HEMOCYTOMETER

BY DR UMEMA ZAFAR
HEMOCYTOMETER
 The equipment used for counting different cells of
blood is called haemocytometer.
INTRODUCTION
 Blood is composed of liquid plasma in which
formed elements are suspended.
 A blood count gives the number of blood cells in a
given sample of blood.
 The RBCs, WBCs and platelets can be counted in a
similar manner by using a specially designed
counting chamber of uniform dimensions. This is
called hemocytometer. The most frequently used
hemocytometer is Neubauer’s Chamber
HISTORY
HEMOCYTOMETER is a device invented by the
19th century French anatomist Louis-Charles
Malassez to perform blood cell counts.
REQUIREMENTS
 Microscope
 Hemocytometer
 RBC pipette
 WBC pipette
CONSTRUCTION OF COUNTING
CHAMBER
 A hemocytometer consists of a thick glass microscope
slide with a grid of perpendicular lines etched in the
middle.
 The grid has specified dimensions so that the area
covered by the lines is known, which makes it possible to
count the number of cells in a specific volume of
solution.
 The most common type of hemocytometer has an “H”
shape engraved in the middle that encloses two
separate mirror-like polished grid surfaces and
provides the cover slip mounting area and are the
counting areas.
CONSTRUCTION OF COUNTING
CHAMBER
 Each half has a counting chamber which consist of a
square, the sides of which are 3 mm each. This
square is divided into 9 squares each is 1mm2.
 Out of these nine squares corner squares are used for
WBC counting while the central square is used for
RBC counting.
3 mm
LOADING THE HEMOCYTOMETER
 Before starting ensure that both the hemocytometer and its
coverslip are clean by removing any dust particles with lens
paper.
 Coverslips that are used for mounting on hemocytometers
are specially made to be thicker than the conventional
microscopy coverslips because they must be able to
overcome the surface tension of a drop of liquid.
 Make sure to first place the coverslip over the counting
surface before loading the cell suspension. Then place the
pipette tip with your sample into one of the V-shaped wells,
and gently expel the sample.
LOADING THE HEMOCYTOMETER
 The area under the coverslip fills by capillary action.
 Enough liquid should be introduced so that the mirrored
surface is just covered, usually around 10 µl, but do not
overfill the surface.
 You can load two samples on one hemocytometer, one into
each of the two grids.
 The loaded hemocytometer is then placed on the microscope
stage and the counting grid is brought into focus at low
power.
 Allow the sample to settle for a couple of minutes and avoid
moving the coverslip as it might introduce air bubbles and
make counting difficult.
 3mm

3mm
WBC SQUARES
 Four corner squares are further subdivided into 16
smaller squares. The area of each smaller square is
¼*1/4= 1/16 mm2
 So total number of small squares used for WBC
counting are 16*4= 64
 The height of central platform is 0.1 or 1/10 mm
less than rest of the slide.
 So volume of WBC square is 1/16 * 1/10 =
1/160 mm3
 1 mm

¼
mm

1mm
RBC SQUARES
 The central 1mm2 is divided into 25 small squares, each
measuring1/5*1/5 = 1/25 mm2 and every small
square is further subdivided into 16 smallest squares.
 The 1/25mm2 squares are bounded by double lines. So
total no. of squares in central 1mm2 area in 25*16
=400 squares.the area of each of these smallest
squares is thus 1/400 mm2.
 The height of central platform is 0.1 or 1/10 mm less
than rest of the slide.
 So volume of RBC square is 1/400*1/10 =
1/4000mm3
1 mm 1/20mm

1/5 mm
PIPETTES
 They are two in number:
1. WBC pipette
2. RBC pipette
CONSTITUENTS OF PIPETTES

 Both the pipettes have:

1. Long narrow stem
2. Mixing chamber
3. Rubber tubing
DIFFERENCE BETWEEN RBC AND WBC
PIPETTES
 RBC pipette has 0.5 and 1 marking below the chamber
and 101 above it
 It has a red coloured bead in mixing chamber which
helps in mixing.
 WBC pipette has 0.5 and 1marking below the chamber
and 11 above it.
 It has a white coloured bead in the mixing chamber
 The volume of bulb is 10 times more than the volume of
stem for WBC pipette and is 100 times more for RBC
pipette.
 RBC PIPETTE

WBC PIPETTE