SIST EN ISO 8199:2019

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

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.

Contents Page
European foreword . 3

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.

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
ISO 8199:2018(E)
© 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
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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

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 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

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 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

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.
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 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

ISO 8199:2018(E)
3.10
relative standard deviation
u
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 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 nature and
numbers of the microorganisms sought, but also on the nature of the water and the reasons for the
examination.
5 General measurement requirements
5.1 Uniformity of temperatures
The following accepted ranges of temperatures and their ranges for incubation or storage are applied,
where appropriate for the intended target organism and unless otherwise required in the specific
standard.
Storage temperatures: (−70 ± 10) °C; (−20 ± 5) °C; (5 ± 3) °C
Incubation temperatures: (22 ± 2) °C; (36 ± 2) °C; (44 ± 0,5) °C
Media tempering temperature: 44 °C to 47 °C
The upper incubation temperature limits shall be followed strictly to ensure optimal growth. The lower
temperature limits may be exceeded for short periods, e.g. due to opening the door of an incubator, but
recovery to the operating temperature should be rapid.
5.2 Incubation times
The following accepted ranges of incubation times are applied, where appropriate for the intended
target organism and unless otherwise required in the specific standard.
Incubation times: (21 ± 3) h; (44 ± 4) h; (68 ± 4) h
5.3 Volumes and masses
Measuring equipment shall be appropriate to the required accuracy and precision. The accepted range
of any measured value is ±2 % where the stated value is critical to method performance and test results,
and ±5 % where the stated value has been shown not to be critical. Examples of critical values having
a direct effect on test results are inoculum and diluent volumes. For tolerances relating to the mass of
ingredients used to prepare culture media, refer to ISO 11133.
NOTE Critical tolerances have been set at 2 % to minimize the uncertainty of test results.
4 © ISO 2018 – All rights reserved

ISO 8199:2018(E)
6 Diluents and culture media
6.1 General
General requirements for preparation, production, sterilization, storage and performance of culture
media are given in ISO 11133.
For preparation of microbiological culture media, unless otherwise stated, add the ingredients to the
volume of water, rather than make the ingredients up to a certain volume.
Before use, check the quality of the culture media, diluents, membrane filters and reagents by following
the procedures described in ISO 11133 and ISO 7704 or as given in the specific standard.
For information on storage of culture media, refer to ISO 11133.
6.2 Quality requirements of ingredients
Use constituents of uniform quality and analytical grade chemicals for the preparation of media.
Other grades of chemicals may be used provided they can be shown to produce equivalent results.
Alternatively, dehydrated complete media or diluents may be used. Follow the manufacturer’s
instructions strictly.
[2]
Refer to ISO 11133 and ISO 3696 for further information on the quality of ingredients and the quality
of water that should be used for media preparation.
6.3 Diluents
The following diluents are commonly used in water microbiology. However, other appropriate diluents
may be used and this list is not exhaustive:
— saline solution;
— peptone diluent;
— peptone saline solution [maximum recovery diluent (MRD)];
— quarter-strength Ringer’s solution;
— phosphate buffer solution.
Follow the formulations and the preparation, storage and performance testing instructions given in
Annex D for these diluents.
7 Sterilization and decontamination
7.1 Sterilization of apparatus and glassware
Sterilize apparatus and glassware not supplied sterile by one of the following methods:
a) in an oven, operating at (170 ± 10) °C for at least 1 h (excluding pre-heating time);
b) in an autoclave, operating at (121 ± 3) °C for at least 15 min.
Some heat labile items may require sterilization by other means (e.g. ultraviolet light or irradiation) but
these are not carried out in the routine laboratory.
ISO 8199:2018(E)
7.2 Sterilization of consumables
Sterile disposable equipment and materials may be used instead of re-usable items (glassware, Petri
dishes, pipettes, bottles, tubes, loops, spreaders, etc.) if the specifications are similar.
If membrane filters are not obtained sterile, these are usually sterilized by moist heat according to
process b) described in 7.1, or by following the manufacturer’s instructions.
7.3 Decontamination of glassware and materials after use
Materials for decontamination and disposal should be placed in appropriate containers, e.g. autoclavable
plastic bags. Autoclaving is the preferred method for all decontamination processes (at least 30 min at
121 °C). The autoclave should be loaded in a way that favours heat penetration into the load (e.g. without
over packing). Take care to loosen caps/lids and open bags to prevent dangerous pressurization of the
container, which could lead to possible breakage, e.g. explosion of glass bottles.
Modern autoclaves may not require caps to be loosened, but follow the manufacturer’s instructions
strictly to avoid dangerous pressurization of the containers.
Alternative methods other than autoclaving may be used.
Autoclave all equipment which has been in contact with microbiological cultures (solid or liquid culture
media), including re-usable containers prior to being washed.
During examination, decontamination by immersion in freshly prepared disinfectant, prepared at the
correct dilution, may be used for small-sized and corrosion resistant equipment (e.g. pipettes).
Pasteur pipettes may be difficult to clean and are usually discarded after a single use.
Most disinfectants have some toxic effects. Wear gloves and eye protection when handling disinfectant
and follow the manufacturer’s instructions.
7.4 Waste management
The correct disposal of contaminated materials does not directly affect the quality of sample analysis,
but it is a matter of good laboratory management. A system for identification and separation of waste
materials and their containers should be established for:
— non-contaminated waste (e.g. uncultured water samples) that can be disposed of using general
waste streams;
— scalpels, needles, knives and broken glass;
— contaminated materials for autoclaving and recycling;
— contaminated materials for autoclaving and disposal, or for disposal only if the material is to be
incinerated.
Materials contaminated with risk category 3 microorganisms and their containers shall be autoclaved
before they are incinerated.
8 Samples and sample handling
8.1 Sampling
Take samples in accordance with ISO 19458. Collect disinfected water samples in sample bottles
containing suitable and sufficient neutralizers.
6 © ISO 2018 – All rights reserved

