ISO 29201:2012

INTERNATIONAL ISO
STANDARD 29201
First edition
2012-01-15
Water quality — The variability of
test results and the uncertainty of
measurement of microbiological
enumeration methods
Qualité de l’eau - Variabilité des résultats d’essais et incertitude de
mesure des méthodes d’énumération microbienne
Reference number
ISO 29201:2012(E)
©
ISO 2012

---------------------- Page: 1 ----------------------
ISO 29201:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 29201:2012(E)
Contents Page
Foreword . v
Introduction .vi
1 Scope . 1
2 Key concepts . 1
2.1 Uncertainty of measurement . 1
2.2 Estimation of the uncertainty of measurement . 1
2.3 Intralaboratory reproducibility . 2
2.4 Combined standard uncertainty . 2
2.5 Relative standard uncertainty . 2
2.6 Relative variance . 3
2.7 Expanded uncertainty and expanded relative uncertainty . 3
3 Microbiological methods . 4
3.1 Common basis . 4
3.2 Quantitative instruments . 4
3.3 Uncertainty structure . 4
3.4 Expression of combined uncertainty . 4
4 Choices of approach . 5
4.1 General . 5
4.2 Choices of evaluation approach . 6
4.3 Choices of expression and use of measurement uncertainty . 7
5 The component approach to the evaluation of operational uncertainty . 7
5.1 General . 7
5.2 Identification of the components of uncertainty . 7
5.3 Evaluation . 7
6 The global approach to the determination of the operational uncertainty . 8
6.1 General . 8
6.2 Evaluation . 9
7 Combined uncertainty of the test result .10
7.1 Basic principle .10
7.2 Operational variability .10
7.3 Intrinsic variability .10
7.4 Combined uncertainty .10
7.5 Borderline cases .10
Annex A (informative) Symbols and definitions . 11
Annex B (normative) General principles for combining components of uncertainty .13
Annex C (normative) Intrinsic variability — Relative distribution uncertainty of colony counts .18
Annex D (normative) Intrinsic variability of most probable number estimates .20
Annex E (normative) Intrinsic variability (standard uncertainty) of confirmed counts .23
Annex F (normative) Global approach for determining the operational and combined uncertainties .26
Annex G (normative) Component approach to evaluation of the combined relative uncertainty under
intralaboratory reproducibility conditions .31
Annex H (normative) Experimental evaluation of subsampling variance .35
Annex I (normative) Relative repeatability and intralaboratory reproducibility of
volume measurements .38
Annex J (normative) Relative uncertainty of a sum of test portions .40
Annex K (normative) Relative uncertainty of dilution factor F .44
© ISO 2012 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO 29201:2012(E)
Annex L (normative) Repeatability and intralaboratory reproducibility of counting .46
Annex M (normative) Incubation effects — Uncertainty due to position and time .50
Annex N (informative) Expression and use of measurement uncertainty .55
Bibliography .61
iv © ISO 2012 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 29201:2012(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 2.
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.
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.
ISO 29201 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
© ISO 2012 – All rights reserved v

