1、BS 8496:2007Water quality Enumeration of micro-organisms in water samples Guidance on the estimation of variation of results with particular reference to the contribution of uncertainty of measurementICS 03.120.30; 07.100.20NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITIS
2、H STANDARDPublishing and copyright informationThe BSI copyright notice displayed in this document indicates when the document was last issued. BSI 2007ISBN 978 0 580 49930 2The following BSI references relate to the work on this standard:Committee reference EH/3/4Publication historyFirst published J
3、anuary 2007Amendments issued since publicationAmd. no. Date Text affectedBS 8496:2007 BSI 2007 iBS 8496:2007ContentsForeword iiIntroduction 11 Scope 12 Normative references 13 Terms and definitions 14 General 35 Procedure 8AnnexesAnnex A (informative) Worked examples 14Bibliography 18List of figures
4、Figure 1 Illustration of hypothetical water sample containing 30 micro-organisms distributed at random throughout 10 equal sized sub-samples 7List of tablesTable A.1 Counts on 10 replicate sub-samples from a sample from each of 5 water sources 14Table A.2 Replicate counts on daily duplicate sub-samp
5、les 16Summary of pagesThis document comprises a front cover, an inside front cover, pages i and ii, pages 1 to 19 and a back cover.BS 8496:2007ii BSI 2007ForewordPublishing informationThis British Standard was published by BSI and came into effect on 31 January 2007. It was prepared by Subcommittee
6、EH/3/4, Microbiological methods, under the authority of Technical Committee EH/3, Water quality. A list of organizations represented on this committee can be obtained on request to its secretary.SupersessionThis British Standard supersedes DD 260:2003, which is withdrawn.Relationship with other publ
7、icationsThis British Standard gives guidance on the interpretation of the requirements in BS EN ISO/IEC 17025:2005 for estimating uncertainty of measurement, in the context of microbial counts in water samples. It is intended to form an adjunct to BS ISO 8199 which gives guidance on the enumeration
8、of micro-organisms in water samples.Use of this documentAs a guide, this British Standard takes the form of guidance and recommendations. It should not be quoted as if it were a specification and particular care should be taken to ensure that claims of compliance are not misleading.Any user claiming
9、 compliance with this British Standard is expected to be able to justify any course of action that deviates from its recommendations.Presentational conventionsThe provisions in this standard are presented in roman (i.e. upright) type. Its recommendations are expressed in sentences in which the princ
10、ipal auxiliary verb is “should”.Commentary, explanation and general informative material is presented in smaller italic type, and does not constitute a normative element.Contractual and legal considerationsThis publication does not purport to include all the necessary provisions of a contract. Users
11、 are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations. BSI 2007 1BS 8496:2007IntroductionBS EN ISO/IEC 17025:2005, 5.4.6.2 specifies that “Testing laboratories shall have and shall apply procedures for estimating uncertainty of
12、measurement”. This is difficult to apply to water microbiology because the distribution and behaviour of microbial cells in water is not uniform. The purpose of the present document is to provide practical guidance on how to interpret and implement these requirements within the context of a water mi
13、crobiology laboratory. It is intended to form an adjunct to BS ISO 8199:2005.1 ScopeThis British Standard gives guidance on interpretation of the requirements specified in BS EN ISO/IEC 17025:2005 with regard to the uncertainty of measurement in microbial counts in water samples. It gives guidance o
14、n the estimation of variation in results between replicate sub-samples, which will include that due to uncertainty of measurement, in order to assess whether the variation due to uncertainty of measurement has been controlled in accordance with the requirements specified in BS EN ISO/IEC 17025:2005.
