In the manual WBC count, 50 µl of EDTA-blood (collected with an Eppendorf
pipette) is mixed together with 950 µl dilution solution (e.g. Türk’s solution,
collected with an Eppendorf pipette).
● This constitutes a dilution of 1:20. The erythrocytes will be lysed, and the
leukocyte nucleus will be stained.
● The counting chamber is immediately filled after mixing. After 2 minutes, one
may begin counting the leukocytes in the 4 large squares.
● Calculation of the leukocyte count is achieved by following the formula below
using these factors:
- Number of leukocytes counted in the big squares.
- Dilution of the cell solution
- Number of counted big squares.
- Volume above a big square.
- Conversion factor which is necessary in order to come to the volume of one
liter. Since we are moving in the µl area, this is equal to 106 (1 µl = 1 x 106 L or 1
L = 1 x 106 µl)
■ #Calculations:
WBC count / µl = Number of WBC counted × Dilution (20) ÷Number of squares
counted (4) ×volume of 1 big square (0.1 µl)
i.e.
WBC count / µl = (Number of WBC counted × 20) ÷ (4 × 0.1 µl)
i.e.
WBC count / µl = (Number of WBC counted × 20) ÷ (0.4 µl)
■ Example with 200 counted WBCs.
WBC count / µl = (Number of WBC counted × 20) ÷ (0.4 µl)
= (200 × 20) ÷ (0.4 µl) =10000 / µl