Turn a hemocytometer count into cells per mL. Enter the cells you counted, the number of large squares and the dilution (for example with trypan blue), and get the concentration, total cells, the volume to seed, and percent cell viability.
Hemocytometer formulas
Each large square of a Neubauer chamber is 1 mm × 1 mm × 0.1 mm deep, a volume of 0.1 µL (10⁻⁴ mL). That is where the 10⁴ factor comes from.
Worked example
You mix cells 1:1 with trypan blue (dilution factor 2) and count 200 live cells in the 4 corner squares. Cells/mL = 200 / 4 × 2 × 10⁴ = 1 × 10⁶ cells/mL. To seed 500,000 cells you need 0.5 mL (500 µL). If you also saw 20 blue (dead) cells, viability = 200 / 220 × 100 = 90.9%.
Counting tips
| Tip | Why |
|---|---|
| Count 20–100 cells per square | Fewer is imprecise, more is hard to count |
| Count cells on the top and left lines only | Avoids counting border cells twice |
| Count both chambers and average | Reduces random error |
| Count within 3–5 minutes of adding trypan blue | Live cells start taking up dye with time |
Frequently asked questions
How do I calculate cells per mL with a hemocytometer?
Divide the cells counted by the number of large squares, multiply by the dilution factor, then multiply by 10,000.
Why multiply by 10,000?
One large square holds 0.1 µL, which is 1/10,000 of a mL. Multiplying by 10⁴ converts cells per square to cells per mL.
What is the dilution factor with trypan blue?
Mixing equal volumes of cells and 0.4% trypan blue is a 1:1 mix, a dilution factor of 2.
How do I calculate cell viability?
Viability = unstained (live) cells / total cells × 100. Dead cells take up trypan blue and look blue.
How much cell suspension do I need to seed a plate?
Divide the number of cells you need by the concentration. Enter it in the optional box to get the volume in µL.
Related tools
Free educational tool by HawkInc. Results are calculated in your browser and rounded for display; check critical work by hand or with a second method.