ENV ISO 13843:2001

Water quality - Guidance on validation of microbiological methods (ISO/TR 13843:2000)

The method is intended for a concentration range to meet the demands of the EC Drinking Water Directive.

Wasserbeschaffenheit - Richtlinie zur Validierung mikrobiologischer Verfahren (ISO/TR 13843:2000)

Anwendungsbereich
Diese Europäische Vornorm befasst sich mit der Validierung mikrobiologischer
Verfahren, mit besonderem Schwerpunkt auf selektive quantitative Verfahren,
bei denen die quantitative Abschätzung auf der Auszählung von Partikeln
beruht, entweder direkt mit Hilfe eines Mikroskops oder indirekt durch Wachs-tum
(Vermehrung) zu Kolonien oder Trübung.Die Grundlagen und Verfahren in diesem Anwendungsbereich sind allgemein
bekannt als presence/absence (P/A), wahrscheinlichste Zahl (most probable
number, MPN), Koloniezählung und direkte (mikroskopische) Zählung.
Diese Europäische Vornorm ist nicht anwendbar auf die Validierung von so
genannten schnellen oder modernen Verfahren, die meist von der Messung von
Produkten oder Änderungen aufgrund mikrobieller Aktivität abhängen, aber
nicht den Nachweis einzelner Partikel ansprechen.

Qualité de l'eau - Lignes directrices pour la validation des méthodes microbiologiques (ISO/TR 13843:2000)

Le présent Rapport technique traite de la validation des méthodes microbiologiques. L'accent est mis sur les méthodes quantitatives sélectives, dans lesquelles l'estimation quantitative repose sur le comptage des particules, soit directement à l'aide d'un microscope, soit indirectement en se basant sur la croissance (multiplication) des colonies ou l'apparition d'une turbidité. Les principes et les modes opératoires entrant dans le domaine d'application sont généralement présence/absence (P/A), le nombre le plus probable (NPP), le comptage de colonies et le comptage (microscopique) direct. Le présent Rapport technique n'est pas applicable à la validation des méthodes dites rapides ou modernes, qui dépendent le plus souvent du mesurage des produits ou des modifications résultant de l'activité microbienne, mais lesquelles ne concernent pas la détection de particules individuelles.

Kakovost vode – Navodilo za validacijo mikrobioloških metod (ISO/TR 13843:2000)

General Information

• Status: Withdrawn
• Publication Date: 30-Aug-2001
• Withdrawal Date: 11-Jul-2017
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230/WG 3 - Water analysis - Microbiological methods
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Completion Date: 12-Jul-2017
• Directive: 98/83/EC - Quality of water intended for human consumption
• Ref Project: SIST-TS ENV ISO 13843:2004 - Water quality - Guidance on validation of microbiological methods (ISO/TR 13843:2000)

Relations

• Replaced By: EN ISO 13843:2017 - Water quality - Requirements for establishing performance characteristics of quantitative microbiological methods (ISO 13843:2017)
• Effective Date: 19-Jul-2017

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TS ENV ISO 13843:2004
01-junij-2004
Kakovost vode – Navodilo za validacijo mikrobioloških metod (ISO/TR 13843:2000)

Water quality - Guidance on validation of microbiological methods (ISO/TR 13843:2000)

Wasserbeschaffenheit - Richtlinie zur Validierung mikrobiologischer Verfahren (ISO/TR

13843:2000)

Qualité de l'eau - Lignes directrices pour la validation des méthodes microbiologiques

(ISO/TR 13843:2000)
Ta slovenski standard je istoveten z: ENV ISO 13843:2001
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST-TS ENV ISO 13843:2004 en,fr,de

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

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SIST-TS ENV ISO 13843:2004
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SIST-TS ENV ISO 13843:2004
EUROPEAN PRESTANDARD
ENV ISO 13843
PRÉNORME EUROPÉENNE
EUROPÄISCHE VORNORM
May 2001
ICS 07.100.20
English version
Water quality - Guidance on validation of microbiological
methods (ISO/TR 13843:2000)

Qualité de l'eau - Lignes directrices pour la validation des Wasserbeschaffenheit - Richtlinie zur Validierung

méthodes microbiologiques (ISO/TR 13843:2000) mikrobiologischer Verfahren (ISO/TR 13843:2000)

This European Prestandard (ENV) was approved by CEN on 7 April 2001 as a prospective standard for provisional application.