15、 It is applicable to examination of sub-samples as part of a quality assurance system. It is applicable to samples of all types of water.2 Normative referencesThe following referenced documents are indispensable for the application of this document. For dated references only the edition cited applie
16、s. For undated references, the latest edition of the referenced document (including any amendments) applies.BS EN ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratoriesDD ENV ISO/TR 13843:2001, Water quality Guidance on validation of microbiological metho
17、ds3 Terms and definitionsFor the purposes of this British Standard, the following terms and definitions apply.3.1 accuracysum of trueness plus precisionNOTE This equates to the degree of conformity between the result of a measurement and the true value of the measurand.3.2 biassystematic errors asso
18、ciated with the methods usedNOTE These errors include those due to dilution, population selection, and calibration.BS 8496:20072 BSI 20073.3 precisioncloseness of agreement between independent test results obtained under stipulated conditions NOTE Precision does not relate to the true value or the s
19、pecified value. It is usually expressed in terms of imprecision and computed as a standard deviation of the test results.DD ENV ISO/TR 13843:2001, 2.253.4 imprecisionrandom errors incurred in applying the methods together with random variation in the test material3.5 limit of detectionlowest number
20、of micro-organisms that need to be present in the test portion of water examined for their presence to be detected by a particular microbiological testNOTE Limit of detection should not be confused with limit of determination which is the lowest average number of micro-organisms in a body of water t
21、hat would result in a 95% probability of giving a positive result when a representative test portion is examined by a particular microbiological test.3.6 measurandspecific quantity that is subjected to measurement3.7 repeatabilitycloseness of the agreement between the results of successive measureme
22、nts of the same measurand carried out under the same conditions of measurement within a short period of time NOTE 1 Modified from International vocabulary of basic and general terms in metrology, 1993 1.NOTE 2 Conditions of measurement include, the method of analysis; the analyst; the measurement in
23、strument and the conditions under which it is used; and the location.NOTE 3 Within a short period of time is normally considered to be within one hour.3.8 reproducibilitycloseness of the agreement between the results of measurements of the same measurand carried out under different conditions of mea
24、surementNOTE 1 Modified from International vocabulary of basic and general terms in metrology, 1993 1.NOTE 2 Conditions of measurement include, the method of analysis; the analyst; the measurement instrument and the conditions under which it is used; the location; and the time.3.9 reference culturec
25、ulture of a micro-organism obtained from a recognized national culture collection 3.10 reference materialmaterial containing a defined population of micro-organisms from one or more reference cultures in numbers expected to fall within a defined range for use in the assessment of a measurement metho
26、d BSI 2007 3BS 8496:20073.11 certified reference materialreference material accompanied by a certificate stating the expected microbial count with an associated confidence interval3.12 uncertainty of measurementUMparameter associated with the result of a measurement, that characterizes the dispersio
27、n of the values that can reasonably be attributed to the measurand Guide to the expression of uncertainty in measurement, 1995 23.13 validationprocess providing evidence that a method is capable of serving its intended purpose in detecting or quantifying a specific microbe or microbial group with th
28、e required level of precision and accuracy NOTE Modified from DD ENV ISO/TR 13843:2001, 4.2.1.3.14 secondary validationprocess providing evidence that a method validated elsewhere performs within the users laboratory according to the specifications determined in the original validationNOTE 1 Also kn
29、own as verification.NOTE 2 Modified from DD ENV ISO/TR 13843:2001, 2.38 and 4.2.3.3.15 samplevolume of water collected from the body of water under investigation3.16 sub-samplevolume of water taken from a sample3.17 test portionvolume of water examined in a particular testNOTE The test portion may c
30、omprise the whole sample or a sub-sample.4 General4.1 Significance of requirements specified in BS EN ISO/IEC 17025BS EN ISO/IEC 17025:2005, 5.4.6.2 specifies: “Testing laboratories shall have and shall apply procedures for estimating uncertainty of measurement. In certain cases the nature of the te
31、st method may preclude rigorous, metrologically and statistically valid, calculation of uncertainty of measurement. In these cases the laboratory shall at least attempt to identify all the components of uncertainty and make a reasonable estimation, and shall ensure that the form of reporting of the
32、result does not give a wrong impression of the uncertainty. Reasonable estimation shall be based on knowledge of the performance of the method and on the measurement scope and shall make use of, for example, previous experience and validation data.”BS 8496:20074 BSI 2007The document EA-04/10 Accredi
33、tation for microbiological laboratories 3, produced by the joint EA/EURACHEM Working Group, supplements BS EN ISO/IEC 17025 and states the following in paragraphs 5.2 to 5.4.NOTE Points of particular importance to the present British Standard are indicated in bold type.“5.2 Microbiological tests gen
34、erally come into the category of those precluding rigorous, metrologically and statistically valid calculation of uncertainty of measurement. It is generally appropriate to base the estimation of uncertainty on repeatability and reproducibility data alone, but ideally including bias (e.g. from profi
35、ciency testing results). The individual components of uncertainty should be identified and demonstrated to be under control and their contribution to the variability of results evaluated. Some components (e.g. pipetting, weighing and dilution effects) may be readily measured and easily evaluated to
36、demonstrate a negligible contribution to overall uncertainty. Other components (e.g. sample stability and sample preparation) cannot be measured directly and their contribution cannot be evaluated in a statistical manner but their importance to the variability of results should be considered also.5.
