Milk - Enumeration of somatic cells - Part 2: Guidance on the operation of fluoro-opto-electronic counters (ISO 13366-2:2006)

ISO 13366-2|IDF 148-2:2006 gives guidance on the operating conditions for counting somatic cells, in both raw and chemically preserved milk, using fluoro-opto-electronic somatic cell counters in which either a rotating disc technique or flow cytometry is applied in the counting section.
The guidance is applicable to the counting of somatic cells in raw cow milk. The guidance is also applicable to raw milk of other species, such as goat, sheep and buffalo, if the specified prerequisites are met.

Milch - Zählung somatischer Zellen - Teil 2: Leitfaden zum Betrieb fluoreszenzoptoelektronischer Zählgeräte (ISO 13366-2:2006)

Dieser Teil von ISO 13366 | IDF 148 gibt Leitlinien zu den Betriebsbedingungen der Zählung somatischer Zellen in Rohmilch und chemisch konservierter Milch. Die Zählung erfolgt mit einem fluoreszenz¬optoelektronischen Zählgerät für somatische Zellen, in der entweder ein Verfahren mit rotierender Scheibe oder die Durchflusszytometrie im Zählbereich eingesetzt wird.
Der Leitfaden ist anwendbar für die Zählung somatischer Zellen in Rohmilch. Der Leitfaden ist auch für die Zählung in Rohmilch anderer Tierarten wie Ziegen, Schafen und Büffeln anwendbar, falls die festgelegten Vorbedingungen eingehalten werden.

Lait - Dénombrement des cellules somatiques - Partie 2: Lignes directrices pour la mise en oeuvre des compteurs fluoro-opto-électroniques (ISO 13366-2:2006)

L'ISO 13366-2 | FIL 148-2:2006 indique des lignes directrices concernant les conditions opératoires pour le dénombrement des cellules somatiques dans le lait cru et le lait contenant des conservateurs chimiques, à l'aide de compteurs fluoro-opto-électroniques utilisant soit une méthode sur disque rotatif, soit la cytométrie en flux pour la partie comptage.
Les présentes lignes directrices s'appliquent au dénombrement des cellules somatiques dans le lait cru de vache. Elles s'appliquent également au dénombrement dans le lait cru d'animaux d'autres espèces, telles que la chèvre, la brebis et la bufflesse, si les conditions préalables spécifiées sont remplies.

Mleko - Ugotavljanje števila somatskih celic - 2. del: Navodilo za uporabo flouro-opto-elektronskih števcev (ISO 13366-2:2006)

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Status
Withdrawn
Publication Date
30-Jan-2007
Withdrawal Date
29-Apr-2007
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
01-Oct-2006
Completion Date
01-Oct-2006

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SLOVENSKI STANDARD
SIST EN ISO 13366-2:2007
01-marec-2007

Mleko - Ugotavljanje števila somatskih celic - 2. del: Navodilo za uporabo flouro-

opto-elektronskih števcev (ISO 13366-2:2006)

Milk - Enumeration of somatic cells - Part 2: Guidance on the operation of fluoro-opto-

electronic counters (ISO 13366-2:2006)
Milch - Zählung somatischer Zellen - Teil 2: Leitfaden zum Betrieb
fluoreszenzoptoelektronischer Zählgeräte (ISO 13366-2:2006)

Lait - Dénombrement des cellules somatiques - Partie 2: Lignes directrices pour la mise

en oeuvre des compteurs fluoro-opto-électroniques (ISO 13366-2:2006)
Ta slovenski standard je istoveten z: EN ISO 13366-2:2006
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 13366-2:2007 en

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

---------------------- Page: 1 ----------------------
EUROPEAN STANDARD
EN ISO 13366-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2006
ICS 67.100.10 Supersedes EN ISO 13366-2:1997
English Version
Milk - Enumeration of somatic cells - Part 2: Guidance on the
operation of fluoro-opto-electronic counters (ISO 13366-2:2006)

Lait - Dénombrement des cellules somatiques - Partie 2: Milch - Zählung somatischer Zellen - Teil 2: Leitfaden zum

Lignes directrices pour la mise en oeuvre des compteurs Betrieb fluoreszenzoptoelektronischer Zählgeräte (ISO

fluoro-opto-électroniques (ISO 13366-2:2006) 13366-2:2006)
This European Standard was approved by CEN on 29 September 2006.

