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Trypan Blue Cell Count Calculator

Count live and dead cells stained with trypan blue on a hemocytometer, correct for the dye dilution, and get viable cells per mL, viability and the total in your suspension.

Formula

DF=Vcells+VdyeVcells\mathrm{DF} = \dfrac{V_{\text{cells}} + V_{\text{dye}}}{V_{\text{cells}}}
viable cells/mL=nlivensquares×DF×104\text{viable cells/mL} = \dfrac{n_{\text{live}}}{n_{\text{squares}}}\times \mathrm{DF}\times 10^{4}
viability=nlivenlive+ndead×100\text{viability} = \dfrac{n_{\text{live}}}{n_{\text{live}} + n_{\text{dead}}}\times 100
live, dead\text{live},\ \text{dead}
unstained and blue-stained cells counted
squares\text{squares}
number of large (1 mm × 1 mm) squares counted
10410^{4}
conversion from cells per 0.1 µL (one square) to cells per mL

How it works

Trypan blue is excluded by cells with an intact membrane and enters cells whose membrane is damaged, which stain blue. Mixing the cell suspension with the dye dilutes it, and the dilution has to be put back: a 1:1 mix is a dilution of two.

The count gives the concentration of viable cells, which is what you need to seed a plate, and the viability, the share of cells that are unstained. A hemocytometer square holds 0.1 µL, so the count per square times 10⁴ is cells per mL. Count enough cells for the statistical error to be acceptable.

Worked example

186 live and 14 dead cells in 4 squares, from a 10 µL sample mixed with 10 µL of 0.4% trypan blue, in 5 mL of suspension.

  1. Dilution = (10 + 10) / 10 = 2.
  2. Viable cells/mL = (186 / 4) × 2 × 10⁴ = 9.3 × 10⁵.
  3. Viability = 186 / 200 × 100 = 93.0%; viable cells in 5 mL = 4.65 × 10⁶.

9.3 × 10⁵ viable cells/mL with 93% viability.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Cells are in a single-cell suspension and evenly distributed on the counting chamber.
  • The count is made within a few minutes of mixing, as live cells also take up the dye with time.
  • Only membrane integrity is measured, which is not the same as the cell's health or ability to grow.

Common mistakes

  • Forgetting the dilution by the dye, which halves the concentration for a 1:1 mix.
  • Counting clumps, or cells on the edge of the square twice.
  • Letting the cells stand in trypan blue, which raises the apparent number of dead cells.

Related tools

Hemocytometer Cell Counting CalculatorCells per mL from a hemocytometer countOpen calculatorCell Viability Calculator (Trypan Blue)Percent viability from live and dead countsOpen calculatorCell Seeding CalculatorSuspension and medium for a plate of wellsOpen calculatorCell Dilution CalculatorDilute a cell suspension to a target densityOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.