1、September 2014 Translation by DIN-Sprachendienst.English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).
2、ICS 67.100.10!%:W“2235852www.din.deDDIN EN ISO 16297Milk Bacterial count Protocol for the evaluation of alternative methods (ISO 16297:2013);English version EN ISO 16297:2014,English translation of DIN EN ISO 16297:2014-09Milch Bestimmung der Gesamtkeimzahl Protokoll fr die Bewertung alternativer Ve
3、rfahren (ISO 16297:2013);Englische Fassung EN ISO 16297:2014,Englische bersetzung von DIN EN ISO 16297:2014-09Lait Dnombrement bactrien Protocole pour lvaluation des mthodes alternatives (ISO 16297:2013);Version anglaise EN ISO 16297:2014,Traduction anglaise de DIN EN ISO 16297:2014-09www.beuth.deIn
4、 case of doubt, the German-language original shall be considered authoritative.Document comprises 19 pages08.14 DIN EN ISO 16297:2014-09 2 A comma is used as the decimal marker. National foreword The text of ISO 16297:2013 has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretari
5、at: AFNOR, France), Subcommittee SC 5 “Milk and milk products” (Secretariat: NEN, Netherlands) and has been taken over as EN ISO 16297:2014 by Technical Committee CEN/TC 302 “Milk and milk products Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involve
6、d in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-12 AA Validierung mikrobiologischer Verfahren. The DIN Standards corresponding to the International Standards referred to in th
7、is document are as follows: ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 ISO 11095 DIN ISO 11095 ISO 16140-1 E DIN EN ISO 16140-1 ISO 16140-2 E DIN EN ISO 16140-2 ISO 21187 DIN EN ISO 21187 National Annex NA (informative) Bibliography E DIN EN ISO 16140-1, Microbiology of food and animal feed
8、 Method validation Part 1: Vocabulary E DIN EN ISO 16140-2, Microbiology of food and animal feed Method validation Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method DIN EN ISO 21187, Milk Quantitative determination of bacteriological quality Guidance
9、 for establishing and verifying a conversion relationship between routine method results and anchor method results DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of meas
10、urement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN ISO 11095, Linear calibration using reference materials EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 16297 May 2014 ICS 67.100.10 English Versi
11、on Milk - Bacterial count - Protocol for the evaluation of alternative methods (ISO 16297:2013) Lait - Dnombrement bactrien - Protocole pour lvaluation des mthodes alternatives (ISO 16297:2013) Milch - Bestimmung der Gesamtkeimzahl - Protokoll fr die Bewertung alternativer Verfahren (ISO 16297:2013)
12、 This European Standard was approved by CEN on 1 May 2014. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical referen
13、ces concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN m
14、ember into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
15、France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHE
16、S KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 16297:2014 EEN ISO 16297:2014 (E) 2 DIN EN ISO 16297:2014-09Contents Page Foreword . 3 I
17、ntroduction . 4 1 Scope 5 2 Normative references 5 3 Terms and definitions . 5 4 Transformation of results . 5 5 Attributes of the alternative method 6 5.1 Description of the method to be evaluated. 6 5.2 Measuring range 6 5.3 Carry-over 7 5.4 Stability . 9 Precision 9 6 The alternative method as an
18、 estimate of the reference method . 10 6.1 Evaluation of factors affecting the estimation . 10 6.2 Measurement protocol 11 6.3 Calculations . 11 6.4 Attributes of the alternative method expressed in units of the reference method 14 7 Rating of the elaborated attribut 14 Annex A (informative) Express
19、ion of precision parameters 15 Bibliography 17 5.5 EN ISO 16297:2014 (E) 3 DIN EN ISO 16297:2014-09Foreword The text of ISO 16297:2013 has been prepared by Technical Committee ISO/TC 34 “Food products” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 1629
20、7:2014 by Technical Committee CEN/TC 302 “Milk and milk products Methods of sampling and analysis” the secretariat of which is held by NEN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November
21、 2014, and conflicting national standards shall be withdrawn at the latest by November 2014. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent r
22、ights. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, Fran
23、ce, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 16297:2013 has been approved by CEN as EN ISO
24、 16297:2014 without any modification. IntroductionAny quantitative measurement in microbiology should consider that there is a requirement for the microbiological state in a sample to be regarded as one point within the co-ordinates of a multidimensional system, which is to be projected on to the on
25、e-dimensional scale of the method applied, i.e. plate count, flow cytometry. Aspects such as flora (types and numbers of microorganisms and their distribution), growth phase, sub-lethal damage, metabolic activity, and history, influence to a greater or lesser extent any parameter that is measured. I
26、t is evident that any projection of an n-dimensional situation on to an one-dimensional scale is bound to provide a picture of the real situation that is rather restricted. In this respect one has to bow to the inevitable, regardless of which method of measurement is preferred.The term reference (or
27、 official or anchor) method in this International Standard means a method internationally recognized by experts, used in legislation or by agreement between the parties. There are requirements for evaluation of an alternative method to refer to the reference method and to be based on the examination
28、 of suitable samples for its intended use.DIN EN ISO 16297:2014-09EN ISO 16297:2014 (E) 41 ScopeThis International Standard gives guidelines for the evaluation of instrumental alternative methods for total bacterial count in raw milk from animals of different species.NOTE The document is considered