ISO 8199:2018(E)
8.2 Sample preparation
8.2.1 Waters and other aqueous matrices
Clean and dirty water should be separated and processed using separate equipment in separate areas
to reduce cross-contamination risk where possible. Alternatively, process batches of clean waters
before dirty water.
Before examination, mix the sample thoroughly by agitation to achieve uniform distribution of
microorganisms and other particles. This can be achieved by inversion of the sample or by a to-and-
fro motion. Depending on the nature of the water and the microbial content anticipated, make any
necessary dilutions at this stage.
For plate counts, ten-fold dilutions are usually used. For membrane filtration (with a smaller surface
area), smaller dilution steps are recommended. For many most probable number (MPN) techniques,
dilutions are an inherent part of the procedure. Refer to 9.2.3 for guidance on dilutions in MPN
[7]
techniques. See ISO 6887-1 for general guidance on the preparation of serial dilutions.
For ten-fold dilutions, aseptically measure nine volumes of the diluent and one volume of the water
sample into sterile dilution bottles or tubes. Alternatively, volumes of diluent pre-sterilized in screw-
capped bottles are used and volumes verified after autoclaving. One or more ten-fold dilutions are made
by transferring one volume of water sample to nine volumes of diluent. Mix the solution thoroughly
with a fresh pipette or by mechanical means and transfer one volume of this dilution to another nine
volumes of diluent. Repeat these steps as many times as required. Prepare sufficient volumes of each
dilution for all the tests to be carried out on each water sample.
For dilutions of other magnitudes, the volume of diluent to volume of sample is adjusted accordingly.
For example, four-fold dilutions can be made as described above for ten-fold dilutions, only in this case
one volume of water sample is mixed with three volumes of diluent. Another approach is to use a ten-
fold dilution series, but filter both 10 ml and 30 ml volumes.
If the concentration of the target organism is expected to be high, hundred-fold dilution steps may
be used by mixing one volume of water sample with 99 volumes of diluent, but such large intervals
between measurements can adversely affect the reliability of the test results.
8.2.2 Swabs
8.2.2.1 General
Swabs are sometimes used for assessing water quality, e.g. investigation of biofilms, and may be tested
by both quantitative and qualitative methods.
Different types of swabs are available for specific purposes, including stick and sponge swabs, with
and without neutralizing agents for any disinfectants present. Where transport times before testing
are likely to be extended, special transport swabs giving some protection to the organisms present
are recommended. All types of swabs should be evaluated before use, as some have been found to be
inhibitory to certain microorganisms. Further guidance on the use of swabs and swabbing techniques
[18]
can be found in ISO 18593 .
NOTE Dry stick swabs are not suitable for microbiological testing because they offer no protection against
dehydration or disinfectant residues for any microorganisms present during transport and storage before
testing.
8.2.2.2 Stick swab preparation
Stick swabs in neutralizing solution are mixed thoroughly, either manually or by vortexing, in the
solution.
Transport swabs in agar are carefully removed from the packaging and immersed in a volume (usually
10 ml) of diluent. The stick is then broken or cut off to allow thorough mixing in the sealed bottle or
ISO 8199:2018(E)
tube. Mix either manually or by other means, such as vortexing, shaking or ultrasonication, to optimize
the recovery of any organisms present.
In both cases, the resulting suspensions are used as the initial dilution and the results are expressed
accordingly.
8.2.2.3 Sponge swab preparation
Sponge swabs are recommended for qualitative testing to ensure a representative sample is taken and a
