HAEMOCYTOMETRY

APPARATUS: Haemocytometer
Pipettes
Neubauer’s Chamber

METHOD
Pipetting
charging
 PRINCIPLE
A measured unit of blood is diluted quantitatively with
diluents by using pipettes

RBC PIPETTE
counting of RBC
stem, bulb, rubber tube, mouthpiece
red bead
Markings: 0.5 and 1
Above the bulb: 101
Mouthpiece is red
 WBC PIPETTE
for counting WBC
marking above is 11
white bead
mouthpiece is white

FUNCTION OF THE BEAD

Identify
mixing
 IMPROVED NEUBAUER’S CHAMBER

2 Central platform – H shaped groove

Depth under the coverslip – 1/10 mm
Square is 3mm x 3mm
Divided into 9 large squares, each has area of 1mm 2
4 large corner squares used for WBC count
Large centre square used for RBC count
Each WBC square is divided into 16 smaller squares
area is 1/16 mm2

RBC square is divided into 25 medium sized squares by

means of triple lines
area of medium sized square is 1/25mm 2
4 corner and central medium sized square is used for
RBC count
area of 5 medium sized square is 1/25 x 5= 1/5mm2
volume of five medium sized square is 1/5 x 1/10mm3
each medium sized square is divided into 16 small squares.
5 x 16 = 80 small squares

area of smallest RBC square 1/20 x 1/20 = 1/400mm 2

depth 1/10
volume of smallest RBC square is 1/400 x 1/10 = 1/4000mm 3

volume of 80 small sized RBC squares is 1/4000 x 80 = 1/50

mm3
 RBC COUNT:
Area of big sized square = 1 x 1 = 1mm2

Area of each medium sized square =1/5 x 1/5 = 1/25mm 2

Area of each small square =1/25 x 1/16 = 1/400mm 2

=0.0025mm2

Depth of the chamber = 1/10 mm

Volume of each small square =1/400 x 1/10 = 1/4000mm 3

Volume of fluid in 80 small square = 1/50mm3

(1/4000x80).
WBC Count :
Area of each big square = 1mm2

Area of each small square = 1/16 mm2

Depth of chamber = 1/10 mm

Volume of fluid in 1 WBC square = 1/10 mm3

Volume of fluid in 4 WBC squares = 4/10 mm3

Volume of fluid in each small square

= 1/16 x 1/10 = 1/160 mm3
PIPETTING
CHARGING THE CHAMBER