Cell Counting with a Hemacytometer

Note: This should be obvious in the tissue culture room, but please use proper PPE (personal protective equipment) as trypan blue is a mutagen.

1. To get a an accurate cell count, make sure you have a single cell suspension. It is a good practice to pipette up and down 10 times using a 1ml micropipettor.
2. Add 10 microliters of 0.4% trypan blue solution to 10 microliters of cell suspension. Pipette up and down a few times and let site for 10 minutes to allow the trypan blue to stain the dead cells.
3. While you are waiting, clean the hemacytometer and cover slip with EtOH and allow to air dry.
4. Put cover-slip on the hemocytometer and add 10 microliters of cell/trypan blue suspension.
5. Focus the microscope on one of the 4 corner large squares of the grid and count the cells in each of these separately. Make sure to count the live (non-trypan stained) and dead (trypan stained) separately. Note that cells that touch the borders of the squares are not counted. Each large square of the hemocytometer has a total volume of 10^-4 ml.
6. For accuracy, the total number of cells counted per square should be between 20 and 50. If more than 50, dilute appropriately. If less than 20 centrifuge cells and resuspend in a smaller volume.
7. Calculate the average number of live cells in the four squares.
8. Calculations:


• % Cell Viability = [Total Viable cells (Unstained) / Total cells (Unstained + Stained)]  X 100.
• Viable Cells/ml = Average viable cell count per square x Dilution Factor x 10,000


9. In the calculations above don't forget the two fold dilution for addition of the trypan blue.
10. Clean the instrument as soon as possible. Let's face it, nobody likes a sloppy lab mate, especially in the tissue culture room. Clean the instrument with dilute bleach solution followed by 70% ethanol or isopropanol. Air dry. Dispose of trypan blue contaminated articles in biohazard.