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Reagent
:- WBC
Diluting fluid.
WBC Diluting fluid
hemolysis the RBC due to acidity. So that the counting of white cells become
easy. The following are the formula of turk solution -----
a)
Acetic
Acid glacial – 2
ml
b)
Distilled
water (q.s) – 100
ml
c)
Aqueous
methylene blue - 10 drops solution (0.3 % w/v)
Note :- Methylene blue solution
is prepaired by dissolving 0.3 gram of methylene blue in 100 ml of distilled water.
The methylene blue solution should be filtered before adding to the acid
solution. Gentain violet ( 1 ml of 1% solution w/v) can be used inplace of
methylene blue.
Equipment :-
a) Sahli’s
Pipette ( 20 micro liter )
b) E
Flask (25 ml) or test tube
c) Microscope
d) Hemocytometer
with Cover slip
e) Rubber
tubing for mouth pipeting, surgical Gauze.
Procedure : -
1)
A
dry cleaned test tube is taken and marked as ( T ).
2)
0.38
ml of diluting fluid is taken into the tube.
3)
With
the help of sahli’s pipette well mixed anti coagulated is drawn up to the
marked of 20 micro liter.
4)
The
pipette is held in the horigental position. To check the appropriate amount of
specimen and the out slide of the pipette is wiped out.
5)
The
Blood is added to the tube containing diluting fluid. The blood pipette is
rinsed 3 times and left inside the tube.
6)
The
blood specimen is mixed gentaly by swirling and left undisturbed for 2 minute
for completing hemolysis . the dilution of the blood 1:20 (in twenty)
7)
The
counting chamber is filled with diluted specimen with the help of pipette
weight for 2 minute.
8)
The
counting chamber is placed on the stage of the microscope. And focused under
low power on one of the ‘W” marked four corner square.
9)
The
white cell are identified by there blue color
with a dark dot in the center (Nucleus).
10)
The
cell are counted systematically applying the margin rule.
11)
The
WBC count on the other side of the chamber an average is made of the count. The
cells count is calculated applying following formula.
No
of WBC (per micro liter) of whole blood.
No of Cells counted X Dilution
=
----------------------------------------
Area counted X Depth Of Fluid
Where
dilution = 2.0
Area
counted = 4 x 1 sq mm
= 4 sq mm
Depth
of fluid = 0.1 mm (constant)
20
Factor = ----------
4 x 0.
200
= ----------
4
= 50
Thus
as long as the dilution and area counted are main constant the above formula
can be simpli9fied as-------
= No of cells
Counted X 50