🦠 Enumeration of somatic cells in raw and chemically preserved milk by the microscopic method (reference method) — 0,01 ml of sample spread over a 1 cm² area of a slide, drying, staining with Newman-Lampert solution (≥ 15 min) or ethidium bromide (fluorescence microscope), counting of nucleated cells in a defined area and conversion with the working factor into cells per millilitre, PN-EN ISO 13366-1

Microbiology PN-EN ISO 13366-1

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN ISO 13366-1:2009 (Polish version), in full (our translation): “A method for counting somatic cells in raw milk and chemically preserved milk is described. The method is intended for preparing standard test samples and determining reference method values required for calibrating methods for the mechanical and automatic determination of the somatic cell count. The method covers: definition, principle, description of reagents, apparatus, preparation of test samples, determination and the way of calculating and expressing the results”. We read the text of the standard in the preview of ISO 13366-1:2008 | IDF 148-1:2008 from the cover to the end of 8.1.1 (page 5); we do not have 8.1.2 (ethidium bromide staining), 8.2 (counting), Clauses 9–11 (calculation, precision, test report) or Annexes A–C.

ACCORDING TO THE TEXT OF ISO 13366-1:2008 (forewords, Clauses 1–8.1.1). The standard was prepared by ISO/TC 34/SC 5 (milk and milk products) and the International Dairy Federation (IDF). The second edition is a technical revision of ISO 13366-1:1997 and also replaces IDF 148A:1995; Part 2 of the series gives guidance on fluoro-opto-electronic counters. Scope: microscopic (reference) method for raw and chemically preserved milk; applicable to cows’ milk provided the stated prerequisites are met; suitable for preparing standard samples and reference values needed for calibrating mechanized and automated methods. Definition (2.1): somatic cells are cells with nuclei, i.e. all leucocytes and epithelial cells, determined according to this standard. Principle (3): a test portion of milk is spread on a slide as a smear, dried, the cells are stained and counted under a microscope; the number of cells in a defined area multiplied by a working factor gives the number of cells per millilitre. Dyes (4.1, with the warning that tetrachloroethane is poisonous and ethidium bromide mutagenic — preparation and staining in a fume cupboard): modified Newman-Lampert solution (Levowitz-Weber modification) — ethanol 95 % 54,0 ml, tetrachloroethane 40,0 ml (xylene in the same volume as an alternative), methylene blue 0,6 g, glacial acetic acid 6,0 ml; the mixture is heated in a water bath at 65 °C, cooled to 4 °C, the acid is added, the solution is filtered and filtered again before use. Ethidium bromide: stock solution 0,25 g in 100 ml of water (up to two months in the dark at 2 °C ± 2 °C), buffer of potassium hydrogenphthalate 0,51 g and potassium hydroxide 0,162 g in 100 ml, working solution 2 ml stock + 8 ml buffer + 0,1 ml Triton X-100 + 90 ml water, prepared directly before use. Phosphate buffer solution PBS (4.2): NaCl 8 g, KCl 0,2 g, Na₂HPO₄·7H₂O 1,15 g, KH₂PO₄ 0,2 g in 1000 ml, pH 7,2 ± 0,1 (a commercial one may be used). Apparatus (5): water baths at 40, 50 and 65 °C (± 2 °C); filter 10–12 µm; microscope with 500–1000× magnification (with fluorescence equipment when ethidium bromide is used); microsyringe of 0,01 ml with a tolerance of not more than 2 %; certified micrometer; slides with a 1 cm² ± 5 % area (95–105 mm²), rectangular or circular, or a template of 20 mm × 5 mm or 11,28 mm diameter; the upper and lower internal widths and the left and right internal heights of the area (for a circular area the vertical and horizontal diameters) should not differ by more than 0,2 mm. Sampling (6) is not part of the method — ISO 707 | IDF 50 is recommended. Storage (7.1): 4 °C ± 2 °C and testing within 6 h of sampling; for longer storage a preservative — boric acid not more than 0,6 g, bronopol not more than 0,05 g, potassium dichromate not more than 0,1 g per 100 ml of sample — and storage at 4 °C ± 2 °C for not more than 6 days. Preparation (7.2): warm in a water bath at 40 °C, mix, cool to the temperature at which the microsyringe was calibrated (e.g. 20 °C); samples above 1 000 000 cells/ml are diluted with buffer to about 500 000 cells/ml (in the text of ISO 13366-1:2008 the factor reads d = Vs/(Vs × Vb) — a multiplication sign in the denominator, although for a dilution factor one would expect the sum Vs + Vb; whether corrigendum AC:2009 changes this we have not checked; record d, Vs and Vb). Determination (8): at least two smears per sample, the better one is counted; slides are dipped in 95 % ethanol, flamed and cooled; staining according to 8.1.1 or 8.1.2 (goats’ milk — Annex B). According to 8.1.1: 0,01 ml of sample with the microsyringe onto the 1 cm² area, spread evenly with the needle over the whole area, also near the edge, dry at room temperature, dip in Newman-Lampert solution for at least 15 min, dry, rinse gently with tap water, dry and protect against dust.

