This document describes methods for counting red blood cells and white blood cells using a hemocytometer. For red blood cell counting, blood is diluted and cells in 5 squares are counted under a microscope. The number of cells is then used to calculate the RBC concentration per microliter. For white blood cell counting, blood is similarly diluted and cells in 4 squares are counted under a microscope to calculate the WBC concentration per microliter. Normal ranges for RBCs and WBCs in human blood are also provided.

1. Total Erythrocyte Counting &
Total Leukocyte Counting
PHYSIOLOGY PRACTICAL

2. Microscope

3. Total Erythrocyte Counting

4. Total Erythrocyte Counting
• Aim: To enumerate the total number of red
blood cells (RBC) of a given blood sample.
• Methods:
– Photoelectric Counting Method
– Electronic Counting Method
– Hemocytometer (Neubauer) Counting Method

5. Total Erythrocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Hemocytometer chamber
– Cover slip
– Light microscope
– RBC pipette
– RBC diluting fluid (Haeyem’s solution or
Physiological saline 0.85% NaCl)

6. Total Erythrocyte Counting

7. Total Erythrocyte Counting

8. Total Erythrocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Blood should be carefully drawn to the 0.5 mark of
the RBC pipette.
– An isotonic solution (Normal saline or Hayem’s
solution) should be drawn to the 101 mark to dilute
the blood.
– The blood and diluting fluid are mixed by shaking
the pipette vigorously in a horizontal position for
2to 3 minutes (to ensure complete hemolysis of
WBC).

9. Total Erythrocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– 2 to 4 drops of mixed fluid are discarded and the
end of the pipette.
– The tip of the pipette is touched to the side of the
hemocytometer chamber and a drop of a fluid will
run under the cover glass.
– Wait for about 2 - 3 minutes as erythrocytes
require settling time to assume a single level.

10. Total Erythrocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Total number of cells in 5 squares in the center of
counting chamber is determined under the high
dry objective of the microscope (40X).

11. Total Erythrocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Calculation:
– RBC/μL or mm3= No. of cells in 5 squares (80 small
squares) x dilution no. x reciprocal of volume.
– Dilution No. = 0.5: 100 = 200
– Reciprocal of volume = 50
– RBC/μL = No. x 200 x 50

12. Total Erythrocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Normal RBC Range in
– Human -Male: 4.7 to 6.1 million cells per μL.
– Female: 4.2 to 5.4 million cells per μL.

13. Total Leukocyte Counting

14. Total Leukocyte Counting
• Aim: To enumerate the total number of
leukocyte (White Blood Cell) of a given blood
sample.
• Materials:
– Hemocytometer chamber.
– Cover slip.
– Light microscope.
– WBC pipette.
– WBC diluting fluid (1% HCl or 1% Glacic Asetic
Acid)[1ml GAA+1ml MB +100ml DW].

15. Total Leukocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Blood should be carefully drawn to the 0.5 mark of
the WBC pipette.
– WBC reagent (Glacial acetic acid 3%) should be
drawn to the 11 mark to dilute the blood.
– The blood and diluting fluid are mixed by shaking
the pipette vigorously in a horizontal position for 2
to 3 minutes (to ensure complete hemolysis of
RBC).

16. Total Leukocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– 2 to 4 drops of mixed fluid are discarded and the
end of the pipette.
– The tip of the pipette is touched to the side of the
hemocytometer chamber and a drop of a fluid will
run under the cover glass.
– Wait for about 2-3 minutes as leukocytes require
settling.

17. Total Leukocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Total number of cells in 4 squares at the corner of
counting chamber is determined under the low
objective of the microscope (10X).

18. Total Leukocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Calculation:
– WBC/μL or mm3= No. of cells in 4 squares (64 small
squares)/4 x dilution No. x reciprocal of chamber
depth.
– Dilution No. = 0.5: 10 = 20
– Depth of the chamber= 1/10 mm
– RBC/μL = No. /4 x 20 x 10

19. Total Leukocyte Counting
• Hemocytometer (Neubauer) Counting
Method:
– Normal WBC Range
– Normal number of WBCs in the blood is 4,500 to
11,000 WBC per microliter (4.5 to 11.0 × 109/L)