The period of validity of this ENV is limited initially to three years. After two years the members of CEN will be requested to submit their

comments, particularly on the question whether the ENV can be converted into a European Standard.

CEN members are required to announce the existence of this ENV in the same way as for an EN and to make the ENV available promptly

at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the ENV) until the final

decision about the possible conversion of the ENV into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,

Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels

© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. ENV ISO 13843:2001 E

worldwide for CEN national Members.
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SIST-TS ENV ISO 13843:2004
Page 2
ENV ISO 13843:2001
CORRECTED 2001-08-15
Foreword

The text of the Technical Report from Technical Committee ISO/TC 147 "Water quality" of the

International Organization for Standardization (ISO) has been taken over as a European

Prestandard by Technical Committee CEN/TC 230 "Water analysis", the secretariat of which

is held by DIN.

This European Prestandard shall be given the status of a national standard, either by

publication of an identical text or by endorsement, at the latest by November 2001, and

conflicting national standards shall be withdrawn at the latest by November 2001.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of

the following countries are bound to announce this European Prestandard: Austria, Belgium,

Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,

Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United

Kingdom.
Endorsement notice
The text of the Technical Report ISO/TR 13843:2000 has been approved by CEN as a
European Prestandard without any modifications.
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SIST-TS ENV ISO 13843:2004
TECHNICAL ISO/TR
REPORT 13843
First edition
2000-06-01
Water quality — Guidance on validation of
microbiological methods
Qualité de l'eau — Lignes directrices pour la validation des méthodes
microbiologiques
Reference number
ISO/TR 13843:2000(E)
ISO 2000
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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
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ii © ISO 2000 – All rights reserved
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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
Contents Page

Foreword.....................................................................................................................................................................iv

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

2 Terms and definitions ...................................................................................................................................1

3 Arrangement of the document .....................................................................................................................8

4 Basic concepts...............................................................................................................................................8

4.1 General............................................................................................................................................................8

4.2 Validation........................................................................................................................................................8

4.3 Detectors ......................................................................................................................................................11

4.4 Performance characteristics ......................................................................................................................11

4.5 Specifications...............................................................................................................................................11

5 Limitations and characteristic features of microbiological methods ....................................................12

5.1 Recovery of the analyte ..............................................................................................................................12

5.2 Sample variance...........................................................................................................................................12

5.3 Particle distribution and overdispersion...................................................................................................12

5.4 Interactions in the detector.........................................................................................................................12

5.5 Robustness ..................................................................................................................................................13

5.6 Spurious errors............................................................................................................................................13

5.7 Control and guidance charts......................................................................................................................13

6 Mathematical models of variation..............................................................................................................14

6.1 Unavoidable basic variation — The Poisson distribution .......................................................................14

6.2 Overdispersion — The negative binomial model .....................................................................................17

6.3 Statistical and practical limits ....................................................................................................................20

6.4 General tests for randomness — Detection of overdispersion ..............................................................21

7 Specifications — Current practice.............................................................................................................21

8 Specifications — Recommended approach..............................................................................................22

9 Determination and expression of performance characteristics .............................................................23

9.1 General..........................................................................................................................................................23

9.2 Categorical characteristics related to specificity and selectivity...........................................................23

9.3 Working limits ..............................................................................................................................................24

9.4 Working range of MPN procedures............................................................................................................25

9.5 Precision.......................................................................................................................................................25

10 Procedures and steps of validation...........................................................................................................26

10.1 General..........................................................................................................................................................26

10.2 Primary validation........................................................................................................................................26

10.3 Secondary validation...................................................................................................................................28

11 Designs for determining specifications ....................................................................................................28

11.1 A general model for basic quantitative specifications ............................................................................28

11.2 Precision of the entire analytical procedure.............................................................................................29

11.3 Categorical characteristics.........................................................................................................................29

11.4 Unplanned data............................................................................................................................................29

Annex A Statistical procedures and computer programs....................................................................................30

Annex B Numerical examples .................................................................................................................................34

Annex C Example of a validation experiment........................................................................................................45

Bibliography..............................................................................................................................................................46

© ISO 2000 – All rights reserved iii
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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.

