Hemocytometer cell count calculator

The formula

$$C = \bar{N} \cdot D \cdot 10^4\ \text{cells/mL}$$

$\bar{N}$ is the average number of cells in one large square, $D$ the dilution factor and $C$ the concentration of your original suspension.

Where it comes from

A hemocytometer is a counting chamber with a grid of known area under a coverslip held at a known height, so every square holds a known volume.

On a standard Neubauer chamber each large square is 1 mm × 1 mm and the chamber is 0.1 mm deep:

$$V_{\text{square}} = 1\ \text{mm} \times 1\ \text{mm} \times 0.1\ \text{mm} = 0.1\ \text{mm}^3$$

One millilitre is one cubic centimetre, which is 1,000 mm³, so

$$V_{\text{square}} = 0.1\ \text{mm}^3 = 10^{-4}\ \text{mL}$$

Concentration is count divided by volume, so cells in the chamber per mL are

$$\frac{\bar{N}}{10^{-4}\ \text{mL}} = \bar{N} \cdot 10^4$$

That is the concentration of what you loaded. If you diluted the cells first, for example 1:1 with trypan blue, the original was $D$ times more concentrated, giving $C = \bar{N} \cdot D \cdot 10^4$.

Worked example

You count 42, 47, 44 and 47 cells in the four corner squares after mixing 1:1 with trypan blue:

$$\bar{N} = \frac{180}{4} = 45, \qquad C = 45 \times 2 \times 10^4 = 9.0 \times 10^5\ \text{cells/mL}$$

Watch out for

• Count enough cells. Counting error scales with $1/\sqrt{N}$, so aim for at least 100 cells in total. That is about 10% error, and 400 cells is about 5%.
• Use one edge rule. Count cells touching the top and left lines but not the bottom and right, or you double count.
• Other chambers differ. This uses the Neubauer large square. Check your chamber’s square size and depth.

Last checked November 11, 2026