“not detected” result is more reliable based on the larger sample. These contain a volume of neutralizer
sufficient to protect any organisms present during transport, which makes their use for quantitative
testing problematic, unless this volume is known and can be included in subsequent calculations.
On receipt at the laboratory, a larger volume (usually 100 ml) of (pre-)enrichment broth is added to the
sponge swab in the original packaging and testing continued according to the specific method.
9 Enumeration (quantitative) methods
9.1 Inoculation of test portions in (or on) solid media
9.1.1 General
A test portion of the water sample or any dilutions prepared is inoculated, either directly or concentrated
on a membrane filter, on the surface of a specified solid culture medium or in a molten medium so that
once incubated, microorganisms form colonies either on or in the medium.
For practical purposes, each colony is considered to have originated from a single microorganism or a
clump of microorganisms present in the test portion at the time of inoculation. Taking into account the
volume of the test portion and the number of colonies formed, the result can therefore be expressed as
a number of colony-forming units (cfu) or colony-forming particles (cfp) in a given volume of the sample,
e.g. 1 ml or 100 ml.
Three procedures are used predominantly for the inoculation of solid media and the choice of technique
depends on several factors. These include the physical and chemical characteristics of the water as
well as the nature of the microorganisms sought, their probable concentration, the effective recovery
of stressed or (sub-lethally) injured microorganisms, and the test precision and sensitivity required.
Indications are given in 9.1.2.2, 9.1.3.2 and 9.1.4.2 of the volumes of water samples that may be used for
each technique.
NOTE 1 Limits of determination and the accuracy of the various techniques are discussed in A.2.
NOTE 2 The nature of the sample and organisms sought are discussed in A.3.
9.1.2 Pour plate technique
9.1.2.1 General
The test portion is mixed with the medium, which has previously been melted and cooled (tempered)
to a temperature close to that of solidification, i.e. 44 °C to 47 °C, so that heat damage to organisms is
minimized. After incubation, the colonies that develop within and on the surface of the medium are
counted.
9.1.2.2 Test portion
The volume of the test portion of the sample, or of a dilution of the sample, can vary between 0,1 ml and
5 ml depending on the size of the Petri dish and the volume of culture medium used. The dilution should
be chosen so that the expected number of colonies formed on plates of 90 mm diameter is less than 300,
and the number of target colonies is greater than 10.
8 © ISO 2018 – All rights reserved

ISO 8199:2018(E)
The acceptable maximum number of target colonies on a plate will depend on the specific method,
colony size, the nature of the colonies (e.g. spreading) and the presence of non-target colonies, and may
be based on the results of verification exercises. As guidance, the maximum number of colonies on a
[8]
90 mm plate (both target and non-target) is typically regarded as 300 (refer to ISO 7218 ). Larger
(such as 140 mm) dishes need separate consideration (see 9.1.7.2).
9.1.2.3 Inoculation
Melt the medium required in boiling water or by any other process (e.g. a steam flow-through
autoclave or microwave oven, if the heating time/temperature combination has been verified for
media preparation). Loosen caps before heating, avoid over-heating and remove the medium as soon
as it has melted. Place the molten medium in a water bath or incubator at 44 °C to 47 °C for sufficient
time, depending on the numbers and volumes of containers, so that the medium will equilibrate to this
temperature throughout. Verify the time required for tempe
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