37、3 It is expected that accredited microbiological testing laboratories will have an understanding of the distributions of organisms within the matrices they test and take this into account when sub-sampling. However, it is not recommended that this component of uncertainty is included in estimates un
38、less the clients needs dictate otherwise. The principal reasons for this are that uncertainty due to distribution of organisms within the product matrix is not a function of the laboratorys performance and may be unique to individual samples tested and because test methods should specify the sample
39、size to be used taking into account poor homogeneity.5.4 The concept of uncertainty cannot be applied directly to qualitative test results such as those from detection tests or the determination of attributes for identification. Nevertheless, individual sources of variability, e.g. consistency of re
40、agent performance and analyst interpretation, should be identified and demonstrated to be under control. Additionally, for tests where the limit of detection is an important indication of suitability, the uncertainty associated with the inocula used to determine the limit should be estimated and its
41、 significance evaluated. Laboratories should also be aware of the incidence of false positive and false negative results associated with the qualitative tests they use.”Theoretically, the uncertainty of measurement (UM) associated with a test result relates to the measurement made on the test portio
42、n examined and is distinct from any uncertainty associated with the sampling process and/or the selection of the test portion. This is an important distinction where the material being sampled is water and the measurement is of numbers of relevant micro-organisms in the test portion. BSI 2007 5BS 84
43、96:2007An estimate of UM, therefore, is meant to demonstrate whether the laboratory uses methods and equipment which ensure a high degree of accuracy, or whether the result is more approximate. The United Kingdom Accreditation Service recommends that any further uncertainty that results from the tes
44、t sample not being fully representative of the whole should normally be identified separately 4.4.2 Comparison of estimates of uncertainty of measurement in chemical analyses and microbiological countsMethods for estimation of UM have been successfully introduced by chemistry laboratories but these
45、do not readily transfer to microbiology because of the assumptions that have to be made regarding homogeneity of the test material. The estimates of UM obtained in chemistry usually involve replicate analyses of sub-samples. In microbiology replicate counts on sub-samples overwhelmingly estimate the
46、 natural variation in numbers of organisms present in the different sub-samples, and are also affected by the different characteristics or physiological states of the organisms being counted.In chemistry laboratories examining water samples the uncertainty of a measured result is considered to be “t
47、he interval on the measurement scale within which the true value lies with a high probability, when all sources of error have been taken into account” 5. The true value applies to the test portion. An estimate of the UM thus reflects the accuracy of the result where accuracy is considered to be the
48、sum of bias plus imprecision.In practice, an estimate of the UM concentrates on precision and might be based on special studies. It would not be practical to make re-evaluations for each routine sample. Any bias arising from systematic errors is expected to have been minimized by quality assurance c
49、hecks, which usually include participation in a proficiency testing or external quality assessment scheme.For estimating precision, and thus for estimating UM in chemistry, attention is paid to each piece of equipment used (some of which will come with a calibration certificate) and each procedure followed. As each stage is considered the contributions to uncertainty may need to be accumulated. Usually the main sources of uncertainty are estimated from repeatability studies which use replicate testing of sub-samp
