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 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

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
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

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.
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

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 ev
...