How to Count Cells With a Hemocytometer

Counting

1. Mix the cells with trypan blue, often 1:1, which stains dead cells blue.
2. Load about 10 µL under the coverslip.
3. Count the unstained cells in the four large corner squares. Count cells touching the top and left lines but not the bottom and right, so none is counted twice.
4. Count the blue cells separately if you want the viability.

The formula

Each large square is 1 mm × 1 mm and 0.1 mm deep, which is 0.1 µL, or 10⁻⁴ mL. So:

cells per mL = average count per square × dilution factor × 10⁴

Counts of 50, 46, 52 and 48 average 49. With a 1:1 trypan blue dilution (factor 2): 49 × 2 × 10⁴ = 9.8 × 10⁵ cells/mL.

Viability is live cells divided by all cells, times 100.

Doubling time

From two counts: doubling time = time × ln 2 ÷ ln(N₂ ÷ N₁). Cells that grow from 1 × 10⁵ to 8 × 10⁵ in 48 hours doubled three times, every 16 hours.

Calculators

The Hemocytometer Calculator gives cells per mL, viability and the volume to seed for a target number of cells. The Cell Doubling Time Calculator works out the doubling time and growth rate.