29、complementary to ISO 16140 and ISO 8196|IDF 128 (see Clause 2 and Reference 1).2 Normative referencesThe following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the refe
30、renced document (including any amendments) applies.ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitionsISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of
31、 repeatability and reproducibility of a standard measurement methodISO 8196-1|IDF 128-1, Milk Definition and evaluation of the overall accuracy of alternative methods of milk analysis Part 1: Analytical attributes of alternative methodsISO 8196-2|IDF 128-2, Milk Definition and evaluation of the over
32、all accuracy of alternative methods of milk analysis Part 2: Calibration and quality control in the dairy laboratoryISO 16140-1, Microbiology of food and animal feed Method validation Part 1: VocabularyISO 16140-2, Microbiology of food and animal feed Method validation Part 2: Protocol for the valid
33、ation of alternative (proprietary) methods against a reference methodISO 21187|IDF 196:2004, Milk Quantitative determination of bacteriological quality Guidance for establishing and verifying a conversion relationship between routine method results and anchor method results3 Terms and definitionsFor
34、 the purposes of this document, the terms and definitions given in ISO 8196-1|IDF 128-1 and ISO 8196-2|IDF 128-2 apply.For the definitions of precision, repeatability and reproducibility, see ISO 5725-1, ISO 5725-2, ISO 8196-1|IDF 128-1, and ISO 16140-1.4 Transformation of resultsA prerequisite for
35、statistics most common in the evaluation of measuring methods is the approximation of a normal distribution of the data. The exponential multiplication of microorganisms usually leads to a right-tailed distribution of quantitative microbiological parameters. Thus, in general, transformation of the r
36、aw data is necessary for approximation of normality. This is usually a common logarithmic transformation or a square root transformation for low bacteria levels. The most appropriate transformation can be checked by comparing histograms. All statistics are then computed from the Milk Bacterial count
37、 Protocol for the evaluation of alternative methods DIN EN ISO 16297:2014-09EN ISO 16297:2014 (E) 5transformed data, unless specified otherwise, and only the final results are re-transformed to give a more expressive idea of the situation to the user (see also Annex A).5 Attributes of the alternativ
38、e methodNOTE The parameters outlined in this clause do not need to be evaluated completely for every alternative method. For example, the measuring range (see 5.2) of the plate loop method is determined by the loop(s) used.5.1 Description of the method to be evaluated5.1.1 DescriptionThe description
39、 of the method under study shall be in line with the checklist in 5.1.2.Most of the information is found in the specification of the method given by the responsible supplier or any other source (author) of the technique specified.5.1.2 Checklista) Principle of the method.b) Parameter or unit.c) Tech
40、nical design of the measurement procedure.d) Field of application:1) purpose: e.g. research, screening, milk grading;2) matrix: e.g. raw milk from cows.e) Supplier(s) of instrument, reagents, standards.f) Specification of the method given by the producer or the author:1) prerequisites for sampling (
41、often compared to the situation of fat analysis);2) possibilities for sample preservation reagent(s), storage condition(s);3) quantitative (units: method under study or reference method) and qualitative (the kind of microorganisms covered) spectrum;4) precision (in units of the method under study or
42、 in reference method units);5) accuracy of the estimate (in reference method units);6) samples per hour;7) list of references.5.2 Measuring range5.2.1 Lower limit of quantificationThe lower limit of quantification is defined as the average of milk without bacteria plus the n-fold of its standard dev
43、iation; generally, n = 10. See also ISO 16140-2.Analyse milk without bacteria or with a very low concentration of bacteria. Transform data by calculating square root from each result. Calculate the mean, x , and the standard deviation, s, of the transformed results. Calculate the lower limit of quan
44、tification as xns+ .DIN EN ISO 16297:2014-09EN ISO 16297:2014 (E) 65.2.2 Upper limit of quantificationThe upper limit of quantification is determined by the highest possible reading of the method or by methodological limitations, e.g. coincidence effects, inaccuracy in the upper measuring range, clo
45、gging of filters. Coincidence is when two or more elements of the measurand are detected simultaneously and identified as only one unit. For example, with flow cytometry, if two bacterial cells pass the detector simultaneously, they are detected as one. The coincidence effect is higher with higher c
46、oncentrations of a measurand.Upper limit of quantification is determined as the highest concentration where the instrument is still linear according to 5.2.3.5.2.3 Linearity of the instrument signalThe relationship between the instrument readings and the expected values shall be linear within the co
47、ncerned range of bacterial counts. Deviations from linearity may stem from non-specific signals and coincidence effects.A linearity check is at first performed visually using appropriate graphs to obtain an impression of the shape of the relationship. Whenever deviation from linearity appears eviden
48、t, a quantitative parameter is calculated to indicate whether the observed trend is acceptable or not.To achieve this, use a high bacterial count milk diluted serially with low bacterial count milk, resulting in a set of at least 10 samples covering the concentration range of interest.Measure all samples at least four times and calculate the average result for each sample. This gives the measured value per sample. Use the measured values for the high count milk and the low count milk to
