The Counting area

EASYCOUNTER YC - total, vital, viable, dead yeast cell counter makes 16 pictures of different areas, for each of the four chambers of CELLCHIP. Device makes a total of 64 images for four chambers, 32 for each test (more than any other instrument on the market). This possibility of device results in higher accuracy of cell count and low coefficient of variations. Our instrument is capable of doing this because of its unique, 3D, multi-image, sequential scanning process in which the CELLCHIP is moved in x and y axis by the internal mechanism. This original mechanism is protected by patent application.

In this way, more than 43 mm² is used to capture the images. After capturing, the images are being processed by the embedded software and the average result, calculated by using the formula from ISO 4833, of all the filmed images is displayed. The whole process, after placing the CELLCHIP in the cartridge, is fully automated.

The Samples volume

Another advantage of the instrument is the use of a large volume of the stained sample, piped into the channel of the chip, which also helps to achieve low values of the coefficients of variation.

The correct dilution of sample

It is known that the accuracy of the assay depends very much on the dilution rate of the starting sample. For the device EASYCOUNTER YC, the most suitable cell concentration to determine the cells count is in the range of 1.105 to 1.107 cells/mL. It can be seen from the table that the coefficients of variation in the cells count are the lowest in this interval.

Table 1. Coefficient of variation (CV) of different cell concentrations in the range from 3x10³ to 100 000x10³ by using the automated EASYCOUNTER YC Fluorescence Image Cytometer and direct microscopic cell counting using Hemocytometer:

Cell suspension, cells/mL	Coefficient of variation, %
Cell suspension, cells/mL	EASYCOUNTER YC	DMCC
3x10³	15	20
6x10³	11	20
8x10³	8	18
20x10³	9	18
50x10³	8	17
80x10³	8	15
200x10³	6	12
500x10³	5	10
800x10³	4	12
1 000x10³	2.8	12
4 000x10³	2.5	16
7 000x10³	2.8	20
10 000x10³	3.2	23
50 000x10³	7	-
100 000x10³	13	-

By knowing that we have developed special guidelines and dilution scheme to achieve optimal concentrations of cells for accurately determination of total cell count and dead cell count (100x10³ – 10 000x10³ cells/ml).

The 2 chamber method of counting cells

EASYCOUNTER YC used 2 separate chambers to measure total and dead cells, as well as total and vital cells. It is know that in the all case the concentration of dead cells are the lower than concentration of total cells. This means that the samples need to dilute in different degree for measuring of total and dead cells. Using this and assuming the most common relations between total cells and dead cells, and total and vital cells - the total cells count is 10 times more diluted than the dead cells count and vital cells count. Using the two chamber method, we always manage to achieve the lowest coefficients of variation.

Other advantage of using 2 chambers is the increased test volume - 8µL for total cell count and 8µL for dead or vital cell count. In this case the total volume tested is 16µL and this increases the results accuracy.

The two chamber method allows using the same dye in both chambers. This completely removes the danger of interference of two dyes, as well as light pollution problem which appears usually when using one chamber, as used by our competitors. A great advantage is the low cost of our consumables, because we synthesize the dye in our laboratory.

For better understanding the advantages of the two chamber method, we apply some examples for determination of cell viability:

EXAMPLE 1

End Results

Viability	Total cells cells/mL	Dead cells cells/mL	Total cells dilutions	Dead cells dilutions
90.2%	1 847 221x10³	181 027x10³	x1000	x100

The results obtained are detailed in the following table and comparison with results of 1 chamber method and with DMCC. Using the dilutions, we can calculate the total and dead cells per mL in each chamber:

