURCE: ISO 6107-6:2004, 103, modified — The domain has been deleted.]
4 Principle

The general principle of these techniques consists of inoculating a test portion of a water sample, or

resultant test sample following membrane filtration or centrifugation, on or into a culture medium

(solid or liquid). It is assumed that after incubation each target microorganism present multiplies,

giving either a colony visible directly on or in the solid medium or changes in the observable properties

of the liquid medium. The choice of a particular culture method depends not only on the nat

...

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