The main task of technical committees is to prepare International Standards. Draft International Standards adopted

by the technical committees are circulated to the member bodies for voting. Publication as an International

Standard requires approval by at least 75 % of the member bodies casting a vote.

In exceptional circumstances, when a technical committee has collected data of a different kind from that which is

normally published as an International Standard ("state of the art", for example), it may decide by a simple majority

vote of its participating members to publish a Technical Report. A Technical Report is entirely informative in nature

and does not have to be reviewed until the data it provides are considered to be no longer valid or useful.

Attention is drawn to the possibility that some of the elements of this Technical Report may be the subject of patent

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

ISO/TR 13843 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.
iv © ISO 2000 – All rights reserved
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SIST-TS ENV ISO 13843:2004
TECHNICAL REPORT ISO/TR 13843:2000(E)
Water quality — Guidance on validation of microbiological
methods
1 Scope

This Technical Report deals with validation of microbiological methods, with particular emphasis on selective

quantitative methods in which the quantitative estimate is based on counting of particles either directly, with the aid

of a microscope, or indirectly, on the basis of growth (multiplication) into colonies or turbidity.

The principles and procedures within this scope are commonly known as the presence/absence (P/A), most

probable number (MPN), colony count and direct (microscopic) count.

This Technical Report does not apply to the validation of the so-called rapid or modern methods which mostly

depend on measuring products or changes due to microbial activity but do not address the detection of individual

particles.
2 Terms and definitions

For the purposes of this Technical Report, the following terms and definitions apply.

2.1
accuracy of measurement

closeness of the agreement between a test result and the accepted reference value

NOTE The term “accuracy”, when applied to a set of test results, involves a combination of random components and a

common systematic error or bias component.
[ISO 3534-1:1993, 3.11]
2.2
analyte
measurand
particular quantity subjected to measurement
NOTE 1 See reference [5].

NOTE 2 In microbiology the analyte is ideally defined as a list of taxonomically defined species. In many cases, in practice

the analyte can only be defined by group designations less accurate than taxonomic definitions.

2.3
analytical portion
test portion
volume of particle suspension inoculated into a detector unit

NOTE Examples of a detector unit are agar plate, membrane filter, test tube, microscopic grid square.

2.4
application range
range of particle concentrations routinely subjected to measurement by a method
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2.5
categorical characteristic

method performance characteristic numerically expressed as a relative frequency based on P/A or +/� classification

2.6
CFU, deprecated
colony-forming unit, deprecated
CFP, deprecated
colony-forming particle, deprecated

NOTE 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 (2.27) and germ (2.13)

are terms with similar meanings but convey the original idea better and apply not only to colony-count methods but also to MPN

and P/A.
2.7
coefficient of variation
relative standard deviation

for a non-negative characteristic, the ratio of the standard deviation to the average

NOTE 1 The ratio may be expressed as a percentage.

NOTE 2 The term "relative standard deviation" is sometimes used as an alternative to "coefficient of variation", but this use is

not recommended.
[ISO 3534-1:1993, 2.35]

NOTE 3 In this Technical Report the term coefficient of variation (CV) is used when the relative standard deviation is

expressed in percent (CV % = 100 RSD).
2.8
collaborative test

method or laboratory performance test where several laboratories join in an experiment planned and co-ordinated

by a leader laboratory

NOTE 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.

Method performance tests produce precision estimates (repeatability, reproducibility) out of data accumulated when several

participating laboratories study identical samples with a strictly standardized method.

2.9
confirmed [verified] colony count
presumptive colony count corrected for false positives
x= pc= c
where
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.
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2.10
control chart

two-dimensional scattergram for monitoring method performance with control values obtained by a Type A study

NOTE In control charts the horizontal axis is usually in the time scale or ordinate scale and the control variable is the mean

or some precision measure (s, CV, RSD).
2.11
detector
particle detector

plate of solid matrix or a tube of liquid containing a nutrient medium for counting or detecting living microbial

particles
2.12
detection set
detector set

combination of plates or tubes on which quantitative estimation of microbial concentration in a sample is based

NOTE The detection set is the set of plates or tubes utilized for numerical estimation of a single value.

