EN ISO 13843:2017

SLOVENSKI STANDARD
01-december-2017
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SIST-TS ENV ISO 13843:2004
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Water quality - Requirements for establishing performance characteristics of quantitative
microbiological methods (ISO 13843:2017)
Wasserbeschaffenheit - Anforderungen zur Bestimmung von Leistungsmerkmalen von
quantitativen mikrobiologischen Verfahren (ISO 13843:2017)
Qualité de l'eau - Exigences pour l'établissement des caractéristiques de performance
des méthodes microbiologiques quantitatives (ISO 13843:2017)
Ta slovenski standard je istoveten z: EN ISO 13843:2017
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 13843
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2017
EUROPÄISCHE NORM
ICS 07.100.20 Supersedes ENV ISO 13843:2001
English Version
Water quality - Requirements for establishing
performance characteristics of quantitative
microbiological methods (ISO 13843:2017)
Qualité de l'eau - Exigences pour l'établissement des Wasserbeschaffenheit - Anforderungen zur
caractéristiqes de performance des méthodes Bestimmung von Leistungsmerkmalen von
microbiologiques quantitatives (ISO 13843:2017) quantitativen mikrobiologischen Verfahren (ISO
13843:2017)
This European Standard was approved by CEN on 5 June 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 13843:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 13843:2017) 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 January 2018 and conflicting national standards shall
be withdrawn at the latest by January 2018.
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 ENV ISO 13843:2001.
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 13843:2017 has been approved by CEN as EN ISO 13843:2017 without any modification.
INTERNATIONAL ISO
STANDARD 13843
First edition
2017-06
Water quality — Requirements
for establishing performance
characteristics of quantitative
microbiological methods
Qualité de l’eau — Exigences pour l’établissement des caractéristiqes
de performance des méthodes microbiologiques quantitatives
Reference number
ISO 13843:2017(E)
©
ISO 2017
ISO 13843:2017(E)
© 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.
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www.iso.org
ii © ISO 2017 – All rights reserved

ISO 13843:2017(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Basic concepts . 6
4.1 General . 6
4.2 Characterization . 7
4.3 Verification . 8
4.4 Method comparison . 8
4.5 Samples . 9
5 Specifications: some guideline values . 9
6 Designs for determining performance characteristics of a method .10
6.1 General considerations .10
6.2 Determination of sensitivity, specificity, efficiency, selectivity, false positive rate
and false negative rate .10
6.2.1 Type of samples to be used .10
6.2.2 Number of samples .11
6.2.3 Procedure .11
6.2.4 Categorical performance characteristics .11
6.2.5 Worked example . . .12
6.3 Determination of the upper limit and consideration of the lower limit of detection .14
6.3.1 Working range .14
6.3.2 Upper limit related to linearity.14
6.3.3 Type and number of samples to be used .14
6.3.4 Worked example . . .15
6.3.5 The lower limit of detection .16
6.4 Assessment of precision: Determination of repeatability and reproducibility .16
6.4.1 General.16
6.4.2 Repeatability .17
6.4.3 Intralaboratory reproducibility .18
6.5 Robustness .20
6.5.1 General.20
6.5.2 Experimental designs for effects due to time and temperature .20
6.6 Relative recovery .21
6.6.1 General.21
6.6.2 Determination of relative recovery .21
6.7 Uncertainty of counting .22
6.7.1 General.22
6.7.2 Experimental design for assessing the uncertainty of counting colonies .22
6.7.3 Example of individual (or personal) uncertainty of counting colonies .22
6.7.4 Example of intralaboratory uncertainty of counting colonies .23
6.7.5 Example of intralaboratory uncertainty of reading MPN .23
7 Designs for single laboratory verification of a method .24
7.1 General considerations .24
7.2 Calculation of sensitivity, specificity, efficiency, selectivity, false positive rate and
false negative rate .24
7.2.1 Type of sample to be used .24
7.2.2 Number of samples .25
7.2.3 Procedure for confirmation .25
7.2.4 Categorical performance characteristics .25
7.3 Determination of repeatability .26
ISO 13843:2017(E)
7.4 Uncertainty of counting .26
7.5 Procedure for single laboratory verification .26
Annex A (informative) Mathematical models of variation.30
Annex B (normative) Assessment of the lower limits .40
Annex C (normative) Assessment of the upper limit .43
Annex D (normative) Determination of the operational variability in repeatability and
intralaboratory reproducibility conditions .44
Annex E (normative) Uncertainty of counting .48
Annex F (normative) Determination of the operational variability (interlaboratory
reproducibility) in a collaborative performance study .50
Annex G (informative) Glossary of principal symbols .58
Bibliography .60
iv © ISO 2017 – All rights reserved

ISO 13843:2017(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 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 the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
This first edition of ISO 13843 cancels and replaces ISO/TR 13843:2000, which has been technically
revised.
