1 Human Physiology-I (PHSL 205) Lab
2nd Lab
Hakami,Hana A- 2010/2011
White blood cell count
Prepared by;
Hakami, Hana A
Viewed by;
Dr.Naseem Siddiqui
2 Human Physiology-I (PHSL 205) Lab
2nd Lab
Hakami,Hana A- 2010/2011
White blood cell
Blood contents are cells and plasma . Blood cells Include 3 main
types are ; thrombocytes , erythrocytes and leukocytes .
Blood plasma is the yellow liquid component of blood, in which
the blood cells in whole blood would normally be suspended. It makes
up about 55% of the total blood volume. It is the intravascular fluid part
of extracellular fluid. It is mostly water (90% by volume) and contains
dissolved proteins, glucose, clotting factors, mineral ions, hormones
and carbon dioxide (plasma being the main medium for excretory
product transportation).
Thrombocytes are cells that play a key role in blood clotting. In
mammals, thrombocytes are anucleated cell fragments called platelets.
The erythrocytes are the most numerous blood cells. They are
also called red blood cells. In man and in all mammals, erythrocytes are
devoid of a nucleus and have the shape of a biconcave lens. In the other
vertebrates (e.g. fishes, amphibians, reptilians and birds), they have a
nucleus.
White blood cells (WBCs), or leukocytes (also spelled
"leucocytes"), are cells of the immune system defending the body
against both infectious disease and foreign materials. there are different
and diverse types of leukocytes exist, but they are all produced and
derived from a multipotent cell in the bone marrow known as a
hematopoietic stem cell. Leukocytes are found throughout the body,
including the blood and lymphatic system.
3 Human Physiology-I (PHSL 205) Lab
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Hakami,Hana A- 2010/2011
WBC (Leukocyte) Manual Counting
Principle
Blood sample is mixed and diluted with weak concentration of
hydrochloric acid (HCl), or acetic acid (in specified known volumes).
Weak acids will lyse red blood cells, and will darken WBC’s to facilitate
counting by the hemacytometer.
Manual WBC counting is used in cases of very low WBC count
(leukopenia) with automated hematology cell counters, and when
automated cell counters are not available.
Sample
EDTA anticoagulated whole venous blood.
Reagent and Supplies To Prepare Diluting Fluid
1- Volumetric Flask 100 cc.
2- Serological pipettes.
3- Concentrated HCL
4- Glacial Acetic Acid
Preparation of Diluting Fluid
Diluting fluid is either:

1% hydrochloric acid in distilled water ( 1 ml Conc. HCL + 99 ml
Dist. water).

2% Acetic Acid in distilled water ( Turk’s solution) (2 ml glacial
acetic acid
+ 98 ml distilled water).
Glassware, Apparatus, Equipment
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Hakami,Hana A- 2010/2011
1- Neubauer improved hemacytometer.
2- Clean cover slip slide (especially made for the hemacytometer).
3- Automatic micropipette (20
l, 380 l are the required
volumes).
4- Gauze 10 x 10 cm
5- Glass/Plastic tubes- (12x75 mm).
6- Handy tally counter.
7- Conventional light microscope.
Procedure
1- Mix the blood sample gently but thoroughly by inversion,
manually or by mechanical rocking mixer.
2- Pipette 0.38 ml (380 l) of diluting fluid into a 12x75 mm tube.
3- Pipette 0.02 ml (20 l) of well mixed blood to be counted
and wipe the tip with gauze into the tube containing diluting
fluid and mix the tube.
4- Let the tube stand for 2-3 minutes to ensure complete RBC
lyses, then mix well.
5- Prepare the clean hemacytometer and cover it with the
designed coverslip.
6- Load one side of the hemacytometer with the aid of
a
capillary tube or micropipette, do not attempt to overload or
underload the hemacytometer.
7- Allow the hemacytometer to sit for several minutes to allow
the WBC’s to settle in the counting chamber, to avoid drying
effect, place the loaded hemacytometer in a covered Petri dish
with a moist gauze, until counting.
8- Place the hemacytometer in the microscope stage.
5 Human Physiology-I (PHSL 205) Lab
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Hakami,Hana A- 2010/2011
9- Focus with x10 objective lens (low power), with lowering the
condenser.
10-The WBC’s are counted in the 9 corner large squares, with the aid
of hand tally counter.
11-Follow the counting pattern shown in the figure below. During
counting, do not count cells that touch the right or bottom
boundaries to ensure unduplicated counting.
Begin
12- The total counted WBC’s in the 9 squares are added together.
Fig. WBC’s are counted in the 9 hemacytometer squares

If the number of cells in a square varies from any other square by
more than 9 cells, the count must be repeated, because this
represents an uneven distribution of cells, which is may be caused
6 Human Physiology-I (PHSL 205) Lab
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by
improper
Hakami,Hana A- 2010/2011
mixing of the dilution or improperly filled
hemacytometer.
Calculations
Total WBC count =
N x D (mm) x DF
A (mm2 )
= c/mm3
Where:
N = Total WBC counted by the counting chamber.
Depth factor in mm = 10
DF = Dilution Factor = 20
A (mm²) = Area counted = 3 mm x 3 mm = 9 mm²
So,
Total WBC count =
N x 10 mm x 20
9 mm2
= c/mm3
Example
20
19
18
21
14
16
19
16
15
Hemacytometer
Squares
N= 20 +19 +18 +21 +14 +16 +19 +16 +15 = Tallied 158 Counted WBC Cell
Total WBC count =
158 x 10 x 20
9 mm2
= 3500 c/mm3 = 3.5 x 109 /L