HEMOCYTOMETER

HEMOCYTOMETER
Hemo: blood
Cyto: cell
Meter: measurement
Thus, it is an instrument used to count the
blood cells.

It includes:
a) Neubauers
slide
b) Diluting
pipette
c) Cover glass

Hemocytometer
Chamber

The four corner squares are further

divided into sixteen smaller squares and
are used for WBC counting.

Four corner squares

are meant for WBC
counting.
Total = 64 small
squares

 Central square is divided into 25 medium

sized square and are separated by triple
line
The medium sized square are further
divided into 16 small square(tiny)
The four corner and central square are used
for platelet and RBC count.

Counting Rule
Do not count cells
touching

Bottom line
Right line
This is to avoid
double counting.

Thomas pipette
Consists of graduated capillary tube, mixing bulb
with glass bead and aspirating tube
Parts: stem, bulb, rubber tube
Thomas pipette are: WBC pipette and RBC
pipette

DIFFERENCES BETWEEN RBC AND

WBC PIPETTE
RBC
pipette

WBC
pipette

1)

It has a red
bead

It has a white
bead

2)

It has
graduations
upto mark 101

It has
graduations
upto mark 11

3)

Size of bulb is
larger

Size of bulb is
smaller

RBC PIPETTE

WBC PIPETTE

For WBC counting

0.5 part of blood is mixed in 10 parts of
fluid
So, 1 part of blood is in 20 parts of fluid
Thus, dilution factor for WBC counting is

20.

FOCUSING
4X to see the general
formation of slide.
10X for WBC counting
40X for RBC/Plt. counting

Calculation
Cell count=N x dilution factor x depth
factor/area
counted

Total leukocyte count

White cell count (WBC)

White cell count (WBC) is the total number of leukocytes
in a volume of blood, expressed as thousands/l.
WBC can be done by manual methods or by automated
cell counters.
Normal Values:

Newborn 9.0-30.0 x 103/l

1 week
5.0-21.0 x 103/l
1 month 5.0-19.5 x 103/l
6-12 months 6.0-17.5 x 103/l
2 years 6.2-17.0 x 103/l
Child/adult
4.8-10.8 x 103/l

Principle of WBCs count test

Free-flowing capillary or well-mixed anticoagulated
venous blood is added to a diluent) at a specific volume in
the thoma pipette.
The diluent lyses the erythrocytes but preserves
leukocytes and stains the nuceli.
The diluted blood is added to the hemacytometer
chamber.

Specimen:

EDTA- anticoagulated blood

Reagents, supplies and equipment:

White blood cells count diluting fluid

Turks' solution which is formed of:
Glacial acetic acid 3 ml
Crystal violet 1 ml
100 ml distilled water.

Equipment
1.

White blood cells count diluting

fluid
2. Thoma white pipette
3. Hemocytometer and cover slip
4. Microscope

haemocytometer chamber

Thoma white pipette

Rubber sucking tube

Hemocytometer

Procedure
1.
2.
3.

4.

Draw the blood up to 0.5 mark in the thoma pipette.

Wipe the outside of the capillary pipette to remove
excess blood that would interfere with the dilution
factor.
Holding the pipette almost vertical place into the fluid.
Draw the diluting fluid into the pipette slowly until the
mixture reaches the 11 mark, while gently rotating the
pipette to ensure a proper amount of mixing.
Place the pipette in a horizontal position and firmly
hold the index finger of either hand over the opening in
the tip of the pipette, detach the aspirator from the
other end of the pipette now the dilution of the blood is
completed

5.
6.
7.

Mix the sample for at least 3 minutes to facilitate

hemolysis of RBCs.
Clean the hemocytometer and its cover slip with an
alcohol pad and then dry with a wipe.
Before filling the chamber, discard the first four to
five drops of the mixture on apiece of gauze to expel
the diluent from the stem.

8. Carefully charge hemocytometer with diluted

blood by gently squeezing sides of reservoir to
expel contents until chamber is properly filled.

Procedure for counting WBCs

1.
2.

3.

Under 10 x magnifications, scan to ensure even

distribution. Leukocytes are counted in all 4 large
squares of counting chamber.
Count cells starting in the upper left large corner
square. Move to the upper right corner square,
bottom right corner square, bottom left corner square
and end in the middle square.
Count all cells that touch any of the upper and left
lines, do not count any cell that touches a lower or
right line.

Calculations
Dilution factor 20
Volume= Area x depth
TLC/uL= No of WBCx Correction for Volume X
dilution
No of large squares (4)
= N x 20 x10
4
= N x 50