1、BRITISH STANDARDBS EN ISO 13366-1:2008Milk Enumeration of somatic cells Part 1: Microscopic method (Reference method)ICS 67.100.10g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g5
2、1g60g53g44g42g43g55g3g47g36g58Incorporating corrigendumAugust 2009 National forewordThis British Standard is the UK implementation of EN ISO 13366-1:2008, incorporating corrigendum August 2009.It is identical to ISO 13366-1:2008. It supersedes BS EN ISO 13366-1:1997 which is withdrawn.The start and
3、finish of text introduced or altered by corrigendum is indic-ated in the text by tags. Text altered by ISO corrigendum August 2009 is indicated in the text by .The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products.A list of org
4、anizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligat
5、ions.BS EN ISO 13366-1:2008This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 March 2008 BSI 2009Amendments/corrigenda issued since publicationDate Comments 30 November 2009 Incorporating ISO corrigendum August 2009ISBN 978 0 580 68734 1EUROP
6、EAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 13366-1February 2008ICS 67.100.10 Supersedes EN ISO 13366-1:1997 English VersionMilk - Enumeration of somatic cells - Part 1: Microscopic method(Reference method) (ISO 13366-1:2008)Lait - Dnombrement des cellules somatiques - Partie 1:Mthode au micros
7、cope (Mthode de rfrence) (ISO13366-1:2008)Milch - Zhlung somatischer Zellen - Teil 1:Mikroskopisches Verfahren (Referenzverfahren) (ISO13366-1:2008)This European Standard was approved by CEN on 3 February 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate
8、the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists i
9、n three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies o
10、f Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN C
11、OMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 13366-1:2008: EForeword This docu
12、ment (EN ISO 13366-1:2008) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration with 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
13、the status of a national standard, either by publication of an identical text or by endorsement, at the latest by August 2008, and conflicting national standards shall be withdrawn at the latest by August 2008. Attention is drawn to the possibility that some of the elements of this document may be t
14、he subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 13366-1:1997. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to i
15、mplement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerl
16、and and the United Kingdom. Endorsement notice The text of ISO 13366-1:2008 has been approved by CEN as a EN ISO 13366-1:2008 without any modification. BS EN ISO 13366-1:2008Reference numbersISO 13366-1:2008(E)IDF 148-1:2008(E)INTERNATIONAL STANDARD ISO13366-1IDF148-1Second edition2008-02-15Milk Enu
17、meration of somatic cells Part 1: Microscopic method (Reference method) Lait Dnombrement des cellules somatiques Partie 1: Mthode au microscope (Mthode de rfrence) BS EN ISO 13366-1:2008IDF 148-1:2008ii iiiContents Page Foreword iv Foreword. v 1 Scope 1 2 Terms and definitions .1 3 Principle1 4 Reag
18、ents.1 4.1 Dye solutions .1 4.2 Phosphate Buffer Solution (PBS).3 5 Apparatus .4 6 Sampling.4 7 Preparation of test sample4 7.1 Storage4 7.2 Preparation .5 8 Procedure .5 8.1 Preparation of the smear and staining 5 8.2 Determination.6 9 Calculation and expression of results.10 9.1 Rectangular shape
19、counting in successive fields .10 9.2 Rectangular shape counting in bands 11 9.3 Circular shape counting in successive fields.11 9.4 Expression of results 12 10 Precision.12 10.1 Repeatability.12 10.2 Reproducibility.12 11 Test report 13 Annex A (informative) Collaborative trial14 Annex B (informati
20、ve) Staining for goats milk 15 Annex C (informative) Poisson distribution .16 Bibliography 17 BS EN ISO 13366-1:2008IDF 148-1:2008iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing Int
21、ernational 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 w
22、ith 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 techni
23、cal 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
24、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 13366-1|IDF 148-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk pro
25、ducts, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. This second edition of ISO 13366-1|IDF 148-1 cancels and replaces the first edition (ISO 13366-1:1997), of which it constitutes a technical revision. ISO 13366 consists of the following parts, under th
26、e general title Milk Enumeration of somatic cells: Part 1: Microscopic method (Reference method) Part 2: Guidance on the operation of fluoro-opto-electronic counters BS EN ISO 13366-1:2008IDF 148-1:2008vForeword IDF (the International Dairy Federation) is a non-profit organization representing the d
27、airy sector worldwide. IDF membership comprises National Committees in every member country as well as regional dairy associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented at the IDF Standing Committees carrying out the technical w
28、ork. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard
29、requires approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 13366-1|IDF 148
30、-1 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team Automated methods of the Standing Committee
31、 on Quality assurance, statistics of analytical data and sampling under the aegis of its project leaders, Mrs. S. Orlandini (IT) and Mr. H.J.C.M. van den Bijgaart (NL). This edition of ISO 13366-1|IDF 148-1 cancels and replaces IDF 148A:1995. ISO 13366 consists of the following parts, under the gene