ISO 13843:2017(E)
Introduction
Methods are considered microbiological when the quantitative estimate is based on counting of
microbial particles either directly with the aid of a microscope or indirectly on the basis of growth
(multiplication) into colonies, turbidity, a colour change or fluorescence. The principles and procedures
within the scope of this document are commonly known as microscopic count, most probable number
(MPN) and colony count. Most of the procedures for the determination of performance characteristics
described in this document are applicable to all three types of method. However, where the procedures
are not applicable, alternative suggestions are made within the body of the document or in Annexes D
and E (for repeatability, reproducibility and uncertainty of counting).
Plaque counts of bacteriophages are in most respects similar to bacterial colony counts.
Some of the ”newer” microbiological methods such as those utilizing fluorescent in situ hybridization
(FISH) or polymerase chain reaction (PCR) can also be covered by this document. However, they may
require special consideration, depending upon how they are used. The issues of importance in these
situations include the mechanism of determining the numbers of microbes present (e.g. standard curve
for qPCR or microscopic count for FISH) and the viability of the organisms detected. If such techniques
are used for confirmation as part of a method then all sections of this document are relevant.
While not essential, during the characterization of microbiological methods it may be beneficial
to generate data using stressed organisms. Various methods can be used to stress organisms, but
the two that are most useful for water are disinfectant stress (usually chlorine injury) and nutrient
depletion caused by organisms being in a low nutrient environment (i.e. drinking water and other
oligotrophic waters) for a period prior to testing. The effect on some of the performance characteristics
of “stressing” organisms is almost totally dependent on the type and degree of stress applied and it is
inappropriate to include such detail in this document. However, there are descriptions in the literature
that laboratories can follow in case they should wish to determine performance characteristics of a
method with stressed cells.
vi © ISO 2017 – All rights reserved

INTERNATIONAL STANDARD ISO 13843:2017(E)
Water quality — Requirements for establishing
performance characteristics of quantitative
microbiological methods
1 Scope
This document deals with characterization of microbiological methods. In terms of this document,
characterization means the study of parameters that can be measured to describe how the method is
likely to perform in a given set of conditions, which can be described as performance characteristics.
The document describes procedures for the determination of performance characteristics which can be
used for subsequent validation or verification of methods.
The emphasis is on selective quantitative methods and this document applies to all types of water. For
methods that are not based upon direct microscopic count, colony count or most probable number, the
applicability of the procedures described in this document should be considered carefully.
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 17994:2014, Water quality — Requirements for the comparison of the relative recovery of
microorganisms by two quantitative methods
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
accuracy
measurement accuracy
closeness of agreement between a measured quantity value and an assigned quantity value of a
measurand
Note 1 to entry: The concept ‘measurement accuracy’ is not a quantity and is not given a numerical quantity
value. A measurement is said to be more accurate when it offers a smaller measurement error.
Note 2 to entry: ‘Measurement accuracy’ is sometimes understood as closeness of agreement between measured
quantity values that are being attributed to the measurand.
[16]
[SOURCE: ISO/IEC Guide 99:2007, 2.13 , modified — “…a true quantity value” replaced by “… an
assigned quantity value; Notes 1 and 2 to entry added]
ISO 13843:2017(E)
3.2
analyte
component represented in the name of a measurable quantity
Note 1 to entry: In water microbiology, the analyte is ideally defined as a list of taxonomically defined species.
In most cases, in practice the analyte can only be defined by group designations less accurate than taxonomic
definitions.
[14]
[SOURCE: ISO 17511:2003, 3.2 ]
3.3
analytical portion
test portion
volume of particle suspension (sample) inoculated into a detector unit (agar plate, membrane filter, test
tube, microscopic grid square)
3.4
bias
measurement bias
estimate of a systematic measurement error, or the systematic difference between the quantitative
assigned value and the average of measurement replicate results
3.5
categorical characteristics
method performance characteristic numerically expressed as a relative frequency based on P/A or
+/‑ classification
3.6
colony-forming unit
CFU
colony‑forming particle
CFP
organism (or cluster of organisms) with the ability to form a colony under certain specified conditions
Note 1 to entry: The term was originally introduced to convey the idea that a colony may originate not only from
a single cell but from a solid chain or aggregate of cells, a cluster of spores, a piece of mycelium, etc. It mistakenly
equates the number of colonies observed to the number of living entities seeded on the medium. Growth unit,
viable particle, propagule and germ are terms with the same meaning but convey the original idea better and
apply not only to colony count methods but also to the most probable number (MPN).
