EN ISO 13366-3:1997

Milk - Enumeration of somatic cells - Part 3: Fluoro-opto-electronic method (ISO 13366-3:1997)

Milch - Zählung somatischer Zellen - Teil 3: Fluoreszenzoptoelektronisches Verfahren (ISO 13366-3:1997)

Dieser Teil von ISO 13366 legt ein Verfahren zum Zählen somatischer Zellen in Rohmilch und chemisch konservierter Milch mit einem fluoreszenzoptoelektronischen Gerät fest. Anmerkung 1: Das Zählen von Zellen in nicht konservierten Proben innerhalb der ersten 24 h nach dem Melken kann bei älteren Geräten (z.B. Fossomatic 90 und 215) zu unzuverlässigen Ergebnissen führen.

Lait - Dénombrement des cellules somatiques - Partie 3: Méthode fluoro-opto-électronique (ISO 13366-3:1997)

Mleko - Ugotavljanje števila somatskih celic - 3. del: Fluorooptoelektronska metoda (ISO 13366-3:1997)

General Information

• Status: Withdrawn
• Publication Date: 14-Jun-1997
• Withdrawal Date: 30-Sep-2006
• ICS: 07.100.30 - Food microbiology
• ICS: 67.100.10 - Milk and processed milk products
• Technical Committee: CEN/TC 302 - Milk and milk products - Methods of sampling and analysis
• Drafting Committee: CEN/TC 302 - Milk and milk products - Methods of sampling and analysis
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 01-Oct-2006
• Completion Date: 01-Oct-2006
• Ref Project: SIST EN ISO 13366-3:1998 - Milk - Enumeration of somatic cells - Part 3: Fluoro-opto-electronic method (ISO 13366-3:1997)

Relations

• Parent: EN ISO 13366-1:1997 - Milk - Enumeration of somatic cells - Part 1: Microscopic method (ISO 13366-1:1997)
• Effective Date: 22-Dec-2008
• Replaced By: EN ISO 13366-2:2006 - Milk - Enumeration of somatic cells - Part 2: Guidance on the operation of fluoro-opto-electronic counters (ISO 13366-2:2006)
• Effective Date: 22-Dec-2008

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Mleko - Ugotavljanje števila somatskih celic - 3. del: Fluorooptoelektronska metoda (ISO 13366-3:1997)Milch - Zählung somatischer Zellen - Teil 3: Fluoreszenzoptoelektronisches Verfahren (ISO 13366-3:1997)Lait - Dénombrement des cellules somatiques - Partie 3: Méthode fluoro-opto-électronique (ISO 13366-3:1997)Milk - Enumeration of somatic cells - Part 3: Fluoro-opto-electronic method (ISO 13366-3:1997)67.100.10SURL]YRGLMilk and processed milk products07.100.30Mikrobiologija živilFood microbiologyICS:Ta slovenski standard je istoveten z:EN ISO 13366-3:1997SIST EN ISO 13366-3:1998en01-avgust-1998SIST EN ISO 13366-3:1998SLOVENSKI
STANDARD



SIST EN ISO 13366-3:1998



SIST EN ISO 13366-3:1998



SIST EN ISO 13366-3:1998



INTERNATIONAL STANDARD IS0 13366-3 First edition 1997-06-I 5 Milk - Enumeration of somatic cells - Part 3: Fluoro-opto-electronic method Lait - Dknombrement des cellules somatiques - Partie 3: Mkthode fluoro-opto-klectronique Reference number IS0 13366-3: 1997(E) SIST EN ISO 13366-3:1998



IS0 13366-3: 1997(E) Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 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. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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. International Standard IS0 13366 was prepared by Technical Committee ISOmC 34, Agricultural food products, Subcommittee SC 5, Milk and milk products, in collaboration with the International Dairy Federation (IDF) and AOAC INTERNATIONAL, and will also be published by these organiz- ations. IS0 13366 consists of the following parts, under the general title Milk - Enumeration of soma tic cells: - Part I: Microscopic method - Par? 2: Electronic particle counter method - Part 3: Fluoro-opto-electronic method Annexes A to D of this part of IS0 13366 are for information only. 0 IS0 1997 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 the publisher. International Organization for Standardization Case postale 56 l CH-1211 Geneve 20 l Switzerland Internet central @I isocs.iso.ch x.400 c=ch; a=400net; p=iso; o=isocs; s=central Printed in Switzerland ii SIST EN ISO 13366-3:1998



