1.
20μl of media containing cells was taken in an
eppendorf to which 20μl of trypan blue was added and mixed well
2.
The cover slip was placed on the haemocytometer
and 10μl of this solution was loaded to one side of the clean haemocytometer
and 10μl more was loaded on to the other side and allowed it to fill by
capillary action.
3.
4×4 = 16 blocks on one corner was found and
cells were counted with 10X objective. Cells were not counted if they touch the
right hand or bottom borders of the square.
4.
The cell number obtained was calculated in a ml
a.
Average of the four squares was calculated
b.
This was multiplied by 104
Extra Information:
·
Haemocytometer was cleaned with ethanol before
use.
·
Dead cells take up trypan blue but not viable
cells.
·
Each large square measure 1mm x 1mm, 0.1mm deep.
·
Each square represents a volume of 0.1mm3 (1.0mm2area
x 0.1mm deep) or 10-4cm3
·
Cell number is usually expressed in cm3
and is determined by multiplying the average of the no. of cells counted by a
conversion factor which is constant for the haemocytometer. The conversion
factor is estimated at 1000, based on the fact each large square represents a
total volume of 10-4cm3
Total cells =(no.of cells counted)/(no.of large squares counted) x conversion factor x dilution factor x
total volume of suspension
