Milk - Enumeration of somatic cells - Part 1: Microscopic method (Reference method) (ISO 13366-1:2008)

ISO 13366-1|IDF 148-1:2008 specifies a microscopic method (reference method) for the counting of somatic cells in both raw and chemically preserved milk.
ISO 13366-1|IDF 148-1:2008 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 automated cell-counting methods.

Milch - Zählung somatischer Zellen - Teil 1: Mikroskopisches Verfahren (Referenzverfahren) (ISO 13366-1:2008)

Dieser Teil der ISO 13366|IDF 148 legt ein mikroskopisches Verfahren (Referenzverfahren) für die Zählung somatischer Zellen sowohl in roher als auch in chemisch konservierter Milch fest.
Dieser Teil von ISO 13366|IDF 148 ist für die Zählung somatischer Zellen in Kuhmilch anwendbar, vorausgesetzt, dass die möglicherweise erwähnten Forderungen erfüllt sind.
Dieses Verfahren ist geeignet für die Herstellung von genormten Untersuchungsproben und für die Bestimmung von Werten für das Referenzverfahren, die für die Kalibrierung mechanisierter und automatisierter Zellzählverfahren verwendet werden.
WARNUNG — Die Anwendung dieser Norm ist möglicherweise mit der Anwendung gefährlicher Stoffe, Verfahrensschritte und Ausrüstungsgegenstände verbunden. Die Norm kann jedoch nicht alle mit ihrer Anwendung verbundenen Sicherheitsprobleme anführen. Es liegt in der Verantwortung des Anwenders dieser Norm, angemessene Sicherheits  und Gesundheitsvorkehrungen zu treffen und vor der Anwendung die Anwendbarkeit einschränkender Vorschriften zu bestimmen.

Lait - Dénombrement des cellules somatiques - Partie 1: Méthode au microscope (Méthode de référence) (ISO 13366-1:2008)

L'ISO 13366IFIL 148-1:2008 spécifie une méthode de comptage au microscope (méthode de référence) des cellules somatiques dans le lait cru et dans le lait additionné de conservateurs chimiques.
L'ISO 13366IFIL 148-1:2008 est applicable au comptage des cellules somatiques dans le lait de vache sous réserve que les conditions préalables éventuellement mentionnées soient satisfaites.
La présente méthode convient pour la préparation d'échantillons étalons et la détermination de valeurs de référence nécessaires pour l'étalonnage des méthodes de comptage de cellules mécanisées et automatisées.

Mleko - Ugotavljanje števila somatskih celic - 1. del: Mikroskopska metoda (Referenčna metoda) (ISO 13366-1:2008)

General Information

Status
Withdrawn
Publication Date
30-May-2008
Withdrawal Date
30-Aug-2008
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
15-Feb-2008
Completion Date
15-Feb-2008

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SLOVENSKI STANDARD
SIST EN ISO 13366-1:2008
01-maj-2008
1DGRPHãþD
SIST EN ISO 13366-1:1998
0OHNR8JRWDYOMDQMHãWHYLODVRPDWVNLKFHOLFGHO0LNURVNRSVNDPHWRGD
5HIHUHQþQDPHWRGD  ,62

Milk - Enumeration of somatic cells - Part 1: Microscopic method (Reference method)

(ISO 13366-1:2008)
Milch - Zählung somatischer Zellen - Teil 1: Mikroskopisches Verfahren
(Referenzverfahren) (ISO 13366-1:2008)
Lait - Dénombrement des cellules somatiques - Partie 1: Méthode au microscope
(Méthode de référence) (ISO 13366-1:2008)
Ta slovenski standard je istoveten z: EN ISO 13366-1:2008
ICS:
67.100.10
SIST EN ISO 13366-1:2008 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
EUROPEAN STANDARD
EN ISO 13366-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2008
ICS 67.100.10 Supersedes EN ISO 13366-1:1997
English Version
Milk - Enumeration of somatic cells - Part 1: Microscopic method
(Reference method) (ISO 13366-1:2008)

Lait - Dénombrement des cellules somatiques - Partie 1: Milch - Zählung somatischer Zellen - Teil 1:

Méthode au microscope (Méthode de référence) (ISO Mikroskopisches Verfahren (Referenzverfahren) (ISO

13366-1:2008) 13366-1:2008)
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 the conditions for giving this European

Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national

standards may be obtained on application to the CEN 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 member into its own language and notified to the CEN Management Centre has the same status as the

official versions.