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 Central Secretariat 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 Central Secretariat has the same status as the official

versions.

CEN members are the national standards bodies of Austria, Belgium, 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

© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13366-2:2006: E

worldwide for CEN national Members.
---------------------- Page: 2 ----------------------
EN ISO 13366-2:2006 (E)
Foreword

This document (EN ISO 13366-2:2006) 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 April 2007, and conflicting national

standards shall be withdrawn at the latest by April 2007.
This document supersedes EN ISO 13366-2:1997.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of

the following countries are bound to implement this European Standard: Austria, Belgium,

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.

Endorsement notice

The text of ISO 13366-2:2006 has been approved by CEN as EN ISO 13366-2:2006 without any

modifications.
---------------------- Page: 3 ----------------------
INTERNATIONAL ISO
STANDARD 13366-2
IDF
148-2
Second edition
2006-10-01
Milk — Enumeration of somatic cells —
Part 2:
Guidance on the operation
of fluoro-opto-electronic counters
Lait — Dénombrement des cellules somatiques —
Partie 2: Lignes directrices pour la mise en œuvre des compteurs
fluoro-opto-électroniques
Reference numbers
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
ISO and IDF 2006
---------------------- Page: 4 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
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© ISO and IDF 2006

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.
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Published in Switzerland
ii © ISO and IDF 2006 – All rights reserved
---------------------- Page: 5 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
Contents Page

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

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

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

2 Normative references ........................................................................................................................... 1

3 Terms and definitions........................................................................................................................... 1

4 Principle................................................................................................................................................. 2

5 Factors affecting the results of measurements................................................................................. 2

5.1 Sample bottles ...................................................................................................................................... 2

5.2 Sampling................................................................................................................................................ 2

5.3 Preservation .......................................................................................................................................... 3

5.4 Sample storage and transport............................................................................................................. 3

5.5 Interfering substances ......................................................................................................................... 3

5.6 Sample quality at analysis ................................................................................................................... 4

5.7 Applied chemicals ................................................................................................................................ 4

5.8 Instrument condition ............................................................................................................................ 4

5.9 Working factor....................................................................................................................................... 5

5.10 Testing volumes.................................................................................................................................... 5

6 Calibration ............................................................................................................................................. 5

6.1 Reference materials.............................................................................................................................. 5

6.2 Calibration procedure........................................................................................................................... 6

7 Sampling................................................................................................................................................ 8

8 Determination........................................................................................................................................ 8

9 Checks on performance in routine operation.................................................................................... 8

9.1 Blank checks ......................................................................................................................................... 8

9.2 Carry-over effect ................................................................................................................................... 8

9.3 Ratio of reagent volume and test sample volume............................................................................. 9

9.4 Pilot checks ........................................................................................................................................... 9

9.5 Additional instrument surveillance..................................................................................................... 9

9.6 Repeatability.......................................................................................................................................... 9

9.7 Intralaboratory reproducibility .......................................................................................................... 10

9.8 External comparisons ........................................................................................................................ 10

10 Specific remarks for the use with milk from different species ...................................................... 10

10.1 General................................................................................................................................................. 10

10.2 Cow milk .............................................................................................................................................. 10

10.3 Goat milk.............................................................................................................................................. 10

10.4 Sheep milk ........................................................................................................................................... 11

10.5 Buffalo milk ......................................................................................................................................... 11

11 Precision.............................................................................................................................................. 11

11.1 Repeatability........................................................................................................................................ 11

11.2 Intralaboratory reproducibility .......................................................................................................... 11

11.3 Reproducibility.................................................................................................................................... 12

12 Test report ........................................................................................................................................... 12

Bibliography ..................................................................................................................................................... 13

© ISO and IDF 2006 – All rights reserved iii
---------------------- Page: 6 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(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 13366-2⎪IDF 148-2 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 edition of ISO 13366-2⎪IDF 148-2 cancels and replaces ISO 13366-2:1997 and ISO 13366-3:1997, which

have been technically revised.

