Practical Report: Manual Red Blood Cell (RBC) Count
Title:
Manual Counting of Red Blood Cells (RBC) using Hemocytometer
Aim:
To determine the number of red blood cells per cubic millimeter of blood by using a hemocytometer.
Principle:
Red blood cells are counted manually using a Neubauer’s counting chamber (hemocytometer) after
diluting the blood with an appropriate diluting fluid. The dilution ensures cells are uniformly distributed and
prevents rouleaux formation. By counting RBCs in a defined area and applying the dilution factor, the total
RBC count per cubic millimeter of blood is calculated.
Normal Values: - Males: 4.5 – 6.0 million/mm³ - Females: 4.0 – 5.5 million/mm³
Apparatus & Materials:
• Hemocytometer (Neubauer’s counting chamber)
• RBC pipette (red bead)
• RBC diluting fluid (Hayem’s fluid / Gower’s fluid / Dacie’s fluid)
• Clean glass slides and coverslip
• Lancet or needle for finger prick
• Spirit swabs / cotton
• Microscope
Procedure:
1. Preparation of Finger: Clean fingertip with spirit swab and allow to dry.
2. Prick: Prick the finger with a sterile lancet and discard the first drop of blood.
3. Filling Pipette: Draw blood up to the 0.5 mark in the RBC pipette.
4. Dilution: Immediately draw diluting fluid up to the 101 mark. Mix thoroughly by rotating the
pipette.
5. Dilution achieved = 1:200.
6. Charging Chamber: Discard first few drops. Place a coverslip on the counting chamber and charge it
by placing a small drop of diluted blood at the edge of the coverslip.
7. Settling: Allow cells to settle for 2 minutes.
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8. Counting: Focus under microscope (high power). Count RBCs in 5 small squares (four corners and
center) of the large central square of the Neubauer chamber.
Observation and/or Results:
• Suppose the total number of RBCs counted in 5 small squares = N.
Calculation: - Area counted = 5/25 = 1/5 sq mm - Depth = 0.1 mm - Volume = 1/5 × 0.1 = 1/50 mm³ - Dilution
= 1:200
Therefore,
RBC count = N × Dilution f actor × (1/V olume)
RBC count = N × 200 × 50 = N × 10, 000
Result: - RBC count = (N × 10,000) / mm³ of blood.
Precautions:
1. Avoid air bubbles while charging the chamber.
2. Ensure the chamber and coverslip are clean and grease-free.
3. Do not overfill or underfill the chamber.
4. Count only those cells inside the designated boundaries (include cells touching top and left borders,
exclude those on bottom and right borders).
5. Mix the pipette properly to avoid uneven distribution of cells.
Clinical Significance:
• Increased RBC count (Polycythemia): Seen in polycythemia vera, high altitude, dehydration.
• Decreased RBC count (Anemia): Seen in blood loss, nutritional deficiencies (iron, folate, B12), bone
marrow suppression, chronic diseases.
RBC count is essential for evaluating anemia, polycythemia, and overall hematological health.
Other Methods:
• Automated Cell Counters: Modern electronic hematology analyzers provide more accurate results
with multiple parameters.
• Spectrophotometric methods (rarely used).
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Conclusion:
The manual RBC count using a hemocytometer provides a reliable estimation of red blood cells in blood.
Although automated methods are now common, manual counting remains a fundamental skill in
physiology practicals and helps in understanding basic hematological principles.