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Alternative methods for determination of cell number

Several other methods are available for quantifying cells in culture, including direct

measurement using anelectronic Coulter counter. This is an automated method of

counting and measuring the size of microscopic particles. The instrument itself consists

of a glass probe with an electrode that is connected to an oscilloscope (Fig. 2.7). The

probe has a small aperture of fixed diameter near its bottom end. When immersed in a

solution of cell suspension, cells are flushed through the aperture causing a brief

increase in resistance owing to a partial interruption of current flow. This will result

in spikes being recorded on the oscilloscope and each spike is counted as a cell. One

disadvantage of this method, however, is that it does not distinguish between viable and

dead cells.

Indirectly, cells can be counted by determining total cell protein and using a protein

versus cell number standard curve to determine cell number in test samples. However,

protein content per cell can vary during culture and may not give a true reflection

of cell number. Alternatively, the DNA content of cells may be used as an indicator

of cell number, since the DNA content of diploid cells is usually constant. However,

the DNA content of cells may change during the cell cycle and therefore not give an

accurate estimate of cell number.

2.5.7 Seeding cells onto culture plates

Once counted, cells should then be seeded at a density that promotes optimal cell

growth. It is essential therefore that when cells are subcultured they are seeded at the

Cell suspension

To vacuum pump

Aperture

Internal electrode

External electrode

Pulse

measurement

system

2 5 5

Fig. 2.7Coulter counter. Cells entering the aperture create a pulse of resistance between the internal

and external electrodes that is recorded on the oscilloscope.

57 2.5 Types of animal cell, characteristics and maintenance in culture