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 2006 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
Foreword

IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National

Committee in every member country. Every National Committee has the right to be represented on 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 13366-2⎪IDF 148-2 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 on 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 13366-2⎪IDF 148-2 cancels and replaces IDF 148A:1995, methods B and C of which have

been technically revised.

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 2006 – All rights reserved v
---------------------- Page: 8 ----------------------
ISO 13366-2:2006(E)
INTERNATIONAL STANDARD
IDF 148-2:2006(E)
Milk — Enumeration of somatic cells —
Part 2:
Guidance on the operation of fluoro-opto-electronic counters
1 Scope

This part of ISO 13366⎪IDF 148 gives guidance on the operating conditions for counting somatic cells, in both

raw and chemically preserved milk, using fluoro-opto-electronic somatic cell counters in which either a rotating

disc technique or flow cytometry is applied in the counting section.

The guidance is applicable to the counting of somatic cells in raw cow milk. The guidance is also applicable to

raw milk of other species, such as goat, sheep and buffalo, if the specified prerequisites are met.

2 Normative references

The 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 referenced

document (including any amendments) applies.

ISO 8196-1⎪IDF 128-1, Milk — Definition and evaluation of the overall accuracy of indirect methods of milk

analysis — Part 1: Analytical attributes of indirect methods

ISO 8196-2⎪IDF 128-2, Milk — Definition and evaluation of the overall accuracy of indirect methods of milk

analysis — Part 2: Calibration and quality control in the dairy laboratory

ISO 13366-1⎪IDF 148-1, Milk — Enumeration of somatic cells — Part 1: Microscopic method (Reference

method)

ISO Guide 34, General requirements for the competence of reference material producers

ISO Guide 43-1, Proficiency testing by interlaboratory comparisons — Part 1: Development and operation of

proficiency testing schemes
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 8196-1⎪IDF 128-1,

ISO 8196-2⎪IDF 128-2 and the following apply.
3.1
reference method
method described in ISO 13366-1⎪IDF 148-1 for the counting of somatic cells
3.2
somatic cells

those cells that show more than a threshold intensity of fluorescence due to the staining of DNA in their nuclei

NOTE The number of somatic cells is expressed in cells per millilitre.
© ISO and IDF 2006 – All rights reserved 1
---------------------- Page: 9 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
4 Principle

Fluoro-opto-electronic counters contain functions for the uptake of reagents and test sample, a mixing section

and a counting section. In the mixing section, the test sample is mixed with a buffer and a stain solution. Part

of the resulting mixture is transferred to the counting section and put onto an object plane. Each stained

particle observed with a fluorescence microscope produces an electrical pulse that is filtered, amplified and

recorded. The resulting pulse height distribution is electronically processed, whereby discrimination is made

between noise signals and pulses that are attributed to stained somatic cells. The discriminator level can

either be fixed or dynamic.

In the mixing section, closely controlled volumes of test sample and buffer/stain solutions are dosed and

mixed. Mixing can take place in a cup, a mixing chamber, a centrifuge, a sample loop or in the tubing leading

to a flow cell.

In the counting section, either disc cytometry or flow cytometry can be applied. In the case of disc cytometry, a

thin film of the mixture is brought through a nozzle to the top of a vertical rotating disc. This rotating surface

acts as a moving object plane for a fluorescence microscope. When using flow cytometry, part of the mixture

is placed in the high-speed flow of a surrounding sheath liquid in a capillary flow cell. Through acceleration,

the mixture forms a thin string in which the somatic cells are dynamically focused and aligned. This string then

passes the objective of a fluorescence microscope.

