WBC COUNT
• Aim: To determine total White blood cells (Leukocytes) count of
blood sample using haemocytometer. White blood cells
(Leukocytes) are nucleated cells that are involved in the defense
mechanism of the body.
Leukocytes are classified as (1) Granulocytes: a) Neurotrophils b) Eosinophils
c) Basophils
(2) Agranulocytes: a) Lymphocytes b) Monocytes.
• Formation of White blood cells (WBC) is known as leukopoiesis. The
number of circulating leukocytes is very precisely controlled. There
is no specific feedback control system like RBCs. The increase in
total leukocyte count of more than 11000/ [Link] (ul) is known as
leukocytosis and decrease or less than 4000 /[Link] (ul) as
leucopenia. The normal ratio of WBC to RBC is 1:700.
• Principle: The blood is diluted with glacial acetic acid solution which
• Requirements:
1. Compound Microscope 2. Naubaur's counting chamber (Haemocytometer)
3. WBC diluting pipette 4. Sterile disposable pricking needle
5. Watchglass 6. Coverslip
7. Cotton swabs 8. 70% alcohol or spirit
9. Turk's fluid(WBC diluting fluid)
• The composition of Turk's fluid (WBC diluting fluid):
It consists of -
• 1. Glacial acetic acid-2 ml (for hemolysis)
• 2. Methylene - 1 ml (1% w/v)
• 3. Distilled water - 97 ml (as solvent)
• Procedure:
1. Clean and dry the Neubaur's chamber, pipette, watchglass and coverslip.
2. Take enough WBC diluting fluid in a watchglass.
3. Sterilize the tip of finger with 70% alcohol or spirit. Let it dry.
4. Make a bold finger prick and allow free flow of blood to get big drop of blood at
the finger tip, keeping the finger in an upright position.
5. Suck the blood into the pipette upto the mark 0.5.
6. Wipe off the excess blood outside the pipette by using cotton.
7. Then suck the diluting fluid in the pipette up to mark 11.
8. Hold the pipette horizontally and then mix the contents of the bulb between the
palms of the hands by rolling for two minutes.
9. Keep the pipette in a horizontal position to prevent any loss of its contents until
the cell counts performed.
12. Charge the Neubauer's counting chamber by allowing a drop of diluted blood from the tip of the pipette
held at an angle of 45°, placed just at the edge of the coverslip.
13. Allow the cells to settle for about 2 to 3 minutes.
14. Placed the charged counting chamber on the stage of microscope and adjust the microscope for
observation under low power (10 X objective).
15. Adjust the Neubauer's chamber under the low power objective and check for uniform distribution of the
cells in the WBC squares. If cells are non-uniformly distributed then clean and recharge the chamber.
16. Count the total white blood cells in the four corners.
17. The white blood cells are identified as round, nucleated, refractile dots.
OBSERVED
• Observations: Countthe white blood cells in four corner
squares i.e. upper left, upper right, lower left and
lower right. Avoid the counting of the same cells
again. Count the white blood cells present in the
square and those touch-any of the upper and left
lines, do not count any cell that touches a lower or
right line (Counting rule 1). The counting should be
done in zig-zag manner (Counting rule 2)
• OBSERVERD WBC COUNT:-
• Dilution of WBCS:
Dilution factor is 1: 20. The volume of bulb is 10 (11-1 = 10).
The first part of the pipette (from the tip of the pipette to mark 1) contains only diluting fluid. Mixing of blood with diluting fluid i.e. dilution
occurs only in the bulb. Thus, 10 volume of diluted blood (only in the bulb) contains 0.5 volume of blood and 9.5 volume of diluting fluid.
Therefore, dilution is of 0.5 in 10. So, the dilution obtained is 1: 20 or 20 times (0.5:10 means 1: 20).
• Calculations:
The WBC counting is done in four corner squares.
The area of the smallest WBC square = 1 x 1 mm³ = 1 mm³
The area of four WBC squares = 4 x 1 = 4 mm³
Volume of four WBC squares =The area of the four WBC squares × depth
= 4 × 1/10
= 4/10 mm³
Dilution factor = 1/20 .
The number of WBC in 4/10 mm³ volume of diluted blood (Total no. of WBCS counted in 4 WBC squares)= n
Therefore, number of WBCs in 1mm³ volume of diluted blood =n:-10/4
Therefore, N=n1+n2+n3+n4
=35+40+30+35
=140
Since, the blood has been diluted 20 times, total number of WBCs in 1mm³ of blood = 140 x 10/4 × 20
= 140 × 50
• Precautions:
1. Neubauer's chamber, pipette, coverslip should
be clean and dried.
2. Prick should be deep enough to allow free flow of
blood.
3. Discard first drop of blood.
4. Avoid sucking of air bubble in the pipette.
5. Blood should be sucked upto the mark of 0.5 and
diluted exactly upto the mark of 11.
6. Wipe off the extra blood from outside the
pipette.
7. Mix thoroughly the contents of the pipette by
holding horizontally between the palms and
rotating it gently to ensure uniform mixing.
8. If the chamber is overcharged or undercharged,
it is discarded, cleaned and recharged.
9. If the distribution of cells is non-uniform, discard
the chamber and recharge.
10. Do not allow the air bubble to enter the
• Clinical Significance: The estimation of total WBCs count is essential in
haematological testing, since the white blood cells are a part of the
body's defence system. They give protection against infection. The
white blood cells are the mobile defenses of the body. . If the
distribution of cells is non-uniform, discard the chamber and recharge.
The total white blood cells count is used to evaluate severity of
infection, spread of disease and body's defence capability. The
abnormal conditions of total WBC count are leukopenia and
leukocytosis. The leucopenia generally occurs in inflammatory
conditions and infections. Acute Leukemia is a malignant disease
characterized by uncontrolled production and accumulation of
immature leukocytes. In chronic leukemia, mature leukocytes
accumulate in the blood stream because they do not die at the end of
their normal life span.
• Normal value:- Leukocyte count is 5000-11000 cells/[Link].
• Conclusion:- The total WBC count is NORMAL
• Results:- The total WBC count is 7900 cells/[Link]