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Aim: To find out the number of red blood cells in one cubic millimetre of blood.
Principles: The number of RBC in a known volume of diluted blood is counted and the number of
cells in one cm of undiluted blood are calculated from this.
Apparatus Required: Hemocytometer, RBC diluting fluid, compound microscope, sterile lancet,
watch glass, cotton, rectified spirit.
Uses of R.B.C. pipette:
- WBC count in leukemias
- Platelet counting
Uses of the bead in the bulb:
- For proper mixing
- To know whether the pipette is dry
- To identify the pipette
- R.B.C diluting fluid: Hayem’s fluid is the commonly used diluting fluid.
- Sodium chloride 0.5 Gm
- Sodium sulphate 2.5 Gm
- Mercuric perchloride 0.25 Gm
- Distilled water 100 ml sodium chloride and sodium sulphate together keep the isotonicity of fluid. Sodium sulphate also prevents the clumping of red cells. Mercuric perchloride fixes the cells and acts as a preservative.
- Other diluting fluids:
- Gower’s fluid
- Toison’s fluid
- Formol Citrate solution
Procedure: Clean and dry the counting chamber and put on the special coverslip provided. Focus on the high power objective and identify the RBC counting area. Clean the RBC pipette first with distilled water, then with absolute alcohol and finally with ether and keep it dry. Take a small quantity of diluting fluid in a watch glass and keep it aside. Clean the fingertip using rectified spirit and make a deep prick with a sterile lancet, so that blood comes out freely without squeezing. Wipe off the first drop which may contain tissue fluid also. Allow a good-sized blood drop to form a hanging drop and
keep the pointed tip of the pipette touching the drop. Suck in blood up to the 0.5 marks carefully, without any air bubble. Excess blood at the tip of the pipette is removed using a blotting paper or piece of cotton. Immediately, diluting fluid from the watch glass is sucked in up to the 101 marks without any air bubble by keeping the pipette in a vertical position.
Then thoroughly mix the blood and diluting fluid in the pipette by gently rolling the pipette held horizontally between the palms and keep aside. Mixing takes place only in the bulb of the pipette. The column of diluting fluid contained in the stem of the pipette does not enter into the dilution (i.e. 101-1 = 100). So that the blood sucked up to the 0.5 marks will have a dilution of 0.5 in 100 or 1 in 200. Now take out the counting chamber for charging discard the first few drops from the pipette, as the stem contains only diluting fluid. Bring one small drop of diluted blood at the tip of the pipette, to the edge of the coverslip on the counting chamber at an angle of about 450 The fluid enters by capillary action under the coverslip and fills the counting chamber. Both areas are filled.
Focus the RBC counting area under high power. Keep the counting chamber undisturbed for about 3 minutes for the cells to settle down in the counting area, and start counting. At least 5 squares, each having 16 smallest squares (preferably 4 corners and 1 central) should be counted to obtain a satisfactory average and a better dispersal value. While counting each small square, cells touching the top and left margin of each square should be omitted and cells touching the bottom and right margin of each square should be counted. Draw a chart of the counting squares in the record and enter the number of cells in each square and when counted.
Calculations: Let the number of cells counted in (5×16) 80 smallest squares be “N”
The number of cells in 1 smallest square is N/80
Side of 1 square = 1/20mm
Side of 1 square = 1/20mm
The volume of diluted blood in 1 square=1/400×1/10=1/4000mm3
Number of cells in 1/4000mm3 diluted blood = N
80 Number of cells in 1 mm3 of diluted blood N 80×1/4000= Nx4000 80
The dilution factor is 1 in 200
(Total diluted volume in the bulb of the pipette is 100 parts, out of which 0.5 is blood. So dilution is 0.5 in 100 i.e.1 in 200)
So numberof cells in 1 mm3 of undiluted blood =Nx 4000×200 = Nxl0000 80
- Counting chamber and pipette should be clean and dry.
- Fingertip and pricking lancet should be sterile.
- Blood should freely come out without squeezing.
- Be careful to prevent clotting of blood inside the pipette.
- While filling the pipette and charging the counting chamber, no air bubble should enter.
- Blood should be taken only up to the 0.5 marks and diluting fluid sucked only up to 101 mark.
- Blood should be properly mixed with the diluting fluid.
- Discard first few drops before charging because it will not contain RBC5.
- While charging the counting chamber, over filling and spilling should be avoided.
- Cells should be settled down and more or less evenly distributed before counting.
- Don’t keep the microscope in tilted position.
- Count from Left to Right and avoid counting of the same cell.