---------------------- Page: 5 ----------------------
ISO 29201:2012(E)
Introduction
Testing laboratories are required to apply procedures for estimating uncertainty of measurement (see
[5]
ISO/IEC 17025 ). Without such an indication, measurement results cannot be compared, either among
[7]
themselves or with reference values (see ISO/IEC Guide 98-3:2008 ).
General guidelines for the evaluation and expression of uncertainty in measurement have been elaborated by
[7]
experts in physical and chemical metrology, and published by ISO and IEC in ISO/IEC Guide 98-3:2008.
[7]
However, ISO/IEC Guide 98-3:2008 does not address measurements in which the observed values are counts.
[7]
The emphasis in ISO/IEC Guide 98-3:2008 is on the “law of propagation of uncertainty” principle, whereby
combined estimates of the uncertainty of the final result are built up from separate components evaluated by
whatever means are practical. This principle is referred to as the “component approach” in this International
Standard. It is also known as the “bottom-up” or “step-by-step” approach.
It has been suggested that the factors that influence the uncertainty of microbiological enumerations are not
[6]
well enough understood for the application of the component approach (see ISO/TS 19036:2006 ). It is
possible that this approach underestimates the uncertainty because some significant uncertainty contributions
[7]
are missed. Reference [19] shows, however, that the concepts of ISO/IEC Guide 98-3:2008 are adaptable
and applicable to count data as well.
Another principle, a “black-box” approach known as the “top-down” or “global” approach, is based on statistical
[6]
analysis of series of repeated observations of the final result (see ISO/TS 19036:2006 ). In the global approach
it is not necessary to quantify or even know exactly what the causes of uncertainty in the black box are.
According to the global philosophy, once evaluated for a given method applied in a particular laboratory,
the uncertainty estimate may be reliably applied to subsequent results obtained by the method in the same
[10]
laboratory, provided that this is justified by the relevant quality control data (EURACHEM/CITAC CG 4 ). Every
analytical result produced by a given method thus should have the same predictable uncertainty. This statement
is understandable against its background of chemical analysis. In chemical analyses the uncertainty of the
analytical procedure and the uncertainty of the final result of analysis are usually the same. The global principle
dismisses the possibility that there might be something unique about the uncertainty of a particular analysis.
The uncontrollable “variation without a cause” that always accompanies counts alters the situation for
microbiological enumerations. The full uncertainty of a test result can be estimated only after the final result
has been secured. This applies to both the global and the component approaches.
The unpredictable variation that accompanies counts increases rapidly when counts get low. The original
global design is therefore not suitable for low counts, and therefore also not applicable to most probable
number (MPN) methods and other low-count applications, such as confirmed counts.
It is often necessary, and always useful, to distinguish between two precision parameters: the uncertainty of the
technical measuring procedure (operational variability), which is more or less predictable, and the unpredictable
variation that is due to the distribution of particles. A modification of the global principle that takes into account
these two sources of uncertainty is free from the low-count restriction. This is the global model detailed in this
International Standard.
In theory, the two quantitative approaches to uncertainty should give the same result. A choice of two approaches
is presented in this International Standard. Offering two approaches is appropriate not only because some
parties might prefer one approach to the other. Depending on circumstances one approach may be more
efficient or more practical than the other.
Neither of the main strategies is, however, able to produce unequivocal estimates of uncertainty. Something
always has to be taken for granted without the possibility of checking its validity in a given situation. The estimate
of uncertainty is based on prior empirical results (experimental standard uncertainties) and/or reasonable
general assumptions.
vi © ISO 2012 – All rights reserved

---------------------- Page: 6 ----------------------
INTERNATIONAL STANDARD ISO 29201:2012(E)
Water quality — The variability of test results and the uncertainty
of measurement of microbiological enumeration methods
1 Scope
This International Standard gives guidelines for the evaluation of uncertainty in quantitative microbiological
analyses based on enumeration of microbial particles by culture. It covers all variants of colony count methods
and most probable number estimates.
Two approaches, the component (also known as bottom-up or step-by-step) and a modified global (top-down)
approach are included.
The aim is to specify how values of intralaboratory operational variability and combined uncertainty for final
test results can be obtained.
The procedures are not applicable to methods other than enumeration methods.
NOTE 1 Most annexes are normative. However, only the annexes relevant to each case are to be applied. If the choice
is the global approach, then all normative annexes that belong to the component approach can be skipped and vice versa.
NOTE 2 Pre-analytical sampling variance at the source is outside the scope of this International Standard, but needs
to be addressed in sampling designs and monitoring programmes.
NOTE 3 The doubt or uncertainty of decisions based on the use of analytical results whose uncertainty has been
estimated is outside the scope of this International Standard.
NOTE 4 The extra-analytical variations observed in proficiency tests and intercalibration schemes are also not
detailed in this International Standard, but it is necessary to take them into consideration in analytical control. The use
of intercalibration data in uncertainty estimation offers the possibility for the bias between laboratories to be included
[12]
(Nordtest Report TR 537 ).
2 Key concepts
2.1 Uncertainty of measurement
[7]
Uncertainty of measurement according to ISO/IEC Guide 98-3:2008 is defined as a “parameter, associated
with the result of measurement, that characterizes the dispersion of the values that could reasonably be attributed
to the measurand”. It is a measure of imprecision. The parameter is expressed as a standard uncertainty or
relative standard uncertainty.
2.2 Estimation of the uncertainty of measurement
[7]
According to ISO/IEC Guide 98-3:2008, the parameter can be evaluated by statistical analysis of series of
observations. This is termed type A estimation of uncertainty.
Any other type of procedure is called type B estimation of uncertainty. The most common type B estimates in
microbiological analysis are those based on assumed statistical distributions in the component approach.
Types A and B may refer to the uncertainty of individual components of uncertainty as well as to the combined
uncertainty of the final result.
Type A evaluations of standard uncertainty are not necessarily more reliable than type B evaluations. In many
practical measurement situations where the number of observations is limited, the components obtained from
type B evaluations can be better known than the components obtained from type A evaluations (ISO/IEC Guide
[7]
98-3:2008 ).
© ISO 2012 – All rights reserved 1