3.7
collaborative method performance
method or laboratory performance test where several laboratories join in an experiment planned and
co‑ordinated by a leader laboratory
Note 1 to entry: Collaborative tests are mainly of two types. Intercalibration exercises are made to allow
laboratories to compare their analytical results with those of other participating laboratories.
Note 2 to entry: Method performance tests produce precision estimates (repeatability, reproducibility) out of data
accumulated when several participating laboratories study identical samples with a strictly standardized method.
3.8
confirmed colony count
verified colony count
presumptive colony count corrected for false positives
Note 1 to entry: Mathematically:
k
pc= c
n
where
2 © ISO 2017 – All rights reserved

ISO 13843:2017(E)
c is the presumptive count;
p is the true positive rate;
n is the number of presumptive positives isolated for confirmation;
k is the number confirmed.
3.9
corroborated count
count obtained when using a secondary confirmation procedure
3.10
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.
3.11
detection set
combination of plates or tubes on which quantitative estimation of sample microbial concentration
is based
Note 1 to entry: The detection set is the set of plates or tubes utilized for numerical estimation of a single value.
EXAMPLE Parallel plates of a suspension, plates from consecutive dilutions, 3 × 5 tube MPN system,
microtitre plate.
3.12
detector
particle detector
plate of solid matrix or a tube of liquid containing a nutrient medium for counting or detecting
biologically active particles
3.13
efficiency
E
fraction of colonies that are correctly assigned as positives and negatives
Note 1 to entry: Mathematically:
ad+
E =
n
where
a is the number of typical colonies confirmed as being the target organism (true positives);
d is the number of atypical colonies confirmed as not being the target organism (true negatives);
n is the total number of colonies tested for confirmation.
3.14
false negative
result indicated by the test method to be negative which has subsequently been shown to contain the
target organism
ISO 13843:2017(E)
3.15
false positive
result indicated by the test method to be positive which was subsequently shown not to contain the
target organism
3.16
germ
entity capable of biological activity (e.g. respiration or reproduction in a nutrient medium)
3.17
limit of determination
lowest analyte concentration per analytical portion where the expected relative standard uncertainty,
equals a specified value
3.18
method-defined count
count obtained by using only the procedures in the described method
3.19
negative binomial distribution
particular “overdispersed” statistical distribution of counts
2 22
Note 1 to entry: Its variance can be expressed as sx=+ux ()x =mean .
Note 2 to entry: In this document, the square of the relative operational standard deviation (u ) is substituted for
the inverse of the exponent (1/k) of the standard equation for the negative binomial distribution.
3.20
outlier
member of a set of values which is inconsistent with other members of that set
Note 1 to entry: An extreme value which normally appears randomly in less than 1 % of repetitive tests, but more
frequently if abnormal situations occur. Statistical test procedures can be used to quantify this probability.
3.21
over-dispersion
variation in excess of Poisson randomness
Note 1 to entry: Detected qualitatively by the Poisson index of dispersion and measured quantitatively by
estimating the parameter u (relative operational standard deviation) of the negative binomial distribution.
3.22
parallel counts
particle or colony numbers in equal analytical portions drawn from the same suspension
3.23
Poisson distribution
fully random distribution of particle numbers when sampling a perfectly mixed suspension
Note 1 to entry: The probability P(k) of observing exactly k units in a test portion when the mean equals µ is
calculated from
k
u
−μ
Pk()= e
k!
4 © ISO 2017 – All rights reserved

ISO 13843:2017(E)
3.24
precision
measurement precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as
standard deviation, variance, or coefficient of variation under the specified conditions of measurement.
Note 2 to entry: The ‘specified conditions’ can be, for example, repeatability conditions of measurement,
intermediate precision conditions of measurement, or reproducibility conditions of measurement (see
[4]
ISO 5725-3 ).
Note 3 to entry: Measurement precision is used to define measurement repeatability, intermediate measurement
precision, and measurement reproducibility.
3.25
proportionality
agreement of observed particle counts with the volume (or dilution) of a series of analytical portions
from a common root suspension
Note 1 to entry: Proportionality is evaluated as the log‑likelihood ratio statistic G with n-1 degrees of freedom.