Using the 2 chamber method with 2 different dilution and same fluorescence dye - SOFIA GREEN (implemented in the instrument)	Using the 1 chamber method with 1 dilution and 2 fluorescence dye (used by our competitors)
Chamber A – for total cell counting When the end total cell result divided by the dilutions we receive the following cell concentration in the chamber. 1 847 221x10³ / 1000 = 1 847x10³cells/mL This concentration is in the optimal interval for the EASYCOUNTER YC from 100x10³ to 10 000x10³ cells per mL (from table 1) and resulting in CV lower than 2.8 for the total count. Chamber B - for dead cells counting. When the dead cell end result divided by the dilutions we receive the following cell concentration in the chamber. 181 027x10³ / 100 = 1 810x10³cells/mL This concentration is again in the optimal interval for the EASYCOUNTER YC from 100x10³ to 10 000x10³ cells per mL (from table 1) and resulting in CV lower than 2.8% for the dead cell count. Accurate measurement of these two parameters ensures accurate Viability.	In this case when using only one chamber, the dilution for total cells and dead cell will be the same. Using AO+PI dyes. Chamber A, where total cells are counted. After dilution x1000, cell concentration in the chamber are following: 1 847 221x10³ / 1000 = 1 847x10³cells/mL CV for the DMCC is more than 12% For other competitor instruments taking just one picture the CV is 7% Again using Chamber A for Dead cells counting. Using the same dilution the total cell count 181 027x10³ / 1000 = 181x10³cells/mL CV for the competitor counting with this dilution will be 10% and for the DMCC more than 15% Meaning that a result the CV for the Viability test is much higher.
Total cells CV lower than 2.8% Dead cell CV lower than 2.8%	Competitors Total cells CV 7% DMCC Total cells CV 12% Competitors Dead cells CV 10% DMCC Total cells CV 15%

Using the 2 chamber method with 2 different dilution and same fluorescence dye - SOFIA GREEN (implemented in the instrument)

Using the 1 chamber method with 1 dilution and 2 fluorescence dye (used by our competitors)

Counting in two chambers of the CELLCHIP

Chamber A – for total cell counting
When the end total cell result divided by the dilutions we receive the following cell concentration in the chamber.

1 847 221x10³ / 1000 = 1 847x10³cells/mL

This concentration is in the optimal interval for the EASYCOUNTER YC from 100x10³ to 10 000x10³ cells per mL (from table 1) and resulting in CV lower than 2.8 for the total count.
Chamber B - for dead cells counting.
When the dead cell end result divided by the dilutions we receive the following cell concentration in the chamber.

181 027x10³ / 100 = 1 810x10³cells/mL

This concentration is again in the optimal interval for the EASYCOUNTER YC from 100x10³ to 10 000x10³ cells per mL (from table 1) and resulting in CV lower than 2.8% for the dead cell count.
Accurate measurement of these two parameters ensures accurate Viability.

In this case when using only one chamber, the dilution for total cells and dead cell will be the same. Using AO+PI dyes.

Chamber A, where total cells are counted. After dilution x1000, cell concentration in the chamber are following:

1 847 221x10³ / 1000 = 1 847x10³cells/mL

CV for the DMCC is more than 12%
For other competitor instruments taking just one picture the CV is 7%
Again using Chamber A for Dead cells counting. Using the same dilution the total cell count

181 027x10³ / 1000 = 181x10³cells/mL

CV for the competitor counting with this dilution will be 10% and for the DMCC more than 15%
Meaning that a result the CV for the Viability test is much higher.

Total cells CV lower than 2.8%

Dead cell CV lower than 2.8%

Competitors Total cells CV 7%
DMCC Total cells CV 12%
Competitors Dead cells CV 10%
DMCC Total cells CV 15%

Here is a second example, for better understanding. This time with different dilution and using the lower dilution rate of the dead cells for the total cells in the one chamber counting method.

