oSIST prEN ISO 13366-1:2026

SLOVENSKI STANDARD
01-september-2026
Mleko - Ugotavljanje števila somatskih celic - 1. del: Mikroskopska metoda
(Referenčna metoda) (ISO/DIS 13366-1:2026)
Milk - Enumeration of somatic cells - Part 1: Microscopic method (Reference method)
(ISO/DIS 13366-1:2026)
Milch - Zählung somatischer Zellen - Teil 1: Mikroskopisches Verfahren
(Referenzverfahren) (ISO/DIS 13366-1:2026)
Lait - Dénombrement des cellules somatiques - Partie 1: Méthode au microscope
(Méthode de référence) (ISO/DIS 13366-1:2026)
Ta slovenski standard je istoveten z: prEN ISO 13366-1
ICS:
67.100.10 Mleko in predelani mlečni Milk and processed milk
proizvodi products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 13366-1
IDF 148-1
ISO/TC 34/SC 5
Milk — Enumeration of somatic
Secretariat: NEN
cells —
Voting begins on:
Part 1:
2026-06-16
Microscopic method (Reference
Voting terminates on:
method) 2026-09-08
ICS: 67.100.10
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
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Reference numbers
ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
DRAFT
ISO/DIS 13366-1:2026(en)
International
IDF 148-1:2026(en)
Standard
ISO/DIS 13366-1
IDF 148-1
ISO/TC 34/SC 5
Milk — Enumeration of somatic
Secretariat: NEN
cells —
Voting begins on:
Part 1:
Microscopic method (Reference
Voting terminates on:
method)
ICS: 67.100.10
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
© ISO and IDF 2026
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
USER PURPOSES, DRAFT INTERNATIONAL
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Reference numbers
Published in Switzerland ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
ii
ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
5.1 Dye solutions .2
5.1.1 Modified Newman-Lampert stain solution (Levowitz-Weber modification) (MB) .2
5.1.2 Ethidium bromide stain solution .3
6 Apparatus . 4
7 Sampling . 5
8 Preparation of test sample . 5
8.1 Storage .5
8.2 Preparation .5
9 Procedure . 5
9.1 Preparation of the smear and staining .6
9.1.1 Preparation of the smear and staining with Newman-Lampert stain solution .6
9.1.2 Staining with ethidium bromide stain solution and preparation of the smear .6
9.2 Determination .6
9.2.1 Reading optimization .6
9.2.2 Counting in successive fields .8
9.2.3 Counting by bands .10
10 Calculation and expression of results .11
10.1 Rectangular shape counting in successive fields .11
10.2 Rectangular shape counting in bands . 12
10.3 Circular shape counting in successive fields . 12
10.4 Expression of results . . 13
11 Precision .13
11.1 Repeatability . 13
11.2 Reproducibility . 13
12 Test report . 14
Annex A (informative) Collaborative trial .15
Annex B (informative) Staining for goats’ milk.16
Annex C (informative) Poisson distribution . 17
Annex D (informative) Preparation of liquid milk sample in counting chamber and digital
imaging .18
Bibliography .24

iii
ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
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
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with the European
Committee for Standardization (CEN) Technical Committee CEN/TC 302, Milk and milk products – Methods
of sampling and analysis, in accordance with the Agreement on technical cooperation between ISO and CEN
(Vienna Agreement). It is being published jointly by ISO and IDF.
This third edition cancels and replaces the second edition (ISO 13366-1|IDF 148-1:2008), which has been
technically revised.
The main changes are as follows:
— ISO 13366-1:2008/Cor 1:2009 is incorporated;
— Smear preparation is better described and xylene is introduced instead of tetrachloroethane;
— Annex D describes a method to analyze liquid milk.
A list of all parts in the ISO 13366 | IDF 148 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
IDF (the International Dairy Federation) is a non-profit private sector organization representing the
interests of various stakeholders in dairying at the global level. IDF members are organized in National
Committees, which are national associations composed of representatives of dairy-related national interest
groups including dairy farmers, dairy processing industry, dairy suppliers, academics and governments/
food control authorities.
ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis and
sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International Standards
using the logos and reference numbers of both organizations.
IDF draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). IDF takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, IDF had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. IDF shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
This document was prepared by the IDF Standing Committee on Laboratory Statistics and Quality Assurance
and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is
being published jointly by ISO and IDF.
The work was carried out by the IDF/ISO Action Team S19 of the Standing Committee on Laboratory Statistics
and Quality Assurance under the aegis of its project leaders Mrs S. Orlandini (IT) and Mrs C. Egger (CH).