Some instruments contain two channels in the counting section. In terms of analytical quality assurance, such

a situation should be considered equivalent to working with two separate units, so the performance should be

evaluated separately for each channel.
5 Factors affecting the results of measurements
5.1 Sample bottles

Sample bottles should be fit for use; i.e. to transfer test samples from the point of sampling to the laboratory

without loss or damage.

Care is to be exercised that sample bottles are leak-proof and that a proper empty volume is left. Too large an

empty volume can facilitate churning; too small an empty volume can cause problems with mixing.

5.2 Sampling
5.2.1 General

Sampling materials (i.e. sample bottles, beakers and sampling devices) should be clean and dry. Where

automatic samplers are used, these should have been properly validated.

Test samples should preferably be cooled immediately after sampling to between 0 °C and 6 °C and be kept

at that temperature until counting (see 5.4) rather than being preserved. Freezing should be avoided. If

preservation is necessary, suitable means for chemical preservation of test samples are described in 5.3.

5.2.2 Bulk milk samples

Thorough mixing of the raw bulk milk to be sampled is essential. Somatic cells will concentrate in the upper

and lower layers in the case of insufficient stirring.
5.2.3 Milk samples from individual animals

The release of somatic cells in the milk during milking is uneven. When the aim is to produce a representative

counting result for a whole milking, it is essential that a representative sample of the whole milking be

obtained. For diagnostic purposes, a sample of a partial milking may suffice.
2 © ISO and IDF 2006 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
5.3 Preservation

If chemical preservation is considered necessary, the test sample (5.2.1) should be preserved as soon as

possible, but in any case within 24 h after sampling. In all cases, the test sample should be kept cool (0 °C to

6 °C) until the addition of the preservative.
Suitable preservatives are the following.

a) Boric acid: its final concentration in the test sample should not exceed 0,6 g/100 ml. Such preserved

samples may be stored at between 6 °C and 12 °C for up to a further 24 h.

b) Sodium azide: its final concentration in the test sample should not exceed 0,024 g/100 ml. Such

preserved samples may be stored at between 2 °C and 10 °C for up to a further 72 h.

c) Bronopol (2-bromo-2-nitro-1,3-propanediol): its final concentration in the test sample should not exceed

0,05 g/100 ml. Such preserved samples may be stored at between 2 °C and 12 °C for up to a further 6 d.

d) Potassium dichromate: its final concentration in the test sample should not exceed 0,2 g/100 ml. Such

preserved samples may be stored at between 2 °C and 12 °C for up to a further 6 d.

Accompanying colour tracers found to be suitable are

⎯ Patent Blue V with a final concentration in the test sample of up to 0,15 mg/100 ml,

⎯ Yellow Orange S (E110) with a final concentration in the test sample of up to 1 mg/100 ml, and

⎯ a mixture of Patent Blue V and Eosin B with a final concentration in the test sample of up to 0,03 mg and

0,45 mg/100 ml, respectively.

Other preservatives and colour tracers may be used provided that their effectiveness and conditions for use

have been soundly validated.

In all cases, properly validate the absence of interference for the counting equipment concerned before

application.

In cases where fluoro-opto-electronic cell counters are combined with milk analysers for the measurement of

other test sample components, care should be taken that the applied preservatives and colour tracers do not

affect the counting result.
5.4 Sample storage and transport

Unpreserved test samples should be stored at between 0 °C and 6 °C and should be counted within 96 h after

the completion of sampling. Avoid freezing the test samples. Storage at higher temperatures and/or over

longer time scales may result in non-representative counts. The measurement of samples after freezing and

thawing may result in lower counts (by 10 % to 20 %). The age of the samples at freezing and the type of

thawing process can influence the counting result.
5.5 Interfering substances

The use of substances that interfere in the counting should be avoided. Substances known to influence the

instrument read-out are

a) preservatives and colour tracers at higher concentrations than specified in 5.3, and

b) Methylene blue at higher concentrations, i.e. > 0,06 mg/100 ml.
© ISO and IDF 2006 – All rights reserved 3
---------------------- Page: 11 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
5.6 Sample quality at analysis

Breakdown of somatic cells (lysis) will result in an increase of smaller cell fragments. The lower intensity of

fluorescence after staining of these particles causes a shift in the pulse height distribution to the left. This will

hamper proper differentiation from noise pulses and therefore result in a lower count.

