Diagnostic Laboratory Section: Materials and Methods

Materials and Methods

Apparatus

Red cell pipette containing 0.5, 1 and 101 graduation with a red bead in the bulb

RBC-pipette

RBC Pipette

After ensuring uniform dispersion of blood by mixing, it is drawn up to 0.5 mark in the RBC pipette. If it is drawn beyond the mark, excess blood is expelled by gently stroking the tip of the pipette on a glass slide.
The blood adhering to the side of the pipette is wiped off using cotton.
By steady suction, diluting fluid is drawn into the pipette exactly up to 101 mark. The pipette is shaken with the hand or using a pipette shaker for 5 min. Air bubbles should be avoided while sucking.
The first few drops of solution in the pipette are discarded and the counting chamber of haemocytometer is charged after placing the special cover slip on the supporting ribs of the haemocytometer. Three minutes are to be allowed for the cells to settle.
Under low power of the microscope, the central square of the 9 large squares is located and under high power the erythrocytes in 5 of the 25 big squares are counted.

Each big square contains 16 small squares. Duplication in counting the cells may be avoided by counting the cells touching the top and left lines of the small square in addition to the cells inside the square. Cells may be counted using the tally counter.
Caution: A variation of more than 20 cells between any of 5 big square, indicates uneven distribution of cells and requires fresh dilution.
The sum of the cells in 5 big squares multiplied by 10,000 gives the total RBC count in millions/cmm.
- 0.5 of blood diluted to 100 i.e., 1 part of blood diluted to 200. Hence, dilution factor is 200
- Area of the RBC chamber is 1 mm x 1 mm x 0.1 mm
- Out of 25 chambers in the RBC chamber, cells in only 5 squares were counted
- Hence 200 x 10 x 5 = 10,000

Result

Last modified: Sunday, 11 September 2011, 4:57 AM