~~ INTERNATIONAL STANDARD @ IS0 IS0 13366-3: 1997(E) Milk - Enumeration of somatic cells - Part 3: Fluoro-opto-electronic method 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 and health practices and to determine the applicability of regulatory limitations prior to use. 1 Scope This part of IS0 13366 specifies a method for counting somatic cells in both raw and chemically preserved milk, using a fluoro-opto-electronic counting instrumentI). NOTE - Counting of cells in unpreserved samples within the first 24 h after milking could give unreliable results with older instruments (e.g. Fossomatic 90 and 215). 2 Definition For the purposes of this part of IS0 13366, the following definition applies. 2.1 somatic cells: Those cells that have a minimum intensity of fluorescence due to the staining of DNA in their nuclei. 3 Principle Mixing of the milk to be examined with a buffer and stain solution. Transference of the mixture in the form of a thin film to a rotating disc, serving as an object plane for a microscope. Each stained cell observed by the microscope produces an electrical pulse that is amplified and recorded. Direct reading of the number of somatic cells in thousands per millilitre. 4 Reagents WARNING - Ethidium bromide is toxic. The preparation and application of the basic and working solutions shall be carried out in a fume cupboard. Use gloves for protection. 1) The Fossomatic counting instrument (250, 300 or 360) supplied by Foss Electric, Hillerod, Denmark is an example of suitable equipment available commercially. This information is given for the convenience of users of this part of IS0 13366 and does not constitute an endorsement by IS0 of the equipment named. SIST EN ISO 13366-3:1998



IS0 13366-3: 1997(E) @ IS0 Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or deionized water or water of equivalent purity. 4.1 Basic solutions 4.1 .l Dye-buffer solution 4.1 .I .l Composition Ethidium bromide Tripotassium citrate Citric acid Deionized water Poly(ethylene glycol) mono-p( 1,1,3,3-tetramethylbutyl) phenyl ether’) 2s g 400 g 1495 g 5 litres 50 ml 1) For example, Triton X-100 concentrate. 4.1 .1.2 Preparation Dissolve the ethidium bromide in 1 litre of water in a 5 litre container. Stir gently until the ethidium bromide is completely dissolved. The process can be speeded up by heating to between 40 “C and 60 “C. Add the tripotassium citrate and citric acid to the ethidium bromide solution. Add 4 litres of water. Stir gently until the solids are completely dissolved. Add the poly(ethylene glycol) ether concentrate while stirring. Even when stored under light-proof, airtight and cool conditions, the solution shall be kept for no longer than 90 days. 4.1.2 Poly(ethylene glycol) mono-p-(1 ,1,3,3=tetramethylbutyI) phe 4.1.2.1 Composition Poly(ethylene glycol) mono-p( 1,1,3,3-tetramethylbutyl) phenyl ether’) Water 1) For example, Triton X-l 00 concentrate. 10 ml 1 litre lyl ether solution 4.1.2.2 Preparation Dissolve the poly(ethylene glycol) ether in 1 litre of pre-heated water at approx. 60 “C. Even when stored under airtight and cool conditions, this solution shall be kept for no longer than 25 days. 4.2 Working solution 4.2.1 Dye-buffer working solution Mix 1 part of the dye-buffer basic solution (4.1 .l) with 9 parts of water. (This should be enough for approx. 2 700 samples.) Do not use working solutions older than 7 days SIST EN ISO 13366-3:1998



@ IS0 IS0 13366=3:1997(E) 4.2.2 Rinsing liquid 4.2.2.1 Composition Poly(ethylene glycol) mono-p( 1,1,3,3-tetramethylbutyl) phenyl ether’) Ammonia solution, 25 % (V/V) Water 1) For example, Triton X-100 concentrate. IOml 25 ml 10 litres 4.2.2.2 Preparation Add the poly(ethylene glycol) ether and the ammonia solution to the water. The composition of the reagents might vary depending on the counting system used. Therefore follow the manufacturer’s instructions exactly. 4.3 Preservatives Boric acid, potassium dichromate, sodium azide or bronopol may be used. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Counting instrument, operating according to the fluorescence optical principle (e.g. Fossomatic). Calibrate in accordance with the manufacturer’s instructions. For calibration it is necessary to use milk samples whose cell count has been made by the microscopic method (details are given in IS0 13366-1). NOTE - Cell count standards are available from the manufacturer. 5.2 Water bath, with circulation, capable of being maintained at a temperature of 40 OC + 1 OC. 5.3 Sample tubes, with leak-proof seal. 6 Sampling 6.1 It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in IS0 707 [Il. 6.2 If automatic samplers are used, they shall be tested properly. 6.3 Prior to testing or preservation, samples should be stored at a temperature of between 2 “C and 6 “C. 6.4 Preservation, if necessary, shall be carried out as soon as possible after sampling, but in any case within 24 h, by addition of one of the following preservatives. 3 SIST EN ISO 13366-3:1998



