Practical Physiology

3rd Lab.

Differential White
Blood Cell Count
(Differential WBC count)

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 Lab. no. 3: Differentail WBC Count
Preparation of a Blood film and staining it with
Leishman’s stain:
4.Clean thoroughly three microscopic slides, two to be
covered with the blood film and one to be used as a
spreader.
5.Clean the finger with spirit, allow it to dry and then
prick (puncture) it with a sterile lancet to obtain a
drop of blood.
6.Touch one end of the two slides to the drop of the
blood, only a small quantity is required. Lay the slide
on a flat surface.
7.Place the edge of the spreader on the surface of the
slide just in front of the drop of blood, and at an
angle of 45º. dlerqader74@yahoo.com
 Lab. no. 3: Differentail WBC Count
2.Draw the spreader back until it makes contact with
the drop. The blood will now run along the full width
of the spreader at the line of junction.
3.When this has happened, push the spreader slowly
and smoothly to the other end of the slide.
-The faster the spreader is moved, the thicker the film;
a properly made film should be only one cell thick
throughout.
• Allow the film to dry in air. You can fasten this by
waving it. (Repeat with the second slide).
• Carefully drop Leishman’s stain∗ on to the blood film
until the film is covered. Allow the stain to act for
one minute. *Leishman’s stain: is a mixture of
methylene blue and eosin in methyl alcohol.
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 Lab. no. 3: Differentail WBC Count
3.Then add as much distilled water to the stain as will
stay on the slide without spilling over.
-This gives a dilution of (1:1 or 1:2). With a teat pipette
suck the diluted stain up and down and mix it
thoroughly. If the dilution of the stain is correct, the
fluid will be covered by a thin greenish scum.
5.The diluted stain should be allowed to act for 7-10
min., then wash it off with distilled water. Make sure
that none of the greenish scum settles on the
surface of the blood film.
6.Continue washing with D.W to differentiate until the
film has a salmon pink color.
-Shake off excess water and allow it to dry in air
without heat. dlerqader74@yahoo.com
Lab. no. 3: Differentail WBC Count

spreader

A drop of Blood 1
2 45º(

-Blood film preparation; arrows indicate the sequence

of movements of the spreader.

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 Lab. no. 3: Differentail WBC Count
Examination of the Blood smear:
Fix the stained slide on the mechanical stage of the
microscope. Examine the blood film first by the low
power objective (X10), then by the high power
objective (X40). Before getting the oil immersion
objective into position add on to the blood film a
drop of cedar wood oil. Dip the oil immersion
objective into the drop of oil and focus using only
the fine adjustment.
Numerous Red Blood cells are seen as circular discs
stained deep pink at the periphery, and light pink in
the center (due to the biconcavity).

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 Lab. no. 3: Differentail WBC Count
In iron deficiency anaemia the central light pink area is
increased in size. Normal RBCs have about 7
microns diameter and hence are used by
comparison to judge the size of the white cells.
Scattered, in groups, here and there are platelets
seen as small purplish─pink bodies (2─4 microns)
in size. Use the mechanical stage of the microscope
to traverse the full length of the film. Move the slide
along its width by (2mm) and again traverse the full
length of the film in the reverse direction. Never stop
a count in the middle of the length of the scan,
otherwise the result will not be accurate.

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 Lab. no. 3: Differentail WBC Count
Identify each white cell in a series of successive
microscopic fields; count a sample of (100) white cells
and determine the number (percentage) of each cell
type.
- A table shows the normal ranges, mean values, and normal
size of each WBC type.

Types of WBCs Normal range Mean Value Normal size

Neutrophils 35-85 % 60 % 10 –14 μ

Lymphocytes 20-50 % 30 % 6 –12 μ

Monocytes 2-6 % 4% 12 –18 μ

Eosinophils 0-4 % 2 % 10 –14 μ

Basophils 0-1 % 0.5 % 10 –14 μ

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Lab. no. 3: Differentail WBC Count

Granular
Leukocytes

Neutrophil Eosinophil Basophil

Agranular
Leukocytes
Small Large Monocyte
Lymphocyte

-A figure showing different types of WBCs.

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 Lab. no. 3: Differentail WBC Count

-A table shows how to determine the number of WBC types (%).

WBCs Neutro. Eosno. Baso. Lympho. Mono.

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№
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Percent %

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