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structures while dead cells will stain blue (see Section 2.5.12). The number of dead

cells can therefore be excluded from the total cell count, ensuring that the seeding

density accurately reflects viable cells.

Calculating cell number

Cell number is usually expressed per cm^3 and is determined by multiplying the

average of the number of cells counted by a conversion factor which is constant for

the haemocytometer. The conversion factor is estimated at 1000, based on the fact that

each large square counted represents a total volume of 10^4 cm^3.

Thus:

cells cm^3 ¼

number of cells counted

number of squares counted

conversion factor

If the cells were diluted before counting then the dilution factor should also be taken

into account.

Therefore:

cells cm^3 ¼

number of cells counted

number of squares counted

conversion factordilution factor

To get the total number of cells harvested the number of cells determined per cm^3

should be multiplied by the original volume of fluid from which the cell sample was

removed,

i.e.:

total cells¼cells cm^3 total volume of cell suspension

Example 1CALCULATIONOFCELLNUMBER

Question Calculate the total number of cells suspended in a final volume of 5 ml, taking into
account that the cells were diluted 1 : 2 before counting and the number of cells
counted with the haemocytometer was 400.

Answer Cells cm^3 ¼

number of cells counted

large squares counted conversion factor

¼^400

4

 1000

¼100 000 cells cm^3

Because there is a dilution factor of 2, the correct number of cells cm^3 is given as:

100 000 2 ¼200 000 cells cm^3

Thus in a final volume of 5 cm^3 the total number of cells present is:

200 000 5 ¼1 000 000cells

56 Cell culture techniques