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
· 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