SIST-TS ENV ISO 13843:2004
(Main)Water quality - Guidance on validation of microbiological methods (ISO/TR 13843:2000)
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
Relations
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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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
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
CV
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
x
presumptive colony count corrected for false positives
k
x= pc= c
n
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.
2 © ISO 2000 – All rights reserved
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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
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 %
0
NOTE 1 Probability of a positive result p(+) = 1 – p .
0
NOTE 2 a) Calculation of x via Poisson distribution:
��
11��
x= ln = ln = ln 20 = 3,00
� �
��
��
��
p 0,05
��
0
b) Calculation of x via negative binomial distribution:
2
��
�u
22
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:
1
x �
2
RSD
� �
b) Calculation of x via negative binomial distribution:
1
x= , given overdispersion factor = u
2
2
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
2
22
=+� �
� 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
u
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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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
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
k
�
��
Pk��=
e
k!
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
2
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
d
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� ��
AB
AB
d= =
xx �x
AB
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
s
RSD =
x
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 ,
R
where
s is the reproducibility standard deviation usually compounded from the between-laboratories standard deviation s and
R L
repeatability standard deviation s :
r
22
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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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
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
F
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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SIST-TS ENV ISO 13843:2004
ISO/TR 13843:2000(E)
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
2
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
...
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