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1、 Reference numbers ISO 13366-1:2008(E) IDF 148-1:2008(E) ISO and IDF 2008 INTERNATIONAL STANDARD ISO 13366-1 IDF 148-1 Second edition 2008-02-15 Milk Enumeration of somatic cells Part 1: Microscopic method (Reference method) Lait Dnombrement des cellules somatiques Partie 1: Mthode au microscope (Mt
2、hode de rfrence) Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13366-1:2008(E) IDF 148-1:2008(E) PDF discl
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5、l Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies and IDF national committees. In the unlikely event that a problem relating to it is found, please inform the ISO Central Se
6、cretariat at the address given below. COPYRIGHT PROTECTED DOCUMENT ISO and IDF 2008 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 permissi
7、on in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 0
8、4 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2008 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25
9、/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13366-1:2008(E) IDF 148-1:2008(E) ISO and IDF 2008 All rights reserved iii Contents Page Foreword iv Foreword. v 1 Scope 1 2 Terms and definitions .1 3 Principle1 4 Reagents.1 4.1 Dye solutions .1 4.2 Phosph
10、ate 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 staining5 8.2 Determination.6 9 Calculation and expression of results.10 9.1 Rectangular shape counting in successive fields .10 9.2 Re
11、ctangular 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 report13 Annex A (informative) Collaborative trial14 Annex B (informative) Staining for goats milk 15 Annex C (i
12、nformative) Poisson distribution .16 Bibliography17 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13366-1:
13、2008(E) IDF 148-1:2008(E) iv ISO and IDF 2008 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
14、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
15、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 Interna
16、tional 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 b
17、e 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 products, and the International Dairy Federation (IDF). It is being pu
18、blished 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 the general title Milk Enumeration of somatic cells: Part 1: Microsco
19、pic method (Reference method) Part 2: Guidance on the operation of fluoro-opto-electronic counters Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permit
20、ted without license from IHS -,-,- ISO 13366-1:2008(E) IDF 148-1:2008(E) ISO and IDF 2008 All rights reserved v Foreword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country
21、 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 work. IDF collaborates with ISO in the development of standard methods of analysis and sampl
22、ing 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 requires approval by at least 50 % of the IDF National Committees casting a vote. Attention
23、 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 148-1 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC
24、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 on Quality assurance, statistics of analytical data and sampling under the aegis of its pr
25、oject 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 general title Milk Enumeration of somatic cells: Part 1: Microscopic method (Reference method)
26、Part 2: Guidance on the operation of fluoro-opto-electronic counters Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -
27、,-,- Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 13366-1:2008(E) IDF 148-1:2008(E
28、) ISO and IDF 2008 All rights reserved 1 Milk Enumeration of somatic cells Part 1: Mthode au microscope (Mthode de rfrence) 1 Scope This part 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
29、of ISO 13366|IDF 148 is applicable for the counting of somatic cells in cows milk, provided that the eventually mentioned prerequisites are met. This method is suitable for preparing standard test samples and determining reference method values that are required for calibrating mechanized and automa
30、ted cell-counting methods. WARNING The use of this standard may involve hazardous materials, operations and equipment. This standard does not 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 a
31、nd health practices and determine the applicability of regulatory limitations prior to use. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 somatic cells those cells with nuclei, that is all leucocytes and epithelial cells, determined accordi
32、ng 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 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 cell
33、s 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 recognized analytical grade, unless otherwise specified, and distilled or deionized water or water of equivalent purity. 4.1 Dye solutions WARNING Tetrachloroet
34、hane is poisonous. Ethidium bromide is mutagenic. Proper actions for deactivation should be taken in case of spilling. Preparation and application of the dye solution shall be carried out in a fume cupboard, using protective equipment. Copyright International Organization for Standardization Provide
35、d by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13366-1:2008(E) IDF 148-1:2008(E) 2 ISO and IDF 2008 All rights reserved 4.1.1 Modified Newman-Lampert stain solution (Levow
36、itz-Weber modification) 4.1.1.1 Components Ethanol, 95 % (volume fraction) 54,0 ml Tetrachloroethane a 40,0 ml Methylene blue 0,6 g Acetic acid, glacial 6,0 ml a Xylene can be used as an alternative in the same volume amount as mentioned for tetrachloroethane. 4.1.1.2 Preparation Mix the ethanol and
37、 the tetrachloroethane and stopper the bottle. Heat the mixture in a water bath (5.1) set at 65 C. Add the methylene 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 a
38、n appropriate filter (5.2) into an airtight bottle and store it as such. Filter the Newman-Lampert stain solution again 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 Dissol
39、ve the ethidium bromide in demineralized water preheated to 40 C. Cool the solution to room temperature. Adjust to 100 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
40、Composition Potassium hydrogenphthalate 0,51 g Potassium hydroxide 0,162 g Demineralized water 100 ml 4.1.2.2.2 Preparation Separately dissolve the potassium hydrogenphthalate and the potassium hydroxide in the demineralized water. Copyright International Organization for Standardization Provided by
41、 IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13366-1:2008(E) IDF 148-1:2008(E) ISO and IDF 2008 All rights reserved 3 The buffer solution can be kept for two months at a max
42、imum when stored in the dark at 2 C 2 C. 4.1.2.3 Ethidium bromide stain working solution 4.1.2.3.1 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 a A high temperature may reduce the staining capability of
43、ethidium bromide. 4.1.2.3.2 Preparation Successively add the ethidium bromide stain stock solution, 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 (P
44、BS) 4.2.1 Components NaCl 8 g KCl 0,2 g Na2HPO47H2O 1,15 g KH2PO4 0,2 g Demineralized water 1 000 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 availa
45、ble phosphate buffer solution with pH = 7,2. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 07/25/2008 03:53:50 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13366-1:2008(E)
46、 IDF 148-1:2008(E) 4 ISO and IDF 2008 All rights reserved 5 Apparatus 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.
47、5.3 Microscope, with a magnification of 500 to 1 000. Objectives for oil 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 ce
48、rtified. 5.6 Slides, premarked with an outline shape (rectangular or circular), with an area of 1 cm2 5 % (95 mm2 to 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 a fixed premarked area or a template, in order to avoid the recalculation of the working factor with