NOTE In several types of instruments, features are available for evaluating the position and the shape of the pulse

height distribution. See the relevant instructions and guidance from the instrument manufacturer.

After the processing of problematic samples, the flow path should be checked and possible cleaned. The

proper functioning of the instrument should be tested before further use. Possible problematic test samples

are
a) milk samples from severely infected udders, i.e. with clots,
b) milk samples with impurities,
c) milk samples with high numbers of erythrocytes,
d) colostrum,
e) late lactation milk, and
f) sour milk.
Where possible, analysis of problematic test samples should be avoided.
5.7 Chemicals used

All reagents used should be of recognized analytical and bacteriological quality. Water used should be

demineralized (remaining conductivity < 10 µS/cm) or water of at least equivalent purity. Follow the

manufacturer's instructions for the preparation of the working solutions, the maximum storage time and

storage requirements.

Local conditions regarding the use and discharge of applied chemicals and effluents should be observed.

5.8 Instrument condition
5.8.1 General points of attention are
a) the functioning of the mixing device and the stirrer,

b) possible disturbances at sample intake and in the flow system due to blocking by impurities, clots or

fouling in the mixing and incubation units, and

c) the condition and the functioning of the light source and the photo multiplier, the gain setting and the

signal quality.
5.8.2 Specific points of attention with disc cytometric counters are
a) the positioning of the film on the rotating disc,

b) the cleanliness of the object plane and the functioning of the cleaning sponge; timely replacement of the

sponge is essential, and
c) proper emptying of the collection vessel for the rinsing liquid.

5.8.3 A specific point of attention with flow cytometric counters is the variation in the behaviour of the

sample string in the flow cell and the sheath liquid flow. Some instrument manufacturers offer special

programme features for checking this, thereby indicating possible solutions in the case of deviations.

4 © ISO and IDF 2006 – All rights reserved
---------------------- Page: 12 ----------------------
ISO 13366-2:2006(E)
IDF 148-2:2006(E)
5.9 Working factor

The working factor is the number by which the actual number of somatic cells counted by an instrument is

multiplied in order to arrive at the somatic cell count of the test sample. In theory, precision characteristics and

accuracy should benefit from a lower working factor.
5.10 Testing volumes

A proper ratio between the volume of buffer/stain solution and that of the test sample is essential for correct

counting.
6 Calibration
6.1 Reference materials
6.1.1 General

Reference materials should be produced under closely controlled conditions; i.e. working with a quality

assurance system and fulfilling the requirements as listed in ISO Guide 34.
Reference materials may be

a) certified reference materials (CRMs) as produced by a recognized official organization,

b) secondary reference materials (SRMs) as prepared by an external supplier, or

c) in-house reference materials (IRMs) as prepared by the laboratory itself, whereby traceability is kept with

CRMs, SRMs or via interlaboratory proficiency studies.

NOTE CRMs for somatic cell counting are not available. Examples of suitable procedures for the preparation of IRMs

are listed below. IRMs with a composition as close as possible to natural milk are preferable.

6.1.2 Preparation of calibration samples
6.1.2.1 Preparation by addition of a bovine leucocyte suspension

a) Mix 1 000 ml of sterilized or UHT milk with low somatic cell count with 1 ml of polypropylene 2000 and

0,4 g of bronopol.
b) Add the required amount of a suitable leucocyte suspe
...

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