EXAMPLES Parallel plates of a suspension, plates from consecutive dilutions, 3 � 5 tube MPN system, microtitre plate.

2.13
germ
living entity capable of producing growth in a nutrient medium
cf. propagule (2.27)
2.14
guidance chart

two-dimensional scattergram for presenting method performance data (quantity or precision) with arbitrary guide

values or guide values obtained by Type B reasoning

NOTE In guidance charts, the horizontal axis is usually the colony count per detector.

2.15
heterogeneous Poisson distribution

distribution arising when the mean of a Poisson distribution varies randomly from occasion to occasion

NOTE 1 See reference [11].
NOTE 2 See also negative binomial distribution (2.19).
2.16
limit of detection

particle number x (per analytical portion) where the probability p of a negative result equals 5 %

NOTE 1 Probability of a positive result p(+) = 1 – p .
NOTE 2 a) Calculation of x via Poisson distribution:
11��
x= ln = ln = ln 20 = 3,00
� �
p 0,05
b) Calculation of x via negative binomial distribution:
p � 1
��0 ��uu
�� 0,05��1 20 1
x�� �
22 2
uu u
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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
2.17
limit of determination

lowest average particle concentration x per analytical portion where the expected relative standard uncertainty,

equals a specified value (RSD)
NOTE a) Calculation of x via Poisson distribution:
x �
RSD
� �
b) Calculation of x via negative binomial distribution:
x= , given overdispersion factor = u
RSD � u
� �
2.18
linearity
linear dependence of the signal on concentration of the analyte
cf. proportionality (2.28)
2.19
negative binomial distribution
a particular "overdispersed" statistical distribution of counts
NOTE 1 Its variance can be expressed as
=+� �
� u
where ��is the mean.

NOTE 2 In this Technical Report the square of the overdispersion factor (u) is substituted for the inverse of the exponent

(1/k) of the standard formula for the negative binomial distribution.
2.20
overdispersion
variation in excess of Poisson randomness

NOTE It is detected qualitatively by the Poisson index of dispersion, and measured quantitatively by estimating the

parameter u (overdispersion factor) of the negative binomial distribution.
2.21
overdispersion factor

additional random uncertainty of determination in excess of the Poisson distribution, measured in terms of relative

standard deviation
2.22
overlap error
crowding error
systematic depression of colony counts due to confluence of colonies

NOTE Quantitatively, overlap error depends primarily on the fraction of available growth space occupied by colonial

growth.
2.23
parallel counts

particle or colony numbers in equal analytical portions drawn from the same suspension

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2.24
Poisson distribution

fully random distribution of particle numbers when sampling a perfectly mixed suspension

NOTE The probability P(k) of observing exactly k units in a test portion when the mean equals � is calculated from

Pk��=
2.25
precision

closeness of agreement between independent test results obtained under stipulated conditions

NOTE Precision does not relate to the true value or the specified value. It is usually expressed in terms of imprecision and

computed as a standard deviation of the test results.
2.26
primary validation
full validation

establishment of the specifications for the performance of a new method and/or experimental verification that a

method meets theoretically derived quality criteria
2.27
propagule

a viable entity, vegetative cell, group of cells, spore, spore cluster, or a piece of fungal mycelium capable of growth

in a nutrient medium
cf. germ (2.13)
2.28
proportionality

agreement of observed particle counts with the volume (or dilution) of a series of analytical portions from a common

root suspension

NOTE Proportionality is computed for statistical evaluation as the log-likelihood ratio statistic G with n–1 degrees of

freedom.
2.29
qualitative method

method of analysis whose response is either the presence or absence of the analyte in a certain amount of sample

NOTE See reference [10].
2.30
recovery

general term 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 detector

2.31
relative accuracy

degree of correspondence between the response obtained by the reference method and the response obtained by

the alternative method on identical samples
NOTE See reference [10].
2.32
relative difference
difference between two measured values divided by their mean
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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
2xx�
xx� ��
d= =
xx �x
dd%1� 00

NOTE For all practical purposes, the same value results from the calculation d =ln(x )–ln(x ).