STATUS (PKN catalogue cards as of 02.10.2026). The card of PN-EN ISO 13366-1:2009 (Polish version, published 17-02-2009, 21 pages, price group L) has no “Withdrawn” header: Food, Agriculture and Forestry Sector, KT 35 Milk and Milk Products, ICS 67.100.10, “Introduces: EN ISO 13366-1:2008 [IDT], ISO 13366-1:2008 [IDT]”, “Replaces: PN-EN ISO 13366-1:2008 – English version”, additional elements: PN-EN ISO 13366-1:2009/AC:2009 and PN-EN ISO 13366-1:2009/Ap1:2009. The card of the English version PN-EN ISO 13366-1:2008 has the “Withdrawn” header (withdrawn 17-02-2009, replaced by the 2009 Polish version). The PKN search for “PN-EN ISO 13366-1” (02.10.2026) also shows PN-EN ISO 13366-1:2000 (Polish version).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read 02.10.2026). The number 13366-1 appears in 7 records at 7 laboratories: AB 485, AB 545, AB 546, AB 585, AB 604, AB 620, AB 685. In the current scopes on the PCA website (read 02.10.2026, issues from 14.10.2025 to 19.08.2026) all give the 2009 edition together with corrections: AC:2009 and Ap1:2009 (AB 545, AB 546, AB 585, AB 604, AB 620, AB 685 — in varying order, AB 685 in three separate lines) or only AC:2009 (AB 485). The “Laboratories” tab searches for entries STARTING WITH “PN-EN ISO 13366-1” and shows all 7 laboratories — checked with the tab query on the production server labcoda.pl on 02.10.2026.

WHAT THE LABORATORIES TEST (PCA entries). Always “Raw milk” (“Raw cows’ milk” at AB 604, “Milk” at AB 485) — “Somatic cell count, microscopic method”. Two laboratories give a range: AB 485 from 100 000 to 2 000 000 cells/ml, AB 685 from 10 000 cells/ml; the other five — no range. AB 485 also has the same characteristic according to Part 2 (PN-EN ISO 13366-2:2007, fluoro-opto-electronic method, from 1000 to 1 000 000 cells/ml) — the arrangement for which the microscopic method is the reference.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Sampling and storage: a sample without preservative must be tested within 6 h, a preserved one — at most 6 days at 4 °C ± 2 °C; the standard gives upper limits for preservatives (boric acid 0,6 g, bronopol 0,05 g, potassium dichromate 0,1 g per 100 ml) and for storage time — a result from a sample outside these limits does not meet the conditions of the method, although in itself it looks ordinary. Measurement: the 0,01 ml volume is dispensed by a microsyringe with a 2 % tolerance, and the standard requires cooling the sample to the temperature at which the microsyringe was calibrated (7.2) — skipping this step changes the dispensed volume on which the conversion to millilitres depends; the slide area is 1 cm² ± 5 % and its opposite dimensions (widths, heights or diameters) should not differ by more than 0,2 mm — a slide with a different area requires recalculation of the working factor; the milk must be spread also near the edge of the area. Calculation: a sample above 1 000 000 cells/ml is diluted with buffer to about 500 000 cells/ml and d, Vs and Vb are recorded, checking the formula for d in the edition with corrigendum AC:2009 (the text of ISO 13366-1:2008 has Vs × Vb in the denominator) — the dilution has to be taken into account in the calculation according to Clause 9, which we do not have. Role of the method: it is the reference method for automated counters (separate entry on PN-EN ISO 13366-2) — microscope values are used to calibrate them, not the other way round. Range: AB 685 gives a range from 10 000 cells/ml, AB 485 from 100 000 cells/ml; the standard itself gives no limits of quantification in the part we read.

