| GU/GIP/PURPH-1 11/ Anatomy, Physiology and Health Education /2013-2014 | gxperiment No:9 | DETERMINATION OF RBC COUNT OF BLOOD fo determine RBC count of my own blood Date: ‘apparatus: Improved Neubauer’s chamber, RBC Pipette, blood lancet Composition of RBC Diluting Fluid: Sodium sulphate- Prevent aggregation of RBC. » Sodium chloride (NaC!) ~ Maintain isotonicity of the fluid and prevents hemolysis. 7 Mercuric chloride (HgClz) - Causes fixation of the cells. Prevents bacterial growth (preservative) > Water- Diluent Principle: The number of red cells in blood is too many and the size of cells is too small. It is therefore not possible to count the cells even under high power. This difficulty is practically overcome by diluting the blood with a suitable dilution fluid to a known degree. The diluted blood is placed in a capillary space ofknown capacity in between counting chamber and cover slip. The number of cells in the small capillary space of known volume is then counted under the high power of the microscope. The count can be calculated by multiplying the number with the dilution factor. Procedure: 1) Take adequate RBC diluting fluid in a watch glass. 2) Prick the finger taking necessary precautions. 3) Then wipe out the first drop of blood and suck the next drop exactly up to 0.5 marks. n suck the RBC fluid into the 4) Immediately thereafter holding the pipette in a horizontal po: pipette up to 101 mark just above the bulb. Keep the pipette horizontally between the palm and hold for 1 min. 5) Focus the RBC square of Neubauer's chamber under the low power objective. Then remove the chamber from microscope and place it on the table, Do not disturb the focus of microscope for charging of Neubauer’s chamber. 6) Hold the pipette at an angle of 45° to the surface of counting chamber and bring the tip of the pipette in contact with the edge of the cover slip, 7) allow a drop to come out of the pipette. 8) Allow some time for the cells to settle down in the counting chamber. Check whether the cells are —— GITAM INSTITUTE OF PHARMACY CSS page 19 Scanned with CamScannerGU/GIP/PURPH-111/ Anatom uniformly distributed or not. If not clean the chamber and recharge it 9) Now focus the chamber adjust high power objective and count the RBC s in four medium size comers and one medium size central square and in further sub squat jnonler to avoid counting the square cell twice the following rules are followed, a, Count any cell which is lying on the upper and left border but not the cells lying on the lower or right border. | CALCULATION: pyjution correction factor (DCF): The volume of blood taken = 0.5ml The volume of pipette = 101 Stem volume = 1 Bulb volume = 101 — 1 = 100 Therefore, DCF = Volume of bulb = 00_ = 200 Volume of blood 0.5 Volume correction factor (V4 RBC square length = Imm RBC square area = 1 sq. mm. .04 mm? Small square area = 1/25 mm? Depth = 0.1 mm Volume= Area x Depth = 0.04x 0.1 = 004mm? Volume in 5 small RBC squares = .004x5 = 0.02 mm’* Volume correction factor (VCF) = 1/0.02 = 50 Tal RBC count (RI + R2+R3 +R4 + RS) x DCF x VCF = Report: The total WBC count of my blood is found to be ~ G.SUHASIN ASSISTANT PROFESSOR Page 20 ~ Scanned with CamScanner