A B
2.33
relative recovery

ratio (A/B) of colony counts obtained by two methods tested on equal test portions of the same suspension, where

B is the reference (when applicable)
2.34
relative standard deviation
RSD

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

RSD =
cf. coefficient of variation (2.7)
2.35
repeatability

closeness of the agreement between the results of successive measurements of the same measurand carried out

under the same conditions of measurement
NOTE 1 See Guide to the expression of uncertainty in measurement [6].

NOTE 2 Repeatability is computed as r =2,8s , where s is the repeatability standard deviation.

r r
2.36
reproducibility

closeness of the agreement between the results of measurements on the same measurand carried out under

changed conditions of measurement
NOTE 1 See Guide to the expression of uncertainty in measurement [6].
NOTE 2 Reproducibility is computed as R =2,8 s ,
where

s is the reproducibility standard deviation usually compounded from the between-laboratories standard deviation s and

R L
repeatability standard deviation s :
ss��s
RrL
2.37
robustness
insensitivity of an analytical method to small changes in procedure
NOTE 1 See reference [23].

NOTE 2 To examine the robustness it is advisable to “abuse” the method in a controlled way.

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2.38
secondary validation

demonstration by experiment that an established method functions according to its specifications in the user's

hands
2.39
apparent selectivity

ratio of the number of target colonies to the total number of colonies in the same sample volume

F =lg(t/n)
where

t is the apparent concentration of presumptive target types estimated by counting colonies;

n is the concentration of total colonies.
2.40
sensitivity

fraction of the total number of positive cultures or colonies correctly assigned in the presumptive inspection

2.41
specificity

fraction of the total number of negative cultures or colonies correctly assigned in the presumptive inspection

2.42
standard uncertainty
uncertainty of the result of a measurement expressed as a standard deviation
NOTE See reference [5].
2.43
type A evaluation

�of uncertainty� method of evaluation of uncertainty by the statistical analysis of a series of observations

EXAMPLE Observations may be e.g. standard deviation, relative standard deviation.

NOTE 1 See references [5] and [6].

NOTE 2 Repeatability and reproducibility are often estimated by carrying out collaborative method performance tests where

several laboratories study "identical" samples provided by a central organizer [15].

2.44
type B evaluation

�of uncertainty� method of evaluation of uncertainty by means other than the statistical analysis of series of

observations e.g. from assumed probability distributions based on experience or other information

NOTE See references [5] and [6].
2.45
uncertainty

�of measurement� parameter, associated with the result of a measurement, that characterizes the dispersion of the

values that could reasonably be attributed to the measurand
NOTE See reference [6].
2.46
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
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EXAMPLE Stipulated conditions may be e.g. the same person or different persons in one laboratory, or different

laboratories.
2.47
validation range

range of mean number of particles per analytical portion for which obeyance of validation specifications (particularly

linearity) have been acceptably demonstrated
NOTE It is usually expressed as the range of "reliable" colony counts.
3 Arrangement of the document

The first part (clauses 4 to 8) of this Technical Report contains informative material on basic principles,

characteristics and limitations of microbiological methods, as well as on general aspects of validation. The second

half (clauses 9 to 11) is the actual validation document, containing specifications and recommended procedures for

their determination.

Old and new concepts and principles are not completely defined in the body of this Technical Report. Three

annexes are attached. Annex A details the statistical formulae most relevant to this document, annex B contains

numerical examples and annex C gives detailed plans for two validation experiments.

Statistical tests in the ordinary sense are not central to the ideas. Mathematical calculations are used mainly for the

purpose of providing convenient summaries of data and statistical distributions provide guidance values. A table of

the � distribution is the guide most frequently consulted.

The two BASIC programmes given in annex A are easily copied into desk-top computers or programmable pocket

calculators to help with the basic calculations most frequently needed.
4 Basic concepts
4.1 General

As far as particle statistics is concerned, microscopic counts obey the same laws as viable counts but they are, with

the exception of microcolony methods, free
...