32、ral title Milk Enumeration of somatic cells: Part 1: Microscopic method (Reference method) Part 2: Guidance on the operation of fluoro-opto-electronic counters BS EN ISO 13366-1:2008IDF 148-1:2008blank1Milk Enumeration of somatic cells Part 1: Mthode au microscope (Mthode de rfrence) 1 Scope This pa
33、rt of ISO 13366|IDF 148 specifies a microscopic method (reference method) for the counting of somatic cells in both raw and chemically preserved milk. This part of ISO 13366|IDF 148 is applicable for the counting of somatic cells in cows milk, provided that the eventually mentioned prerequisites are
34、 met. This method is suitable for preparing standard test samples and determining reference method values that are required for calibrating mechanized and automated cell-counting methods. WARNING The use of this standard may involve hazardous materials, operations and equipment. This standard does n
35、ot purport to address all of the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 2 Terms and definitions For the purposes of thi
36、s document, the following terms and definitions apply. 2.1 somatic cells those cells with nuclei, that is all leucocytes and epithelial cells, determined according to the procedure described in this part of ISO 13366|IDF 148 3 Principle A test portion of milk to be examined is spread over a slide to
37、 form a smear. The smear is dried. During this process, the cells are stained. Subsequently, the stained cells are counted using a microscope. The number of cells counted in a defined area are multiplied by a working factor, to give the number of cells per millilitre. 4 Reagents Use only reagents of
38、 recognized analytical grade, unless otherwise specified, and distilled or deionized water or water of equivalent purity. 4.1 Dye solutions WARNING Tetrachloroethane is poisonous. Ethidium bromide is mutagenic. Proper actions for deactivation should be taken in case of spilling. Preparation and appl
39、ication of the dye solution shall be carried out in a fume cupboard, using protective equipment. BS EN ISO 13366-1:2008IDF 148-1:20082 4.1.1 Modified Newman-Lampert stain solution (Levowitz-Weber modification) 4.1.1.1 Components Ethanol, 95 % (volume fraction) 54,0 ml Tetrachloroethanea40,0 ml Methy
40、lene blue 0,6 g Acetic acid, glacial 6,0 ml aXylene can be used as an alternative in the same volume amount as mentioned for tetrachloroethane. 4.1.1.2 Preparation Mix the ethanol and the tetrachloroethane and stopper the bottle. Heat the mixture in a water bath (5.1) set at 65 C. Add the methylene
41、blue under a fume cupboard and carefully mix. Cool the mixture in a refrigerator to 4 C. Then add the glacial acetic acid and carefully mix again. Pass the obtained solution through an appropriate filter (5.2) into an airtight bottle and store it as such. Filter the Newman-Lampert stain solution aga
42、in before use. 4.1.2 Ethidium bromide stain solution 4.1.2.1 Stain stock solution 4.1.2.1.1 Composition Ethidium bromide 0,25 g Demineralized water 100 ml 4.1.2.1.2 Preparation Dissolve the ethidium bromide in demineralized water preheated to 40 C. Cool the solution to room temperature. Adjust to 10
43、0 ml with demineralized water. The ethidium bromide stain stock solution can be kept for two months at a maximum when stored in the dark at 2 C 2 C. 4.1.2.2 Buffer solution 4.1.2.2.1 Composition Potassium hydrogenphthalate 0,51 g Potassium hydroxide 0,162 g Demineralized water 100 ml 4.1.2.2.2 Prepa
44、ration Separately dissolve the potassium hydrogenphthalate and the potassium hydroxide in the demineralized water. BS EN ISO 13366-1:2008IDF 148-1:20083The buffer solution can be kept for two months at a maximum when stored in the dark at 2 C 2 C. 4.1.2.3 Ethidium bromide stain working solution 4.1.
45、2.3.1 Components Ethidium bromide stain stock solutiona (4.1.2.1) 2 ml Buffer solution (4.1.2.2) 8 ml Triton X-100 0,1 ml Demineralized water 90 ml aA high temperature may reduce the staining capability of ethidium bromide. 4.1.2.3.2 Preparation Successively add the ethidium bromide stain stock solu
46、tion, the buffer solution and the Triton X-100 to the demineralized water and carefully mix. Freshly prepare the ethidium bromide stain working solution directly before use. 4.2 Phosphate Buffer Solution (PBS) 4.2.1 Components NaCl 8 g KCl 0,2 g Na2HPO47H2O 1,15 g KH2PO40,2 g Demineralized water 1 0
47、00 ml 4.2.2 Preparation Dissolve the salts in demineralized water. Adjust to 1 000 ml with the remaining demineralized water. Adjust the pH to 7,2 0,1. NOTE It is also possible to use a commercially available phosphate buffer solution with pH = 7,2. BS EN ISO 13366-1:2008IDF 148-1:20084 5 Apparatus
48、Usual laboratory equipment and, in particular, the following. 5.1 Water baths, capable of maintaining a temperature of 40 C 2 C, 50 C 2 C and 65 C 2 C. 5.2 Filter, resistant to the solvents used, with a pore size of 10 m to 12 m. 5.3 Microscope, with a magnification of 500 to 1 000. Objectives for o
49、il immersion can be used. When using ethidium bromide, the microscope shall have fluorescence equipment. 5.4 Microsyringe, for dispensing a fixed volume of 0,01 ml of milk, with a maximum tolerance of 2 %. 5.5 Micrometer, to be certified. 5.6 Slides, premarked with an outline shape (rectangular or circular), with an area of 1 cm2 5 % (95 mm2to 105 mm2), or a standard slide with a template of dimensions 20 mm 5 mm or having a diameter, d, of 11,28 mm. 5.6.1 Selection of slides Preferably, work with
