Haemocytometer Calculation
The volume of the numbered squares is 0.1 mm3 (0.1 µl) and the average number of cells in one square must be multiplied by 10^4 and by a factor to correct for the dilution (if any) to calculate cells/ml.
Number of cells/ml = Average number of cells/grid x Dilution factor x 10^4.
Trypan blue is a vital stain used to selectively color dead tissues or cells blue. It is a diazo dye.
Live cells or tissues with intact cell membranes are not colored Since cells are very selective in the compounds that pass through the membrane, in a viable cell Trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue color under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method.
Procedure for calculating cell count and % viability of BHK-21 cell line
- 100uL of the BHK-21 cell suspension was aseptically transferred into an eppendorf tube inside laminar air flow unit.
- Mix the cell suspension with 100 uL of Trypan Blue.
- Fill the haemocytometer chamber with the cell suspension and observe under phase contrast microscope in 10X magnification.
- Count the cells in each of the four grids bordered by triple lines on any two side of the square.
- the percentage of vialbility can be calculated using the formula
% viability = (No of viable cells / Total no of cells) * 100
Example
Quadrant
|
No of Viable cells
|
No of non viable cells
|
1st
|
26
|
2
|
2nd
|
23
|
5
|
3rd
|
30
|
1
|
4th
|
20
|
4
|
Total
|
99
|
12
|
Average
|
24.75
|
3
|
Cell Concentration
Number of cells/ml = Average number of cells/grid x Dilution factor x 10^4.
= 24.75 * 2 * 10^4
= 4.9*10^5
% viability = (No of viable cells / Total no of cells) * 100
% viability = (24.75/ 27.75) * 100 = 89.18%
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