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contain 16 small squares each; each large square measures 1 mm1 mm and is

0.1 mm deep (see Fig. 2.6).

Thus, with a coverslip in place, each square represents a volume of 0.1 mm^3

(1.0 mm^2 area0.1 mm depth) or 10^4 cm^3. Knowing this, the cell concentration

(and the total number of cells) can therefore be determined and expressed per cubic

centimetre. The general procedure involves loading approximately 10ml of a cell

suspension into a clean haemocytometer chamber and counting the cells within the

four corner squares with the aid of a microscope set at 20magnification. The count

is mathematically converted to the number of cells per cm^3 of suspension.

To ensure accuracy, the coverslip must be firmly in place and this can be achieved

by moistening a coverslip with exhaled breath and gently sliding it over the haemo-

cytometer chamber, pressing firmly untilNewton’s refraction rings(usually rainbow-

like) appear under the coverslip. The total number of cells in each of the four 1-mm^3

corner squares should be counted, with the proviso that only cells touching the top

or left borders but not those touching the bottom and right borders are counted.

Moreover, cells outside the large squares, even if they are within the field of view,

should not be counted. When present, clumps should be counted as one cell. Ideally

100 cells should be counted to ensure a high degree of accuracy in counting. If the

total cell count is less than 100 or if more than 10% of the cells counted appear to

be clustered, then the original cell suspension should be thoroughly mixed and the

counting procedure repeated. Similarly, if the total cell count is greater than 400, the

suspension should be diluted further to get counts of between 100 and 400 cells.

Since some cells may not survive the trypsinisation procedure it is usually advisable

to add an equal volume of the dye trypan blue to a small aliquot of the cell suspension

before counting. This dye is excluded by viable cells but taken up by dead cells. Thus,

when viewed under the microscope, viable cells will appear as bright translucent

1 mm 1 mm 1 mm

1 mm

1 mm

1 mm

0.25 mm

Fig. 2.6Haemocytometer.

55 2.5 Types of animal cell, characteristics and maintenance in culture