🦠 Somatic cells in raw milk — fluoro-opto-electronic counters (disc or flow cytometry)

Microbiology PN-EN ISO 13366-2

Overview

ISO 13366-2 | IDF 148-2:2006 gives guidance on the working conditions for the somatic cell count in raw and chemically preserved milk with fluoro-opto-electronic counters in which the counting section uses either the rotating-disc technique or flow cytometry; it applies to cow's milk and also to goat's, sheep's and buffalo's milk if certain preconditions are met. The edition replaced ISO 13366-2:1997 and ISO 13366-3:1997 and IDF 148A:1995 (methods B and C). Somatic cells are defined as cells showing fluorescence above the threshold as a result of the staining of the DNA in their nuclei; the number is expressed in cells per ml. The reference method is the microscopic method of ISO 13366-1 | IDF 148-1; the calibration and the quality control are based on ISO 8196-1/-2 (accuracy of indirect methods), on reference materials (ISO Guide 34) and on proficiency testing (ISO Guide 43-1). Regulation (EC) No 853/2004 (annex III, section IX) lays down for raw cow's milk a somatic cell count criterion of ≤ 400 000/ml (rolling geometric average) alongside a plate count at 30 °C of ≤ 100 000/ml, allowing derogations with the agreement of the authorities for certain cheeses. Instruments such as the Fossomatic (FOSS) declare conformity with ISO 13366-2 / IDF 148-2:2006 and MicroVal certification for the counting of somatic cells in raw cow's milk.

Method principle

Fluoro-opto-electronic counters have functions for taking up the reagents and the sample, a mixing section and a counting section. In the mixing section strictly controlled volumes of the sample and of the solution of buffer and dye are dispensed and mixed (in a cup, a chamber, a centrifuge, a sample loop or the tubing leading to the flow cell); part of the mixture reaches the object plane in the counting section. Every stained particle observed with the fluorescence microscope produces an electrical pulse, which is filtered, amplified and recorded; the distribution of the pulse heights is processed electronically with a distinction between the noise and the pulses attributed to cells — the discrimination threshold may be fixed or dynamic. In disc cytometry a thin film of the mixture is applied by a nozzle to the top of a vertical rotating disc, which constitutes a moving object plane; in flow cytometry part of the mixture is introduced into a fast stream of sheath fluid in a capillary flow cell, where, after being accelerated, it forms a thin thread with dynamically focused cells passing in front of the objective. Two-channel instruments are treated as two separate instruments. The result = the count × the working factor (a lower factor favours precision); the ratio of the volume of buffer/dye to that of the sample is important. Factors influencing the result: the bottles (tight, with a proper empty volume — too large a one favours churning, too small a one makes mixing difficult), the sampling (thorough mixing of bulk milk — the cells collect in the upper and the lower layer; with samples from individual animals a representative sample of the whole milking), cooling to 0–6 °C without freezing, counting within 96 h (freezing and thawing lower the result by 10–20 %), preservation within 24 h (boric acid ≤ 0.6 g/100 ml — 24 h at 6–12 °C; sodium azide ≤ 0.024 g/100 ml — 72 h at 2–10 °C; bronopol ≤ 0.05 g/100 ml — 6 days at 2–12 °C; potassium dichromate ≤ 0.2 g/100 ml — 6 days at 2–12 °C; colour markers: Patent Blue V ≤ 0.15 mg/100 ml, Yellow Orange S ≤ 1 mg/100 ml, a mixture of Patent Blue V + Eosin B), interfering substances (preservatives and markers above the limits, methylene blue > 0.06 mg/100 ml), lysis of the cells (a shift of the distribution of the pulses to the left — an underestimate), problematic samples (milk from acute mastitis with clots, contaminated milk, milk with a large number of erythrocytes, colostrum, milk from the end of lactation, sour milk), the state of the instrument (the stirrer, the patency of the path, the light source and the photomultiplier; in disc counters — the position of the film, the cleanliness of the object plane and of the cleaning sponge; in flow counters — the behaviour of the sample thread and the flow of the sheath fluid). Reagents of analytical and bacteriological quality, demineralised water < 10 µS/cm.

Applications

• Routine assessment of raw milk in milk testing laboratories (purchase) — the criterion of ≤ 400 000 cells/ml under Regulation (EC) No 853/2004
• Assessment of the performance of dairy cows — samples from individual animals (diagnosis of the state of the udder, mastitis)
• Goat's, sheep's and buffalo's milk once the preconditions of the standard are met
• Dairy laboratories — control of the raw material on reception; combination with composition analysers (CombiFoss) taking into account the influence of the preservatives
• Proficiency testing and calibration against the microscopic method of ISO 13366-1

Key parameters

Standard

Standard number

PN-EN ISO 13366-2:2007 (ISO 13366-2:2006 | IDF 148-2:2006)

Title (PL)

Mleko — Oznaczanie liczby komórek somatycznych — Część 2: Przewodnik obsługi liczników fluoro-opto-elektronicznych

Title (EN)

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

Step-by-step procedure

1. Sampling

   Use clean, dry, tight bottles with a proper empty volume; mix bulk milk thoroughly (the cells collect in the upper and the lower layer); from individual animals take a representative sample of the whole milking (for diagnostic purposes — from part of the milking).

2. Cooling or preservation

   Cool the samples at once to 0–6 °C and do not freeze them; count them within 96 h. If preservation is necessary — add the preservative within 24 h at a permitted concentration (boric acid, azide, bronopol or dichromate) with a colour marker if appropriate and store them under the conditions given; check that there is no interference for the instrument in use.

3. Check of the instrument

   Check the stirrer, the patency of the path (clots, dirt, deposits), the light source, the photomultiplier and the amplification; in disc counters — the position of the film, the cleanliness of the object plane and the replacement of the sponge, the emptying of the vessel for the rinsing fluid; in flow counters — the behaviour of the sample thread and the flow of the sheath fluid.

4. Calibration

   Calibrate against the microscopic method of ISO 13366-1 using reference materials (CRMs are not available; IRMs prepared from a suspension of leucocytes or by the microfiltration of milk, 5–8 levels) in accordance with ISO 8196-1/-2; assess each channel of a two-channel instrument separately.

5. Measurement

   The instrument dispenses and mixes the sample with the buffer/dye, applies the mixture to the disc or introduces it into the flow cell and counts the pulses above the threshold; avoid problematic samples (clots, colostrum, sour milk) and after them check and clean the flow path.

6. Result

   Report the somatic cell count per ml (the count × the working factor); for the legal assessment of raw milk calculate the rolling geometric average and compare it with 400 000/ml; take part in proficiency testing (ISO Guide 43-1).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Sodium azide and potassium dichromate (preservatives) — toxic; the azide forms explosive metal azides in the drainage installations; the dichromate is carcinogenic
• Fluorescent dyes binding DNA — mutagenic, gloves; waste reagents in accordance with the local regulations
• Raw milk may contain pathogens (Listeria, Salmonella, staphylococci) — hygiene at work

Frequently asked questions