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.

Worked examples

• No dilution: counts of 120, 132, 118 and 126 average 124. Cells per mL = 124 × 1 × 10⁴ = 1.24 × 10⁶ cells/mL.
• 1:1 trypan blue, with dead cells: live counts average 62 and dead counts average 8. Live cells per mL = 62 × 2 × 10⁴ = 1.24 × 10⁶ cells/mL; viability = 62 ÷ (62 + 8) = 89%.
• Too dense, diluted 1:10 first: counts average 45 after a 1:10 dilution and then 1:1 with trypan blue (total factor 20). Cells per mL = 45 × 20 × 10⁴ = 9 × 10⁶ cells/mL in the original suspension.
• Total cells and volume to seed: 1.24 × 10⁶ cells/mL in 10 mL is 1.24 × 10⁷ cells. To seed 5 × 10⁵ cells, take 5 × 10⁵ ÷ 1.24 × 10⁶ = 0.40 mL.

Getting the dilution factor right

The dilution factor is the total volume divided by the volume of cell suspension in it. Equal parts cells and trypan blue is 2; 100 µL of cells in 900 µL of medium is 10; doing both is 20. Counting fewer than about 20 or more than about 200 cells per square gives poor estimates, so dilute dense suspensions and count more squares for sparse ones.

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.