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Saturday, July 19, 2008

CELL COUNTING USING HAEMOCYTOMETER / HEMOCYTOMETER


Cell Counting using Haemocytometer Hemocytometer or Haemocytometer is a device used to count cells.Cell Counting using haemocytometer is a technique used to count cells in a given sample. A known volume of cell culture is counted using a haemocytometer and the total number of cells is calculated. Haemocytometer is a device that was originally used to count blood cells. But now it is also used to count many other types of cells. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The simplest dimension for us to use is the 1mm2 area when a special cover slip is placed over the haemocytometer.

Haemocytometer Calculation

The volume of the numbered squares is 0.1 mm3 (0.1 ┬Ál) 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


  1. 100uL of the BHK-21 cell suspension was aseptically transferred into an eppendorf tube inside laminar air flow unit.
  2. Mix the cell suspension with 100 uL of Trypan Blue.
  3. Fill the haemocytometer chamber with the cell suspension and observe under phase contrast microscope in 10X magnification.
  4. Count the cells in each of the four grids bordered by triple lines on any two side of the square.
  5. 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%