MANUAL PHYSIOLOGY PRACTICAL - Repository:The Medical ...

FM/UOJ
Method:
Take some diluting fluid in a watch glass and keep it on the table. Attach the rubber
tubing (with a mouth piece) to the WBC pipette. Make a finger prick and wipe out the first
drop and allow a second drop to from. Hold the pipette horizontally and draw blood in the
pipette up to 0.5 mark. Wipe any blood on the exterior of the pipette tip and insert the pipette
vertically and draw the diluting fluid up to 11 mark. Draw the diluting fluid carefully and do
not exceed the mark because it cannot be reversed. Close the tip of the pipette with the right
index finger and remove the rubber tube. Cover the other end of the pipette by the right
thumb so that the pipette is held in the right hand such that its both ends are closed. Shake the
pipette thoroughly for about one minute. (Now the pipette contains blood diluted 20 times
and haemolysed).
Place the counting chamber on the table with the cover-slip in place. Discard two or
three drops from the pipette in order to eliminate blood-less fluid in the stem of the pipette.
Then, carefully controlling the escape of the fluid with a finger tip applied to the Upper end
of the pipette, allow a small drop to from at the tip of the pipette. Apply this drop to the area
between the cover-slip and the platform. (The counting chamber will be charged by the
surface tension pulling the fluid inwards. This prevents any air entering the counting area,
flooding with excess fluid and spilling over the cover-slip). Examine the counting area under
the microscope for general distribution of cells. If uneven distribution is seen, clean the
counting chamber and re-charge the counting chamber.
Place the counting chamber under the microscope and identify the engraved lines
under the lowest magnification (“x 3.5”). Identify the WBC counting areas and move one
counting area to the center of the field. Turn to the next magnification (“x 10”) and start
counting the cells systematically. The small squares are there to avoid recounting the same
cell or missing any cell; start from the top left square and move across the last square then
come down to the next row and so on; in order to avoid re-counting the cells on the lines,
adopt a convention to ignore cells touching the upper and left line and to include cells
touching the lower and right lines. Count the cells in all four WBC counting areas.
Calculation:
Let the total number of cells counted in all 4 areas be C
The area of one large square
= 1 sq.mm.
The depth of the film of fluid
= 1/10 mm.
The volume of fluid in all 4 squares
= 4 x 1/10 = 0.4 cu.mm.
Assume the number of cells in all areas
= C
Number of cells in 1 cu. mm. diluted blood = C
0.4
The dilution is 1 in 20.
Therefore, Cells in 1 cu. mm. undiluted blood
= Cx20
0.4
=50C
Blood
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