WHAT WE DO NOT STATE. We do not have 8.1.2 and 8.2 or Clauses 9–11: the step-by-step ethidium bromide staining, the number of fields or strips to count, the formulae for the working factor (rectangle — successive fields or strips, circle — successive fields), the repeatability and reproducibility limits or the content of the test report; nor do we have Annexes A (collaborative trial), B (staining of goats’ milk) and C (Poisson distribution). We therefore give no precision figure and no minimum number of cells to count.

Method principle

0,01 ml of milk (diluted with buffer when the count exceeds 1 000 000/ml) is spread over a 1 cm² area of a slide, dried and stained (Newman-Lampert or ethidium bromide); nucleated cells are counted under a microscope in a defined area and multiplied by the working factor, giving the number of cells per millilitre.

Applications

• Raw milk — somatic cell count by the microscopic method; 7 laboratories in PCA scopes
• Reference values and standard samples for calibrating automated counters (PN-EN ISO 13366-2)
• Chemically preserved milk (boric acid, bronopol, potassium dichromate) stored for at most 6 days

Key parameters

Standard

Standard number

PN-EN ISO 13366-1:2009 (wersja polska; EN ISO 13366-1:2008 IDT, ISO 13366-1:2008 IDT) z PN-EN ISO 13366-1:2009/AC:2009 i PN-EN ISO 13366-1:2009/Ap1:2009

Title (PL)

Mleko — Oznaczanie liczby komórek somatycznych — Część 1: Metoda mikroskopowa (Metoda odniesienia)

Title (EN)

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

Step-by-step procedure

1. Sample

   Store at 4 °C ± 2 °C; test within 6 h or add preservative and test within at most 6 days. PN-EN ISO 13366-1:2009 current (PKN cards as of 02.10.2026).

2. Preparation

   Warm in a 40 °C water bath, mix, cool to the calibration temperature of the microsyringe; dilute samples > 1 000 000 cells/ml with buffer to approx. 500 000/ml and record d, Vs, Vb.

3. Smear

   At least two smears: 0,01 ml onto the 1 cm² area of a degreased (95 % ethanol, flaming) slide, evenly over the whole area including the edge; dry at room temperature.

4. Staining

   Newman-Lampert solution at least 15 min, drying, rinsing with tap water, drying (8.1.1); or ethidium bromide according to 8.1.2 (we do not have this subclause).

5. Counting and result

   Count the cells under the microscope in the better smear and convert with the working factor into cells/ml according to Clause 9 (we do not have the formulae).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Tetrachloroethane is poisonous and ethidium bromide mutagenic — prepare the solutions and stain in a fume cupboard, with protective equipment; deactivate spills (4.1)
• It is recommended to restrict potassium dichromate as a preservative to samples requiring long storage (7.1)
• TECHNICAL NOTE: this is the reference method for calibrating automated counters; sample without preservative — within 6 h, with preservative — at most 6 days at 4 °C ± 2 °C

Frequently asked questions