In order to provide a standard environment for experimental studies, tissue culture techniques require a consistent number of cells to be cultured. Too few cells may result in insufficient material, while too many cells may challenge the cells in such a away to affect the outcome of the experiment.

Many laboratories use automated cell counters like Coulter counters or flow cytometers when processing large numbers of samples. However, when checking the numbers of cells for serial dilutions or setting up cultures, it is sometimes easier to return to traditional methods of cell counting.

The haemocytometer is a modified and calibrated microscope slide designed to allow operators to quickly estimate the concentration of cells in a sample. The cells present in a known volume are counted and then this value converted to a number per mL. The name refers to its original use in counting blood cells – blood was diluted to a point where the cells could be reliably counted and this was factored up based on the dilution.

View of a haemocytometer

The cell suspension is introduced to a space of known depth (0.1mm) beneath a coverslip and counted within a grid of area 1mm2 (see over). This gives the number of cells per 0.1mm3 (or 0.1µL). Multiplying by 10 gives the number of cells per µL and multiplying by 10,000 gives the number of cells per mL.

Close up of the grid on a haemocytometer

Close up view of a grid with cells

In the above example, there are 24 cells within the grid (do not count any cells outside the grid).

24 cells per 0.1 µL

=  240 cells per µL

 

=  2.4 x 105 cells per mL

In this example to deliver 1 x 106 cells, one would have to measure out 4mL of cell culture suspension.

ie.           4 x 2.4 x 105  =  9.6 x 105, which is approximately equal to 1 x 106

If the cell count is too high, try counting the cells in a fraction of the larger squares.

e.g.        If you counted 5 of the larger squares and arrived at a number of 100 cells

 

100 x 5 (there are 25 larger squares in total)

  500 cells per 0.1µL

 

  5000 cells per µL

 

  5 x 106 cells per mL

In this example, you would have to deliver 200µL of cell suspension to provide 1 x 106 cells

Setting up the haemocytometer:

  • Wet the raised glass rails with the tip of a moistened finger

Wetting the glass rails with a moistened fingertip

  • Carefully slide the coverslip over the raised glass rails

Sliding the coevrslip onto a haemocytometer

  • Draw up 10µL and deliver into the gap between the coverslip and the countring chamber

Adding the cell suspension to a haemocytometer