3.

Total RBC count

Objective
To determine the number of RBCs per cubic millimeter of blood.

Requirements
1. Microscope
2. Improved Neubauer’s chamber& cover slips
3. RBC pipette
4. Disposable lancet
5. Spirit& cotton
6. RBC diluting (Hayem’s) fluid
7. Watch glass

Constituents of RBC diluting fluid and their functions:

Principle
Since RBCs are millions in number, it becomes difficult to count all the cells. By diluting known
quantity of blood before counting and multiplying the result by the dilution factor, we can derive
the number of cells.

Procedure (steps)
1. Before starting the procedure, check the requirements for the experiment.
2. Clean the RBC pipette (block free), Neubauer’s chamber and cover slip and ensure that
these are clean and dry.
3. Pour Hayem’s fluid (adequate amount) in the watch glass.
4. Sterilize the tip of either middle or ring finger of your non dominant hand with spirit.
5. Allow the sterilized area to get air dried. Do not blow over the sterilized area.
6. After the sterilized area completely dries, prick the finger to a depth of ~3mm with a sterile
lancet.
7. Allow blood to flow freely. Do not squeeze your finger.
8. Discard the first drop of blood by wiping it with sterile dry cotton.
9. Let second drop of blood (medium size) appear on the finger. Dip tip of the RBC pipette
into it. Ensure that the tip of pipette is not touching the finger or held too high as if it is just
touching only the surface of blood drop.
10. Suck blood gently into the pipette up to 0.5 mark. Wipe sides of tip of the pipette taking
care not to touch its lumen. This is followed by sucking Hayem’s fluid upto 101 mark
taking care that no air bubble enters the pipette.
11. Gently mix the blood and diluting fluid by rotating pipette between the palms in horizontal
plane.
(Note: mixing occurs only in the bulb portion of the RBC pipette).
12. After mixing the contents, discard fluid in the stem portion (3 drops). Place cover slip over
the Neubauer’s chamber so that it covers the ruled area in the central platform on both
sides.
13. Keeping pipette at 30°- 45°, charge Neubauer’s chamber between cover slip and the central
platform, so that fluid spreads uniformly without any air bubble. Fluid should not flow into
the groove / gutter or over the cover slip.
14. Place the charged chamber on stage of the microscope and wait for RBC’s to settle down.
15. Switch on the microscope; ensure that rays of light pass through the central platform of
Neubauer’s chamber.
16. Adjust microscope for observing under low power. Iris diaphragm should be partially open,
condenser at lowest position and intensity of light at minimum.
17. Focus RBC counting area in Neubauer’s chamber under low power objective. (RBC
counting area is identified by triple lined border in the center of counting area. There are 25
medium sized RBC squares each of which are further divided into 16 small squares).
18. Now change the objective to high power (40X) and focus the RBCs by making necessary
microscopic adjustments (iris diaphragm partially open, condenser at mid position and intensity
of light at medium level). The RBCs are seen as dark greenish - black disc.
19. Count the cells in all 4 corner and center medium sized RBC squares by the rule of `L’ i.e.
count the cells present on the left and lower border of the RBC squares. This is done to
avoid recounting of cells that are in two squares.
Precautions
1. The pipette, Neubauer’s chamber and cover slip should be clean and dry.
2. Pricking should be 3mm deep to ensure free flow of blood.
3. Do not squeeze your finger as this may dilute your blood with tissue fluid giving a false
low count.
4. Blood in the stem portion of the pipette should be immediately diluted with Hayem’s fluid
to avoid clotting.
5. Never touch tip of the pipette for wiping the extra blood as this may withdraw contents of
the pipette giving a false low count.
6. If you have sucked blood slightly more than 0.5 mark touch tip of the pipette on any dry
area of your palm till the blood comes down to 0.5 units. To avoid this always suck from a
medium sized blood drop.
7. Avoid sucking air bubbles into the pipette. This can be avoided by dipping tip of the pipette
deep into the blood drop.
8. Discard 3 drops of fluid before charging the Neubauer’s chamber as the stem of the pipette
contains only diluting fluid.
9. Do not over charge or under charge the Neubauer’s chamber.
10. Do microscopic adjustments properly to focus the cells under low power or high power.
Observation

Calculation
Side of 1 medium sized RBC square = 1/5mm
Depth of the counting chamber = 1/10mm
Volume of fluid in 1 medium sized RBC square = 1/5 x 1/5 x 1/10mm3= 1/250 mm3
Volume of fluid in 5 medium sized RBC squares=5 x 1/250 mm3 = 1 /50 mm3
Let the number of RBC counted in 1/50mm3 of diluted blood be ‘N’
Therefore 1mm3 of diluted blood contains N x 50 cells
Dilution is 1 in 200
Therefore 1mm3 of undiluted blood contains N x 50x200 cells or Nx10,000 cells
Here N=
Therefore, the total number of RBCs in 1mm3 of undiluted blood = _________ cells

Result
The total number of RBC present in my blood is __________ cells/mm3

Inference:
Questions
1. What is the function and composition of each component of Hayem’s fluid?
2. Justify the dilution factor used in this experiment.
3. Give the normal values of RBC’s in adult males, adult females, newborns and children.
4. List the functions of RBCs.
5. Name physiological conditions where RBC count is increased.
6. Name pathological conditions where RBC count is increased.
7. Name physiological conditions where RBC count is decreased.
8. Name pathological conditions where RBC count is decreased.