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RBC Count Procedure Using Haemocytometer

This document describes the procedure for determining total red blood cell count using a visual/manual method with a haemocytometer. The key steps are: 1) Diluting a blood sample 20μl in 4ml of Hayem's fluid for dilution. 2) Filling the Neubauer counting chamber and allowing cells to settle for 2 minutes. 3) Counting the number of red blood cells in the central area consisting of 25 groups of 16 small squares. 4) Using the counted number to calculate the red blood cell concentration per cubic mm of blood based on the dilution and chamber specifications.

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Hasan W Dandan
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3K views5 pages

RBC Count Procedure Using Haemocytometer

This document describes the procedure for determining total red blood cell count using a visual/manual method with a haemocytometer. The key steps are: 1) Diluting a blood sample 20μl in 4ml of Hayem's fluid for dilution. 2) Filling the Neubauer counting chamber and allowing cells to settle for 2 minutes. 3) Counting the number of red blood cells in the central area consisting of 25 groups of 16 small squares. 4) Using the counted number to calculate the red blood cell concentration per cubic mm of blood based on the dilution and chamber specifications.

Uploaded by

Hasan W Dandan
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOC, PDF, TXT or read online on Scribd
  • Experiment No 2

Experiment No: 2

Date: ____________ (G)

Aim: To determine the total red blood cell count (visual method) Apparatus: 1. Haemocytometer, containing a. Neubauers counting chamber b. Cover slip c. Red Cell Pippette with bead (with 3 markings 0.5, 1, 101) 2. Pricking needle/ lancet 3. Microscope 4. Spirit lamp 1. RBC 2. Spirit 3. Hayems dilution Fluid Principle: RBC can be counted either visually or electronically. Because the visual method (using a Haemocytometer) is tedious & may have error of up to 20 %, it has been superseded by the more convenient and accurate electronic method. The Red Cell concentration varies widely and is different in adult males and females. In healthy males, the red cell count averages 5.5 million cells/ cubic mm of blood and ranges from about 4.5 to 6.5 million. In females, the red cell count averages 4.8 and ranges from 3.9 to 5.5 million / cubic mm of blood Procedure: 1. Into a 75 x 12 mm tube take 4 ml of Hayems fluid. 2. Add 20 l (0.02 ml) of well mixed blood. 3. After sealing the tube with rubber, mix in a mechanical mixer or mix by hand for 2 minutes by tilting the tube through an angle of about 120. 4. Prepare the special Neubauer counting chamber by fixing a special thick cover glass. 5. Without delay fill the chamber by the diluted blood using a capillary tube. 6. The chamber should be left undisturbed for at least 2 min. for cells to settle, but not much longer. 7. Count the red cells in the central area which consists of 25 groups of 16 small squares separated by closely ruled triple lines. (Counting should include the cells which touch the top and right hand margins and omitting from the count those which touch the bottom and left hand margins of the small squares).

Reagents:

Observation: The following observations were made in the central area: Number of Big Square 1 2 3 4 5 Total [N] = Calculation: 1 big square = 16 small squares Total small squares = 16 x 5 = 80 Area of 1 small square =1/20 x 1/20 = 1/400mm2 Depth of the chamber = 1/10 mm Therefore, The volume of fluid over one small square = area x depth = 1/400 x 1/10 = 1/ 4000 mm3 Volume of fluid over 80 small squares = 80 x 1/4000 = 80 / 4000 mm3 N = Number of cells counted in 80 small squares If N cells are counted in 80 / 4000 mm3 of diluted blood, then number of cells in 1 mm3 of diluted blood = N 80 / 4000 = (N x 4000)/ 80 Since blood is diluted 1: 200, 1mm3 of blood contains = (N x 4000 x 200) / 80 = N x 10000 cells = = Result: The given sample of blood contains _____________ number of cells per cubic mm of blood. Precautions: 1. Care should be taken while puncturing the finger & taking the sample. 2. Use sterile needles or lancets. Number of cells

The Neubauers Chamber

Questions 1-What is the Dilution fluid used for red blood cell count and what is the
benefit of using it? 2-What is the normal value of RBC in a healthy males and females? 3-What is the square used in the Neubauers Chamber for RBC count?

Experiment No: 2 Date: ____________ (G) Aim: To determine the total red blood cell count (visual method) Apparatus: 1. Haem
Observation: The following observations were made in the central area: Number of Big Square Number of cells 1 2 3 4 5 Total [
The Neubauer’s Chamber
Questions 1-What is the Dilution fluid used for red blood cell count and what is the benefit of using it? 2-What is the norm

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