Manual cell counting

To count the cells manually you need to use a hemocytometer. A hemocytometer is a microscope slide that is specially designed to enable cell counting. The slide has a sink in its middle; the area of the sink is marked with a grid. A drop of a cell culture is placed in the sink. Looking at the sample under the microscope, the researcher uses the grid to manually count the number of cells in a certain area. The depth of the sink is predefined. Thus the volume of the counted culture can be calculated and with it the concentration of the cells.

Figure 1: Manual hemocytometer, showing in detail the grid to count cells.

Use the following procedure:

• Focus on the grid lines of the hemocytometer with a 10X objective.
• Using a hand tally counter, count the cells in one set of 16 squares. When counting, employ a system whereby cells are only counted when they are set within a square or on the right-hand or bottom boundary line.
• Move the hemocytometer to the next set of 16 corner squares and carry on counting until all four sets of 16 corners are counted.
• Take the average cell count from each of the sets of 16 corner squares and multiply by 10,000 (10⁴).
• If you dilute the cells (using medium or trypan blue) you need to multiply by the dilution factor as well.
• The final value is the number of viable cells/mL in the original cell suspension.