Blood pipette
(Sahli’s) 50 micro liter and 20 micro liter
(Dissolved thorough with a stirrer and fluid is ready to use)
2) Hemocytometer
/Neubauer chamber
Reagent : -
Red Cell
Diluting Fluid
The Red cell diluting fluid is isotonic to Red
cells, i.e it prevents hemolysis. The flowing are the formula of dacies
fluid……………
Tri sodium citrate
- 3.013 g
Commercial
formal dehyde 37% formalin -
1.0 ml
Distilled
water
- 100 ml
(Dissolved thorough with a stirrer and fluid is ready to use)
Procedure :-
1) A test tube is taken
and marked as the patient identity number
2) 4.0 ml of RBC diluting
fluid is taken in to the tube
3) 20 micro liter of well
mixed anti coagulated blood specimen is drawn the sahli’s pipette
4) The pipette is hold
horigently and out side is wiped carefully to check the perfect amount of blood
present in the pipette.
5) The blood specimen is
added in to the RBC diluting fluid . the solution is well mixed by swinging it
is left undisturbed for 2 minutes.
6) The hemocytometer is
placed the flat surface of the work bench. Then the cover slip is placed on the
counting chamber.
7) The well mixed
diluting Blood solution is taken in the pipette
and allowed to seep in to the counting chamber by capillary action . it
is made sure that three is no air bubble and there is no overfilling beyond the
ruled area.
8) The counting chamber
is left on the bench for 3 minute to allow the cells to settle.
9) The counting chamber
is placed on the stage of the microscope . first switched to low power objective
and then switched carefully to high dry objective (40x) . the vision of the
field is set in the middle smaller
squares of the RBC chamber ( 16 middle small squares)
10) The number of RBC is
counted seen on the 5 small square of which each square measure 0.2 x 0.2 =
0.04 square mm/mm2
11) A total of all the
cells counted in five square are calculated . the same procedure is applied to
the other side counting chamber is made finally. The calculation is made.
Calculation
: -
No of RBC counted x
Dilution
No of RBC (Per micro l) =
--------------------------------------
Area counted x Depth of fluid
Note : -
Area
x Depth of fluid = Volume of fluid in the counting chamber.
Now , Dilution = 201
Area counted =
0.2x0.2x5
= 0.04x5
= 0.2 square / mm2
Depth of fluid = 0.1 mm
Dilution
Hence, -------------------------------------
Area counted x Depth
of fluid
201
= ---------------------
0.2 x 0.1
= 10,050
Dilution
Factor =
----------------------------------------------
Area counted X
Depth of fluid
= 10.000
So, the
formula can be rewrite as No of RBC (per micro liter)
No of
Cells counted in 5 square X 10.000
5.00000 cell/mm3