---------------------- Page: 7 ----------------------
ISO 29201:2012(E)
2.3 Intralaboratory reproducibility
A somewhat abstract expression of uncertainty, intralaboratory reproducibility, is frequently considered
[6]
the most appropriate parameter of the uncertainty of measurement, see ISO/TS 19036:2006. It is also
known as intermediate reproducibility or intermediate precision, e.g. [time + equipment + operator]-different
[2]
intermediate precision standard uncertainty as defined by ISO 5725-3. The idea is to evaluate how much the
analytical result might have varied if the analysis had been made by another person in the same laboratory
using different equipment and batches of material and different analytical and incubation conditions than those
actually employed. The value of intermediate precision estimated never belongs to any actual analytical result,
but is assumed to give a general estimate of reasonable uncertainty for the application of a method in one
particular laboratory.
Intralaboratory reproducibility is estimated either by combining separate components of uncertainty determined
under intralaboratory reproducibility conditions (component approach) or by special experiments in which the
analytical conditions are varied by design (global approach).
2.4 Combined standard uncertainty
2.4.1 General
The final test results of microbiological analyses are calculated from intermediate observed values. The
main intermediate observation is the count. Most of the other observed values are connected with volume
measurements.
[7]
Combined standard uncertainty, as defined in ISO/IEC Guide 98-3:2008, is the “standard uncertainty of
the result of a measurement when that result is obtained from the values of a number of other quantities, equal
to the positive square root of a sum of terms, the terms being variances or covariances of these other quantities
weighted according to how the measurement result varies with changes in these quantities”.
NOTE 1 Observation of covariances is only necessary if significant correlations occur between components of
uncertainty. Otherwise a simple root sum of variances is sufficient (see 2.4.2 and 2.5).
NOTE 2 In cases of microbiological enumeration, it can be assumed that all components of uncertainty are independent,
i.e. statistically uncorrelated. In such instances, the combined standard uncertainty is the positive square root of the sum
[7]
of component variances, i.e. the root sum of squares (Annex B). (ISO/IEC Guide 98-3:2008. )
2.4.2 Significant property of combined uncertainties
[10]
According to EURACHEM/CITAC CG 4 , “Unless there is a large number of them, components (standard
uncertainties) that are less than one-third of the largest need not be evaluated in detail”. This statement implies
that in borderline cases, even a single component might provide an adequate estimate of the combined
uncertainty. To decide when a component is unimportant, its approximate size should be known in relation to
other components. Generally at least two, usually more, components are significant and should be included.
EXAMPLE The combined uncertainty of two components, one three times the other, is calculated as
22
uy() =+31 ==10 31, 6 .
c
Without the smaller component, the estimate would be 3,00. Ignoring the smaller component underestimates the
combined uncertainty in this case by about 5 %. For the sake of caution, setting a four-fold difference as the limit might
be recommended.
2.5 Relative standard uncertainty
2.5.1 General
The formula for the final results of microbiological analyses involves only multiplication and division. Under
such conditions, the combined standard uncertainty should be calculated from components expressed as
[7]
relative standard uncertainties (ISO/IEC Guide 98-3:2008 )(see Annex B).
2 © ISO 2012 – All rights reserved

---------------------- Page: 8 ----------------------
ISO 29201:2012(E)
With both type A and type B estimates, the symbol chosen to represent the relative standard uncertainty is u .
rel
NOTE 1 Relative standard uncertainty is often expressed as a percentage. The term commonly used for this expression
is coefficient of variation (CV), C .
V
NOTE 2 When it is important to stress that the standard uncertainty has been calculated by the type A process, the
symbol used is s.
NOTE 3 Any systematic or random variation that takes place in the process before the final suspension, such as
subsampling, matrix, and dilution effects, influence the target concentration in the final suspension proportionally. Relative
variances of these components are therefore additive. Such effects after inoculation as incubation, and reading, can be
more complicated statistically and are not well enough known. Proportionality can still be the best simple approximation.
Systematic errors in these influences are usually treated as if they were random effects.
2.5.2 Logarithms and relative standard uncertainty
“Global” estimates of experimental standard uncertainty are traditionally made by calculation with common
logarithms. When using such estimates in further calculations together with other estimates, it is necessary
to express all components of uncertainty on the same scale of measurement, either by converting relative
standard uncertainties into logarithms or logarithms into relative standard uncertainties.
In most cases, absolute standard uncertainty calculated in natural logarithmic scale and the relative standard
uncertainty in interval scale can be assumed to be numerically equal. Values calculated in common logarithms
can be converted to natural logarithms and vice versa by use of appropriate coefficients. The mathematical
relationships between relative standard uncertainty and standard uncertainty on different logarithmic scales
are shown in B.9.
2.6 Relative variance
[7]
The square of the relative standard uncertainty is called the relative variance (ISO/IEC Guide 98-3:2008).
2.7 Expanded uncertainty and expanded relative uncertainty
Especially when the test result is used for assessing limits concerned with public health or safety, it is pertinent
to give an uncertainty value that encompasses a large fraction of the expected range of the observed values.
The parameter is termed the expanded uncertainty, for which the symbol is U.
The value of U is obtained by multiplying the combined uncertainty with a coverage factor k:
Uk= uy
()
c
The value of k is typically in the range 2 to 3. On the relative scale
Uk= uy
()
relc,rel
For normal distributions, about 95 % of the results are covered by the expanded uncertainty interval m ± U , where
m is the mean, when the coverage factor k = 2 is chosen. When k = 3, coverage corresponds to about 99 %.
Microbiological test results almost never fit a normal distribution perfectly. Distributions are often markedly
asymmetrical (skewed). When there are sufficient reasons for assuming distributions to be other than normal
(e.g. Poisson or negative binomial or log–normal distributions) and plausible estimates of the relevant
parameters are available, upper and lower 95 % boundaries can be based on these distributions. Annex N
gives more details about estimation and use of expanded uncertainty.
© ISO 2012 – All rights reserved 3