IS0 13366-3: 1997(E) @ IS0 a) Boric acid (HsB03): Add the boric acid to the test sample. The final concentration of this preservative in the sample shall not exceed 0,6 g per 100 ml. Such a preserved test sample may be stored at a temperature of between 6 “C and 12 “C for up to a further 24 h. b) Potassium dichromate (K2Cr207): Add the potassium dichromate to the test sample. The final concentration of this preservative in the test sample shall not exceed 0,2 g per 100 ml. Such a preserved test sample may be stored at a temperature of between 6 “C and 12 “C for up to further 72 h. Local conditions regarding the discharge of effluents shall be observed for samples preserved with potassium dichromate. c) Sodium azide: Immediately after sampling, add the sodium azide to the test sample. The final concentration of this preservative in the sample shall not exceed 0,024 g per 100 ml. Such a preserved test sample may be stored at temperature of between 2 “C and 6 “C. Examination should be carried out within 48 h of sampling. d) Bronopol (2-bromo-2-nitropropan-1,3-diol): Immediately after sampling, add the bronopol to the test sample. The final concentration of this preservative in the sample shall not exceed 0,05 g per 100 ml (preferably 0,02 g per 100 ml). Such a preserved test sample may be stored at a temperature of between 2 “C and 6 “C. Examination should be carried out within 72 h of sampling. NOTES 1 A test sample already preserved with boric acid may be further preserved for up to 48 h using potassium dichromate. 2 The time of storage of test samples with added bronopol can increase up to 5 days under good conditions and with verification of the quality of the cells using a modern metering device. However this involves the immediate addition of the preservative and keeping the sample in a cold place until testing. 7 Preparation of test sample 7.1 Store the unpreserved test sample for at least 24 h after milking, at a temperature of between 2 “C and 6 “C. If examination of the unpreserved sample has nonetheless to be performed within 24 h after milking, the test sample shall be pretreated by the addition of potassium dichromate (6.4) and left to stand for at least 3 h. 7.2 Heat both the unpreserved and the preserved samples in the water bath (5.2) set at 40 “C and keep them at room temperature for no longer than 30 min. 8 Procedure 8.1 Test portion Further dilution of the test sample and preparation of the test portion take place automatically in the instrument (5 1) . . 8.2 Determination Ensure that cell counting is carried out within 30 min of the end of heating (7.2) and before the temperature is below 30 “C. Ensure that the instrument stirrer is operating correctly so as to obtain as homogeneous a distribution of the cells as possible. If no instrument stirrer is available, thoroughly mix the test portion immediately before counting. 9 Expression of results Express the number of somatic cells in thousands per millilitre of milk. NOTE - For a discussion of the use of cell-count standard samples, see annex C. 4 SIST EN ISO 13366-3:1998



@ IS0 IS0 13366-3: 1997(E) 10 Precision Details of an interlaboratory test on the precision of the method are summarized in annex A. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given. 11 Test report The test report shall specify: - the method in accordance with which sampling was carried out, if known; - the method used; - the test result(s) obtained; and - if the repeatability has been checked, the final quoted result obtained. It shall also mention all operating details not specified in this part of IS0 13366, or regarded as optional, together with details of any incidents which may have influenced the test result(s). The test report shall include all information necessary for the complete identification of the sample. SIST EN ISO 13366-3:1998



IS0 13366-3: 1997(E) Annex A (informative) Results of interlaboratory test An interlaboratory test (37 participating laboratories) gave the results shown in table A.1 for I- (repeatability limit) and R (reproducibility limit) in thousands of cells per millilitre. Table A.1 Milk sample Mean number of cells per millilitre + r SR R 2 210 13,7 38,9 36,7 103,7 4 438 21,2 59,9 51,3 145,0 6 609 32,6 92,3 89,4 253,0 It should be noted that under practical conditions the geometric mean of several (e.g. three) determinations is used. NOTE - For the targets of precision, see annex B. 6 SIST EN ISO 13366-3:1998



@ IS0 IS0 13366-3: 1997(E) Annex B (informative) Quality control in the laboratory B.1 Purpose The purpose of quality control procedures is to ensure close agreement between cell counts determined in the routine way and the “true” cell count of the samples. Poor agreement may be due to random errors in individual determinations (such as may arise from inadequate mixing or inaccurate pipetting) or it may be due to systematic errors or bias (such as that introduced by incorrect calibration of instruments). The magnitude of both kinds of error may vary with the true cell count of the sample. Figure B.l illustrates the effect of both random and systematic errors on the relationship between true and observed cell counts. Repeatability is a measure of the variation between replicate determinations in one laboratory using the same sample. Reproducibility is a measure of the variation between determinations carried out in different laboratories using the same sample. Neither repeatability nor rep
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