CEN members are the national standards bodies of 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 COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels

© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13366-1:2008: E

worldwide for CEN national Members.
---------------------- Page: 2 ----------------------
EN ISO 13366-1:2008 (E)
Contents Page

Foreword..............................................................................................................................................................3

---------------------- Page: 3 ----------------------
EN ISO 13366-1:2008 (E)
Foreword

This document (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 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 the 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 implement 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, Switzerland 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.
---------------------- Page: 4 ----------------------
INTERNATIONAL ISO
STANDARD 13366-1
IDF
148-1
Second edition
2008-02-15
Milk — Enumeration of somatic cells —
Part 1:
Microscopic method (Reference method)
Lait — Dénombrement des cellules somatiques —
Partie 1: Méthode au microscope (Méthode de référence)
Reference numbers
ISO 13366-1:2008(E)
IDF 148-1:2008(E)
ISO and IDF 2008
---------------------- Page: 5 ----------------------
ISO 13366-1:2008(E)
IDF 148-1:2008(E)
PDF disclaimer

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© 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 permission in writing from either ISO or IDF at the respective

address below.
ISO copyright office International Dairy Federation

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Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88
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Published in Switzerland
ii © ISO and IDF 2008 – All rights reserved
---------------------- Page: 6 ----------------------
ISO 13366-1:2008(E)
IDF 148-1:2008(E)
Contents Page

Foreword............................................................................................................................................................ iv

Foreword............................................................................................................................................................. v

1 Scope ......................................................................................................................................................1

2 Terms and definitions ...........................................................................................................................1

3 Principle..................................................................................................................................................1

4 Reagents.................................................................................................................................................1

4.1 Dye solutions .........................................................................................................................................1

4.2 Phosphate Buffer Solution (PBS).........................................................................................................3

5 Apparatus ...............................................................................................................................................4

6 Sampling.................................................................................................................................................4

7 Preparation of test sample....................................................................................................................4

7.1 Storage....................................................................................................................................................4

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 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 trial........................................................................................................14

Annex B (informative) Staining for goats' milk ..............................................................................................15

Annex C (informative) Poisson distribution ...................................................................................................16

Bibliography ......................................................................................................................................................17

© ISO and IDF 2008 – All rights reserved iii
---------------------- Page: 7 ----------------------
ISO 13366-1:2008(E)
IDF 148-1:2008(E)
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 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 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 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 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 products, 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 the general title Milk — Enumeration of somatic cells:

⎯ Part 1: Microscopic method (Reference method)
⎯ Part 2: Guidance on the operation of fluoro-opto-electronic counters
iv © ISO and IDF 2008 – All rights reserved
---------------------- Page: 8 ----------------------
ISO 13366-1:2008(E)
IDF 148-1:2008(E)
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 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 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 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 148-1 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 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 general title Milk — Enumeration of somatic cells:

⎯ Part 1: Microscopic method (Reference method)
⎯ Part 2: Guidance on the operation of fluoro-opto-electronic counters
© ISO and IDF 2008 – All rights reserved v
---------------------- Page: 9 ----------------------
ISO 13366-1:2008(E)
INTERNATIONAL STANDARD
IDF 148-1:2008(E)
Milk — Enumeration of somatic cells —
Part 1:
Méthode au microscope (Méthode de référence)
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 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 automated 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 and 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 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 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 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 application of the dye solution shall

be carried out in a fume cupboard, using protective equipment.
© ISO and IDF 2008 – All rights reserved 1
---------------------- Page: 10 ----------------------
ISO 13366-1:2008(E)
IDF 148-1:2008(E)
4.1.1 Modified Newman-Lampert stain solution (Levowitz-Weber modification)
4.1.1.1 Components
Ethanol, 95 % (volume fraction) 54,0 ml
40,0 ml
Tetrachloroethane
Methylene blue 0,6 g
Acetic acid, glacial 6,0 ml
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 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 an 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

Dissolve 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 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.
2 © ISO and IDF 2008 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 13366-1:2008(E)
IDF 148-1:2008(E)

The 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
4.1.2.3.1 Components
Ethidium bromide stain stock solution (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 high temperature may reduce the staining capability of
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 (PBS)
4.2.1 Components
NaCl 8 g
KCl 0,2 g
1,15 g
Na HPO⋅7H O
2 4 2
KH PO 0,2 g
2 4
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 available phosphate buffer solution with pH = 7,2.

© ISO and IDF 2008 – All rights reserved 3
---------------------- Page: 12 ----------------------
ISO 13366-1:2008(E)
IDF 148-1:2008(E)
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 µm to 12 µm.

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 certified.
2 2

5.6 Slides, premarked with an outline shape (rectangular or circular), with an area of 1 cm ± 5 % (95 mm

to 105 mm ), 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 each counting.
...

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