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Hemocytometer Cell Counting Calculator

Convert a hemocytometer count into cells per mL and total cells, with the dilution factor applied and the statistical uncertainty of the count shown.

Formula

cells/mL=ncellsnsquares×DF×104\text{cells/mL} = \dfrac{n_{\text{cells}}}{n_{\text{squares}}}\times \mathrm{DF}\times 10^{4}
Ntotal=cells/mL×VsuspensionN_{\text{total}} = \text{cells/mL}\times V_{\text{suspension}}
nsquaresn_{\text{squares}}
number of 1 mm × 1 mm large squares included in the count
DF\mathrm{DF}
2 when the sample is mixed 1:1 with trypan blue
10410^{4}
converts the 0.1 µL volume over one large square to 1 mL

How it works

An improved Neubauer chamber holds the sample at a depth of 0.1 mm. Each large square is 1 mm on a side, so the volume above it is 0.1 mm³, or 0.1 µL. The average count per large square multiplied by 10⁴ is therefore the number of cells in 1 mL.

Counting is a sampling process. For n cells counted, the relative uncertainty is about 1/√n, so 100 cells give roughly ±10% and 400 cells roughly ±5%.

Worked example

212 cells are counted across 4 large squares, in a sample mixed 1:1 with trypan blue. The suspension volume is 10 mL.

  1. Average per square = 212 / 4 = 53.
  2. cells/mL = 53 × 2 × 10⁴ = 1.06 × 10⁶.
  3. Total = 1.06 × 10⁶ × 10 mL = 1.06 × 10⁷ cells.

The suspension is at 1.06 × 10⁶ cells/mL, with a counting uncertainty of about ±7%.

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

Assumptions

  • An improved Neubauer chamber with a 0.1 mm depth and 1 mm large squares.
  • The suspension is well mixed and single-cell, with no clumps.
  • The coverslip is seated correctly, so the chamber depth is as specified.

Common mistakes

  • Counting cells that touch all four borders of a square. Count those on two borders (for example top and left) and ignore the other two.
  • Leaving out the dilution factor from mixing with trypan blue.
  • Counting too few cells. If the total is under about 100, count more squares or concentrate the sample.

Related tools

Cell Viability Calculator (Trypan Blue)Percent viability from live and dead countsOpen calculatorTotal Cell Count CalculatorTotal and viable cells in a suspensionOpen calculatorCell Dilution CalculatorDilute a cell suspension to a target densityOpen calculatorCell Seeding CalculatorSuspension and medium for a plate of wellsOpen calculator

Related equipment

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