The number of somatic cells (SCC) is one of the internationally recognized standards for milk quality control and is also a useful indicator for mastitis presence. There are several different methods for determination the total number of somatic cells in milk (SCC). Each method is based on different feature and exploitation characteristics, but none of them is capable of determining the total number of somatic cells. In case of perfect conditions it is possible to determine the SCC of a milk sample, but there are several theoretical and practical problems, that make it impossible, especially when automation and speed of analysis are required. Because of the random positions of the cells in the sample, each result, unless total sample is tested, may only indicate a part of the SCC. SOFIA GREEN fluorescent dye.




Determination the number of the somatic cells in raw milk


When determining the SCC it is necessary to maintain the requirement of ISO 13366-1:2008 "Milk – Enumeration of somatic cells – Part 1: Microscopic method (Reference method) » standard. Since the cells in milk are located based on the Poason law, there is a determined minimum quantity of cells, which needs to be counted in order to achieve the needed level of exactness (page 6 of the standard). For authentic determination of SCC it is necessary to be counted not less quantity of somatic cells than the mentioned in the following table:

Concentration of somatic cells thousands/mL Coefficient of variation, CV% Min. quantity of counted cells
< 150 10 100
150 - 250 7 200
250 - 400 6 300
≥ 400 5 400

The Poisson distribution is based on the formula: M = V = s2,

Where:
M – average value – (number of counted cells)
V – Dispersion
S – Standard deviation
The coefficient of variation (CV) would be equal to CV = (S/M) ∗ 100%

In order to achieve the needed precision, with the different methods of counting, some limitations appear, linked with the volume of the effectively measured milk. This way, if the measured sample contains 100 000 somatic cells in 1 ml, in order to achieve the needed precision (CV<10) there must be counted not less than 100 cells, in no less than 1 μL undiluted milk. In order to achieve higher precision of measurement – for example CV<5%, it is necessary the amount of measured milk to be substantially greater – to count at least 400 cells that may be contained in 4 μL milk.

An important moment is the correct identification of the cell, if it occurs, and rejection of each object or particle in the milk, which look like but is not a somatic cell. This is an important factor for determination the precision of each method for counting SCC. A key moment is the possibility of measuring the size of the mentioned cells. The question of selectiveness is further complicated by the fact that most milk samples are analyzed in a place, away from the place of milking, after a period of time. Since the milk is a biological medium with active enzymes and microorganisms, this may lead to a change in the number of cells and their morphology, even if they’re preserved.

As a solution to the above, there is general agreement as the reference method for determining the "true" number of somatic cells in milk to accept direct microscopic analysis (DMSCC) using methylene-blue or fluorescent dyes. The method is protected in IDF / ISO standard 148. There are some aspects of this method, though, that limit its feasibility from practical point of view, like long procedure and intensive training of the operator, in order to provide objective selectiveness or precision.

Significant disadvantage of direct microscopic analysis (DMSCC) is the tendency to color artifacts and with the potential problem of cell aggregation and limited sample volume gives rise to uncertainty in the number of cells. Especially clearly this is expressed in the milk with a low content of the somatic cells (up to 300 000 / ml).The accuracy, precision and repeatability of the results of the DMSCC method depend a lot on the operator’s training and abilities, notwithstanding the used equipment or protocol.

There are other methods for determining the SCC. Typical methods are counting the SCC by:

Similar Standard Method, based on Direct Fluorescent image low magnification microscopic recognition;

Fluorescent flow cytometry ISO 13366-2:2006 (IDF 148-2: 2006) Milk -- Enumeration of somatic cells -- Part 2: Guidance on the operation of fluoro-opto-electronic counters;

Based on disc cytometry – fluorine-optical counter with spinning disc (FossoMatic4000TM by Foss Electric). In milk testing laboratories the number of somatic cells is usually determined by automatic electronic machines, which may be exact and reliable instruments.

With them the SCC is quickly and cheaply determined.

There are also indirect methods, where the SCC is determined after the interaction of the milk with certain reagents, for example the use of California mastitis tests. They’re with lower accuracy and repeatability.

Milkotronic Ltd. has developed a portable Lactoscan SCC based on direct fluorescent, low magnification microscopic somatic cell counting. Lactoscan SCC uses a very sensitive fluorescent dye Sofia Green, LED optics и CMOS technologies for capturing in order to make the cells analysis more accurate, reliable and fast. Mostly, the objective selectiveness of Lactoscan SCC is combined with the permanent high stability of the different mechanical, electronic, optical and chemical components of the system, which provides almost identical results during the whole duration of machine’s use. On top of that, the production of the machines provides high level of uniformity between the devices. This is a unique feature of Lactoscan SCC which actually offers identical results when we consider the measurement of one and the same sample, notwithstanding which machine is used, what is the location, who is using the machine, and in what moment the analysis is being made, in case that the characteristics of the samples have not changed.

In order to count the somatic cell with Lactoscan SCC, the milk sample is mixed with the dying reagent, containing fluorescent dye Sofia Green. Only 8 μL from the dyed sample is pipetted on the measuring chamber of disposable LACTOCHIP. The chip is loaded into the device and for a period between few seconds and 2 minutes, depending on the measuring mode, the analysis is done. Lactoscan SCC system focuses automatically on the chip and the dyed cells are captured by the sensitive CMOS camera. The analysis algorithm of digital images determines the number and dimension of the fluorescent cells and counts their concentration. The results are automatically shown on the display, also on printer, with possibility to save the results and generate reports from the results.

MILKOTRONIC LTD
4, Narodni Buditeli Street
8900 Nova Zagora
BULGARIA
tel/fax: + 359 457 670 82
office@autocellcount.com

Copyright © 1997-2018 Autocellcount.com Milkotronic Ltd. All rights reserved. W