3.26
recovery
general term used for the number of particles estimated in a test portion or sample, with the
understanding that there is a true (although unknown) number of particles of which 100 % or less are
“recovered” by the employed methodology
[12]
Note 1 to entry: Another similar term commonly used is productivity (see ISO 11133 ).
3.27
relative recovery
ratio of colony counts obtained by two methods tested on equal test portions of the same suspension
3.28
relative operational standard deviation
u
operational variability, expressed as a relative standard uncertainty, associated with the technical
steps of the analytical procedure
Note 1 to entry: The relative operational standard deviation is often expressed in percent.
3.29
relative operational variance
u
over-dispersion constant, the square of relative operational standard deviation
3.30
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
3.31
relative variance
u
rel
square of relative standard deviation
ISO 13843:2017(E)
3.32
repeatability
measurement repeatability
measurement precision under a set of repeatability conditions of measurement
3.33
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
3.34
reproducibility
measurement reproducibility
measurement precision under reproducibility conditions of measurement
[2] [3]
Note 1 to entry: Relevant statistical terms are given in ISO 5725‑1 and ISO 5725-2 .
3.35
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
3.36
robustness
insensitivity of an analytical method to small changes in procedure
Note 1 to entry: To examine the robustness it is advisable to “abuse” the method in a controlled way.
3.37
sensitivity
fraction of the total number of positive cultures or colonies correctly assigned in the presumptive
inspection
3.38
specificity
fraction of the total number of negative cultures or colonies correctly assigned in the presumptive
inspection
3.39
standard uncertainty
uncertainty of the result of a measurement expressed as a standard deviation
3.40
uncertainty of counting
relative standard deviation 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)
3.41
verification
performance of a second characterization by a different laboratory to confirm the results of the original
characterization
4 Basic concepts
4.1 General
As far as particle statistics are concerned, microscopic counts obey the same laws as viable counts but
they are, with the exception of microcolony methods, free from the biological problems associated with
growth. Differential stains, specifically labelled complexes or other agents used for finding the target
6 © ISO 2017 – All rights reserved

ISO 13843:2017(E)
do not change the basic principles. The same principles as those used with selective colony methods
can be applied. For a more detailed understanding of the theory and application of the formulae used in
this document, the mathematical basis for the variation encountered in all of these types of method is
described in Annex A.
4.2 Characterization
The characterization of a microbiological method is largely based upon the examination and expression
of the performance characteristics of that method.
Characterization is a process of providing information about the likely performance of that procedure
under a specific set of circumstances. It is not the intention of this document to provide guideline values
for each of the specified performance characteristics but rather to give guidance on which parameters
should be determined and how best to derive them for comparative purposes. Methods that have “poor”
performance characteristics may still be useful.
Characterization is an exploratory process with the aim of establishing the likely set of performance
characteristics of a new, modified or otherwise inadequately characterized method. It should result in
numerical and descriptive specifications for the performance and include a detailed and unambiguous
description of the target of interest (such as positive colony, tube or plaque). However the values
generated should not be used as limits since they may change depending on the laboratory, matrix or
even specific samples.
Characterization is performed by a single laboratory in the first instance to determine the likely
performance of a test method in a specific laboratory.
A collaborative method performance study can be performed as an additional step to evaluate the
interlaboratory performance characteristics.
NOTE A laboratory developing an in‑house method or a variant of an existing standard could carry out the
steps of characterization.
It is imperative that technicians involved in the characterization of a method have considerable
experience with other microbiological methods.
The performance characteristics covered by this document are listed in Table 1.
Table 1 — Performance characteristics described in the document
Parameter Definition
a, b, c e
Sensitivity fraction of the total positives correctly assigned in the presumptive count
a, b, c f
Specificity fraction of the total negatives correctly assigned in the presumptive count
a, b
False positive rate fraction of positive results (e.g. typical colonies) that are subsequently shown to
be due to non-target organisms
a, b
False negative rate fraction of negative results (e.g. atypical colonies) shown to be target organisms
a, b, c
Selectivity ratio of the number of target colonies to the total number of colonies in the sam-
ple volume
a, b
Efficiency fraction of total colonies correctly assigned in the presumptive count
a
Required for determination of the performance characteristics.
b
Required for single laboratory verification.
c Guidance specification given.
d
Methods for interlaboratory reproducibility and precision are described in Annex F. Use of these methods should be
considered when interlaboratory performance is paramount, for example when methods are being developed for regulatory
compliance.
e
Positives may be colony counts, positive reaction vessels (MPN) or cell counts.
f
Negatives may be atypical colonies, negative reaction vessels (MPN) or cells without the specific characteristics
required.
