Aim: To determine the total number of erythrocytes in the given sample of blood.
Requirements: Neubauer's counting chamber, RBC pipette, Thomas cover slip, watchglass, RBC dilution fluid, materials for capillary puncture, spirit, Xylol, microscope.
RBC dilution fluid:( Hayem's fluid)
| Ingredients | Quantity | Purpose |
|---|---|---|
| Sodium chloride | 1gm | Provides isotonicity & prevents hemolysis |
| sodium sulphate | 5.5gn | Provides isotonicity & prevents rouleax formation. |
| Mercuric chloride | 0.5gm | Prevents bacterial growth & fixes RBC. |
| Distilled water | 200ml | Diluent. |
Procedure:
- The Thomas cover slip, counting chamber and the lenses of microscope are cleaned first with the help of Xylol and then absorbent cotton.
- The counting chamber is adjusted and observed for RBC squares under low power objective lens of microscope, keeping the Thomas cover slip resting on the platform of the slide.
- The objective of the microscope is raised and then it is adjusted for high power objective lens to view the chamber of RBC square.
- The pipette is cleaned and dried. The ring finger is sterilized with the spirit and it is priced boldly with the help of pricking needle.
- The first drop of blood is discarded and the second drop is sucked into the RBC pipette up to the mark 0.5. Immediately the RBC dilution fluid is sucked up to the mark101. The pipette us brought to a horizontal position and the finger is placed over a tip of the pipette. A simple knot is given to the rubber tube.
- The pipette is rolled between the palms to mix the blood with the dilution fluid for one minute.
- Few drops( 2-3) are discarded and then the pipette is held at an angle of 45 degree to the surface of counting chamber and the tip of the pipette is placed at the narrow slit between the counting chamber and the cover slip. A drop is allowed to come out from the pipette. The fluid will run into the capillary space because of the capillary action and gets filled. Bubbles must be avoided and no fluid should overflow into the grooves of the counting chamber. If fluid overflow then whole procedure is repeated. When the counting chamber is successfully charged, the preparation is set-aside for 3minute on the stage of the microscope for the corpuscles to settle.
- The RBC chamber is located and RBC's are counted in 16 smallest squares by using high power lens. This is repeated in four other such chambers.
Note: Count the cells within the squares and on the left and top borders. The cells present on the right and lower borders are not counted. There should be a uniform counting.
Observation: (DO it by yourselves)
Calculation:
Dilution Factor:
0.5 part of blood is diluted to 100
parts so dilution is 0.5 to 100 times
i.e, 1 to 200 times
So our dilution factor is 200
Volume Correction Factor:
Each , R' section has an area of 0.04 sq.mm and depth of 0.1mm
Area of 1,R' section X depth of 1 ,R' = Volume of 1,R' section
1.4 sq.mm X 0.1mm = 0.004 cu mm
therefore, Total volume of 5,W' sections = Volume of 1, R'
section X 5
= 0.004 X 5 = 0.02 cubic ,millimeter
Volume correction factor = ( Volume desired)/ (Volume used)= 1.0/0.02 = 50
No. of RBC = No of cells counted X Dilution Factor X Volume correction factor
No. of WBC = _____ X 200 X 50
= _____ X 10,000
= ___________ cells/cubic mm
Report:
The total RBC count of my own blood was found to be ____________cells/cu.mm.
Discussion:
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