EXAMPLE 2

End Results

Viability	Total cells cells/mL	Dead cells cells/mL	Total cells dilutions	Dead cells dilutions
97.9%	562 048x10³	11 803x10³	x100	x10

The end results for this example are shown in the table above. Using the dilutions, we can calculate the cells per mL of the yeast suspension that was counted in each chamber:

Using the 2 chamber method with 2 different dilution and same dye (implemented in the instruments)	Using the 1 chamber method with 1 dilution and 2 dye (used by our competitors)
Chamber A – for total cell counting. When the dead cell end result divided by the dilutions we receive the following cell concentration in the chamber. 562 048x10³ / 100 = 5 620x10³cells/mL This concentration is in the optimal interval for the EASYCOUNTER YC and resulting in CV lower than 2.8 for the total count. Chamber B – for the dead cell counting. Dead cell end result divided by the dilutions and receive the following concentration in the chamber. 11 803x10³ / 10 = 1 180x10³cells/mL This concentration is again in the optimal interval for the EASYCOUNTER YC and resulting in CV lower than 2.8% for the dead cell count. Accurate measurement of these two parameters ensures accurate Viability.	In this case when using only one chamber, the dilution for total cells and dead cell will be the same. Using AO+PI dyes. Chamber A, where Total cells are counted. The concentration of cells in chamber after dilution is: 562 048x10³ / 100 = 5 620x10³cells/mL CV for the DMCC is more than 23% For other competitor instruments taking just one picture the CV is 12% Again using Chamber Afor Dead cells count. Using the same dilution as dilution for the total cell count: 11 803x10³ / 100 = 118x10³cells/mL CV for the competitor counting with this dilution, taking just one picture is more than 10% and for the DMCC is more than 15%
Total cells CV lower than 2.8% Dead cell CV lower than 2.8%	Competitors Total cells CV more than 12% DMCC Total cells CV 23% Competitors Dead cells CV 10% DMCC Total cells CV 15%

Using the 2 chamber method with 2 different dilution and same dye (implemented in the instruments)

Using the 1 chamber method with 1 dilution and 2 dye (used by our competitors)

Chamber A – for total cell counting.
When the dead cell end result divided by the dilutions we receive the following cell concentration in the chamber.

562 048x10³ / 100 = 5 620x10³cells/mL

This concentration is in the optimal interval for the EASYCOUNTER YC and resulting in CV lower than 2.8 for the total count.
Chamber B – for the dead cell counting.
Dead cell end result divided by the dilutions and receive the following concentration in the chamber.

11 803x10³ / 10 = 1 180x10³cells/mL

This concentration is again in the optimal interval for the EASYCOUNTER YC and resulting in CV lower than 2.8% for the dead cell count.
Accurate measurement of these two parameters ensures accurate Viability.

In this case when using only one chamber, the dilution for total cells and dead cell will be the same. Using AO+PI dyes.

Chamber A, where Total cells are counted. The concentration of cells in chamber after dilution is:

562 048x10³ / 100 = 5 620x10³cells/mL

CV for the DMCC is more than 23%
For other competitor instruments taking just one picture the CV is 12%
Again using Chamber Afor Dead cells count. Using the same dilution as dilution for the total cell count:

11 803x10³ / 100 = 118x10³cells/mL

CV for the competitor counting with this dilution, taking just one picture is more than 10% and for the DMCC is more than 15%

Total cells CV lower than 2.8%

Dead cell CV lower than 2.8%

Competitors Total cells CV more than 12%
DMCC Total cells CV 23%
Competitors Dead cells CV 10%
DMCC Total cells CV 15%

End Result

Assuming all this information, the using of the EASYCOUNTER YC is much better because:

> Large counting area 43mm² *
> The tested volume is 2 times larger (5µL for the total cell count and 5µL for the dead cell count, total - 10µL *)
> Lowest price per test, because used one dye (SOFIA GREEN), which is synthesized in our laboratory.
> The best results guaranteed by the use of lysing reagent for the total cell count.
> The two chambers utilize 2 different dilutions, making the cells per mL in the optimal concentration interval for the EASYCOUNTER YC.

Download Excel Table for calculating CV