ISO 13843:2017(E)
Table 1 (continued)
Parameter Definition
a
Upper limit upper end of the working range for which the method is useful (i.e. the maximum
countable colonies per plate, or other detection systems)
a, b, c
Repeatability precision under repeatability conditions (same operators, same operating condi-
tions, short period of time, .)
a d
Reproducibility precision under intralaboratory reproducibility conditions
a
Robustness measure of the capacity of a test to remain unaffected by small but deliberate
variations in testing conditions (e.g. temperature)
a
Relative recovery efficiency with which a method recovers target organisms from a sample when
compared to another procedure
(This comparison shall be done where an alternative method for the same organ-
ism exists. Comparison with an ISO reference method is preferred.)
a, b
Uncertainty of counting relative standard deviation of replicate counts of the target obtained by repeated
counting (plates, fields, tubes, etc.) under stipulated conditions (same person,
different person, same laboratory, etc.)
a
Required for determination of the performance characteristics.
b
Required for single laboratory verification.
c Guidance specification given.
d
Methods for interlaboratory reproducibility and precision are described in Annex F. Use of these methods should be
considered when interlaboratory performance is paramount, for example when methods are being developed for regulatory
compliance.
e
Positives may be colony counts, positive reaction vessels (MPN) or cell counts.
f
Negatives may be atypical colonies, negative reaction vessels (MPN) or cells without the specific characteristics
required.
While interlaboratory reproducibility and precision do not form part of the performance characteristics
described in the body of this document, in certain situations knowledge of these parameters is highly
desirable. Such situations include when methods are being used for regulatory compliance or when
data from a variety of laboratories are being compared for any of a number of reasons. For this reason,
suggested methods to determine interlaboratory reproducibility are described in Annex F.
4.3 Verification
Verification takes place when a laboratory proceeds to implement a method developed elsewhere.
Verification focuses on gathering evidence that the laboratory is able to generate performance data
similar to those established in primary characterization. It is not helpful to establish limits on the
various components of method characterization since these can vary dependent on many aspects of the
method, type of sample and performing laboratory. The verification data should be used to establish
the type and quality of data likely to be generated by the laboratory with a given procedure and any
given sample type.
Typically, verification uses selected and simplified forms of the same procedures used in method
characterization, but possibly extended over a longer time. Natural samples are the optimal test
materials and the work need only address those aspects of the method performance that are of interest
to the laboratory. The requirements for single laboratory verification are described in Clause 7.
4.4 Method comparison
Method performance consists of many aspects. There is neither a single test of method comparison
nor numerical criteria for it. One method may be superior in specificity but inferior in recovery. All
the collective information about robustness, precision and specificity gained during characterization
tests can be used for method comparison. The methods only need to be tested in parallel for recovery
comparisons.
8 © ISO 2017 – All rights reserved

ISO 13843:2017(E)
It is necessary to apply two methods in parallel on the same samples when developing an in‑house
method, and also when collecting information to justify the use of an alternative method. Relative
recovery studies of an alternative method against a reference method organized according to
ISO 17994 involve preferably a wide range of samples and participation by a number of laboratories
allowing the expansion of the sample range over large geographical areas. However, sometimes it may
be necessary to verify the result of an alternative method recovery study under ecological conditions
or in a geographical area not represented in the earlier collaborative trial. When a laboratory only
needs to confirm the comparison result of a method already tested and officially accepted, it can
take full advantage of the previous test results. The laboratory should have access to the report of
the collaborative comparison. Accordingly, it should have at its disposal estimates of the mean and
standard deviation of the relative difference. Formula (3) given in ISO 17994:2014, 5.4.3 can be applied
to estimate the recommended number of samples. However, whatever the result of the calculation, the
number of samples should not be less than thirty.
A method giving the highest recovery of confirmed target organisms is obviously the best when
confirmation is required for routine use. A method giving somewhat lower recovery but not requiring
confirmation may be preferable. If high false negative rates or false positive rates observed in
characterization cannot be corrected by more refined target colony definitions or other procedures,
the method may be deemed invalid. The comparison of two microbiological methods should include
a comparison of their performance characteristics (i.e. characterization) together with a side by side
comparison of recovery, using naturally contaminated or spiked samples as specified in ISO 17994.
4.5 Samples
It is a popular concept that the characterization and comparison of methods sh
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