In order to count the WBC, the lysing of RBC or erythrocytes should be done.
For this 1.5% –2% acetic acid solution with a small quantity of crystal violet was
used as diluting fluid. Acetic acid destroys the erythrocytes, leaving behind the
leucocytes. Crystal violet was used as a stain. These stains stain the nucleolus
of leucocytes. This enables the leucocytes to be easily identifiable, and to be
counted. The number of leucocytes in a dilute fluid can be counted by using
a neubauer counting chamber.
- EDTA: Ethylene diamine tetra acetic acid was used as anticoagulant.
- WBC diluting solution: This was prepared by using 1.5 mL of 1.5%–2%
acetic acid solution of crystal violet and 98.5 mL of distilled water.
- WBC pipette
- Neubauer counting chamber
- Cover glass and microscope
Using a syringe, venous blood was drawn and poured into a vial containing
anticoagulant (EDTA) and mixed well. EDTA acts by chelating and preserves
the cellular elements. With the help of a WBC pipette, this well-mixed venous blood was drawn.
Both the fluid and blood are mixed gently, avoiding bubbling. The tube is
kept aside for 5 minutes without disturbing it. In the mean time, a cover slip
is placed on the counting chamber at the right place. The fluid/blood mixture
is shaken and transferred, using a fine-bore posture pipette, onto the counting
chamber. This is called “charging the chamber”. While charging the chamber,
care is taken not to overflow. Whenever there is an overflow, it is washed and
dried and recharged again. After charging, the cells are allowed to settle down
to the bottom of the chamber for 2 minutes. It is seen that the fluid does not
get dried up. For counting, the under part of the chamber was cleaned and
placed on the stage of the microscope using 10X or a low-power objective. The
leucocytes are counted uniformly in the 4 larger corner squares and cells present
on the outermost lines are counted on one side, and those present on the lines
opposite are not counted.
Present determination of total WBC count shows that there are about 7100
of blood. This is well within the normal range (6000 to 8000 WBC/cm3
In some cases of parasitic infection, the total WBC count increases. However,
in the case of leukemia, the number of WBC increases to more than 15,000
of blood. Normally, it would be about 4000 to 7000 WBC/mm3 of
blood. If the WBC count decreases, then that condition is called leukemia.
Number of cells in 1 mm3 of blood
Cells Counted × Dilution Factor × Chamber Depth
Area of chamber counted
Cells counted × 20 × 10
= Cells counted × 50.