---------------------- Page: 9 ----------------------
ISO 29201:2012(E)
3 Microbiological methods
3.1 Common basis
Microbiological enumeration methods based on culture are technical variants of the same basic principle.
The analysis often begins with the mixing of a measured portion of the laboratory sample into a suitable liquid
medium to produce a homogenate called the initial suspension. It may have to be diluted further to produce
a final suspension of appropriate density for detection and enumeration of the target microorganism. In water
analysis, the water sample is the initial suspension and, when dilution is unnecessary, also directly serves as
the final suspension.
3.2 Quantitative instruments
Measured portions of the final suspension are transferred into a detection instrument for quantitative evaluation.
The detection instruments in microbiological analyses vary from a single Petri dish to systems of many parallel
plates in different dilutions and to most probable number (MPN) systems of diverse complexity.
3.3 Uncertainty structure
A complete microbiological analytical procedure consists of five or six successive steps:
a) subsampling and mixing;
b) dilution;
c) delivery of test portions(s) into the detection system of nutrient media;
d) development during incubation;
e) counting and possibly confirming the (presumptive) target organisms.
The operational variability consists of the effects of these technical steps. They are individually estimated for
use in the component approach. When estimating the uncertainty of the final result, the uncertainty due to
random distribution of particles in suspension is additionally taken into account (5.2). In the traditional global
approach all operational components and the random distribution of particles are estimated together.
3.4 Expression of combined uncertainty
3.4.1 Two‑component model
For many practical and illustrative purposes it is sufficient to consider the uncertainty of microbiological test
results to consist of two groups of components.
Combined uncertainty of measurement is obtained by combining the operational variability and the intrinsic
variability (distribution uncertainty).
In microbiological contexts both variances are to be expressed as relative (or logarithmic) variances. The
symbols used in this connection in this International Standard are:
2 2
uy()=+uu (1)
c,relo,rel d,rel
where
4 © ISO 2012 – All rights reserved

---------------------- Page: 10 ----------------------
ISO 29201:2012(E)
u (y) is the combined relative standard uncertainty;
c,rel
u is the relative operational variability (experimental relative standard uncertainty);
o,rel
u is the relative intrinsic variability (relative distribution uncertainty).
d,rel
Equation (1) is applied in both the modified global and the component approaches to construct the combined
relative uncertainty of measurement of the final result.
NOTE Subscripts can be used to indicate the experimental conditions or level of uncertainty (r for repeatability, Rʹ for
intermediate or intralaboratory repeatability and R for interlaboratory repeatability).
3.4.2 Operational variability (technical uncertainty)
Operational variability is the combination of all the uncertainties associated with the technical steps of the
analytical procedure. It includes the variability of the subsampling, mixing, and dilution of the laboratory sample
to prepare the final test suspension. It also includes the possible effects of incubation and the uncertainty of
reading the result. Bias components are involved but form parts of random variation.
3.4.3 Intrinsic variability (distributional uncertainty)
Intrinsic variability is the unavoidable variation without a cause that is associated with the distribution of particles
in the final suspension and in the detection instrument. In microbiological suspensions it is usually believed to
follow the Poisson distribution. When partial confirmation is practised or the MPN principle is used, the intrinsic
variation increases considerably and no longer follows the Poisson distribution (Annexes D and E).
NOTE The intrinsic variability can be decreased by using replicate plates and for MPN estimates by increasing the
number of parallel tubes.
4 Choices of approach
4.1 General
The tradition of evaluation, presentation and use of measurement uncertainty is short in microbiology.
Different parties still have different interpretations and understanding of the meaning and use of measurement
uncertainty. Because of this fluid state, there is no unique right way of determining, expressing and using the
uncertainty of measurement.
This International Standard is primarily intended to provide guidelines for laboratories on how to get started
with esta
...