v
ISO/DIS 13366-1:2026(en)
DRAFT International Standard
IDF 148-1:2026(en)
Milk — Enumeration of somatic cells —
Part 1:
Microscopic method (Reference method)
1 Scope
This document specifies a microscopic method (reference method) for the counting of somatic cells in both
raw and chemically preserved milk.
This document 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 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
somatic cells
those cells with nuclei, that is all leucocytes and epithelial cells, determined according to the procedure
described in this part of ISO 13366-1 | IDF 148-1
3.2
microscope field
circular area visible when looking through a microscope's eyepiece at a specific magnification
3.3
cell cluster
two or more cells loosely grouped together and detected or interpreted as a single larger object
3.4
polymorphonuclear
type of white blood cell characterized by a multi-lobed or segmented nucleus, a feature that distinguishes it
from other white blood cells
ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
3.5
nuclear fragment
small piece of a cell's nucleus, formed when the nucleus breaks apart
4 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.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or deionized
water or water of equivalent purity.
5.1 Dye solutions
WARNING — HIGHLY HAZARDOUS dye solutions
Handle with extreme care. Tetrachloroethane is toxic, and ethidium bromide is mutagenic. In case
of a spill, follow proper deactivation procedures immediately. Prepare and use the dye solution only
in a properly equipped laboratory, in a fume hood, and always wear appropriate personal protective
equipment (PPE), including gloves, lab coat, and eye protection.
5.1.1 Modified Newman-Lampert stain solution (Levowitz-Weber modification) (MB)
5.1.1.1 Components
Ethanol, 95 % (volume fraction)
12,9 ml
(CAS 1330-20-7)
Methylene blue 1,4 % solution
50,0 ml
(CAS 7220-79-3 )
Xylene
46,6 ml
(CAS 1330-20-7)
Mix and cool down to 4 °C ± 2 °C in the refrigerator, overnight.
NOTE Alternatively Levowitz-Weber Xylene stain solution is commercially available.
5.1.1.2 MB working stain solution
MB (5.1.1.1) 109,5 ml
Glacial Acetic acid
7,0 ml
(CAS 64-19-7)
Add the glacial acetic acid and carefully mix again. Pass the obtained solution through an appropriate filter
(6.2) into an airtight bottle and store it as such.
When working with acetic acid, avoid using plastic equipment such as beakers, volumetric pipettes, or
pipette tips.
NOTE Acetic acid can degrade plastics, leading to contamination of the MB staining solution and negatively
affecting the staining quality of milk preparations.

ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
5.1.1.3 Preparation
Filter the MB working stain solution (5.1.1.2) with a filter pore size of 10 µm to 12 µm again before use.
Keep the solution in an airtight glass bottle.
The MB working stain solution solution can be kept for up to 2 years if stored in a dark and cool place (4 °C).
5.1.2 Ethidium bromide stain solution
5.1.2.1 Stain stock solution
5.1.2.1.1 Composition
Ethidium bromide
0,25 g
(CAS 1239-45-8)
Demineralized water 100 ml
5.1.2.1.2 Preparation
Dissolve the ethidium bromide in demineralized water preheated to 40 °C ± 2 °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 4 °C ± 2 °C.
Ethidium bromide solution is also commercially available. Handling the solution is less hazardous than
handling the powder
5.1.2.2 Buffer solution
5.1.2.2.1 Composition
Potassium hydrogen phthalate 0,51 g
(CAS 877-24-7)
Potassium hydroxide 0,162 g
(CAS 1310-58-3)
Demineralized water 100 ml
5.1.2.2.2 Preparation
Separately dissolve the potassium hydrogen phthalate and the potassium hydroxide in the demineralized
water.
The buffer solution can be kept for two months at a maximum when stored in the dark at 2 °C ± 2 °C.

ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
5.1.2.3 Ethidium bromide stain working solution
5.1.2.3.1 Components
a
Ethidium bromide stain stock solution (5.1.2.1) 2 ml
Buffer solution (5.1.2.2) 8 ml
b
Polyethylene glycol trimethylnonyl ether 0,1 ml
Demineralized water 90 ml
a
A high temperature may reduce the staining capability of ethidium
bromide.
b
Tergitol 15 S 9 is an example of a suitable product available
commercially. This information is given for the convenience of users of this
document, and does not constitute an endorsement by ISO and IDF of this
product.
5.1.2.3.2 Preparation
TM1)
Successively add the ethidium bromide stain stock solution, the buffer solution and the Tergitol to the
demineralized water and carefully mix.
Freshly prepare the ethidium bromide stain working solution directly before use.
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 Water baths, capable of maintaining a temperature of 40 °C ± 2 °C, 50 °C ± 2 °C and 65 °C ± 2 °C.
6.2 Filter, resistant to the solvents used, with a pore size of 10 μm to 12 μm.
6.3 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, e.g Annex D.4.3.
6.4 Microsyringe, for dispensing a fixed volume of 0,01 ml of milk, with a maximum tolerance of 5 %.
6.5 Micrometer, certified.
6.6 Slides, premarked with an outline shape (rectangular or circular), with an area of 1 cm ± 5 %
2 2
(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.
6.6.1 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.
6.6.2 Shapes
For a rectangular shape, the upper and lower internal widths, on the one hand, and the left and right internal
heights, on the other hand, should not differ by more than 0,2 mm.
For a circular shape, the vertical and horizontal internal diameters should not differ by more than 0,2 mm.
TM
1) Tergitol is an example of a suitable product available commercially. This information is given for the convenience
of users of this document, and does not constitute an endorsement by ISO and IDF of this product.

ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
6.7 Laboratory table, levelled horizontally.
6.8 Template, to streak the sample correctly on a given area.
6.9 Glass stain box.
7 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this part of ISO 13366|IDF 148. A recommended sampling
method is given in ISO 707|IDF 50.
If using automatic samplers, they shall have been validated properly.
8 Preparation of test sample
8.1 Storage
Prior to testing or preservation, store the test samples at a temperature of 4 °C ± 2 °C.
Analyse the test samples within 6 h after sampling. In the case of longer storage, add chemical preservatives
such as boric acid, bronopol or potassium dichromate. The final concentration of boric acid shall not exceed
0,6 g per 100 ml of test sample. The final concentration of bronopol shall not exceed 0,05 g per 100 ml of test
sample. The final concentration of potassium dichromate shall not exceed 0,1 g per 100 ml of test sample.
Store the thus preserved test samples at a temperature of 4 °C ± 2 °C for no longer than 6 days.
For environmental reasons, it is recommended to restrict the use of potassium dichromate to samples that
require a long shelf life only.
8.2 Preparation
Heat the test sample (8.1) in a water bath (6.1) set at 40 °C ± 2 °C. Mix the test sample carefully. Cool the
sample to the temperature at which the microsyringe (6.4) has been calibrated, for example to 20 °C.
Dilute test samples with an estimated somatic cell count of above 1 000 000 cells/ml with UHT skimmed
milk to obtain a somatic cell count of about 500 000 cells/ml for each diluted test sample.
V
s
d (1)
VV
sb
where
d is the dilution factor to obtain an appropriate somatic cell account in the test sample of about
500 000 cells/ml;
V is the volume, in ml, of the test sample;
s
V is the volume, in ml, of the buffer used for diluting the test sample.
b
Record the required dilution factor, d, the volume of test sample, V , and the volume of buffer, V , used to
s b
obtain the required dilution.
9 Procedure
Prepare, for each test sample, at least two smears and count the best one (for example a smear not damaged
by the dyeing process). Dip the slides (6.6) in ethanol (of volume fraction 95 %). Flame and cool. Write on the
slide only with a pencil.
ISO/DIS 13366-1:2026(en)
IDF 148-1:2026(en)
9.1 Preparation of the smear and staining
Follow either 9.1.1 or 9.1.2 for preparation of the smear and staining.
NOTE Staining for goats' milk is described in Annex B.
9.1.1 Preparation of the smear and staining with Newman-Lampert stain solution
Using the microsyringe (6.4), take 0,01 ml of the test sample (eventually diluted) (see 8.2). Rinse the
microsyringe with the test sample. If necessary, carefully and gently clean the outside of the microsyringe
which has been in contact with the test sample.
Place the mixture on a clean slide with an area of 1 cm (6.6). Using the needle, spread the test sample evenly
over the entire area defined, while ensuring that the area close to the perimeter is evenly covered. Dry the
smear at
...