The formula
cells/mL = (cells counted ÷ squares counted) × dilution × chamber factor
The chamber factor turns cells per square into cells per mL. It is 1 mL divided by the volume of liquid over one square.
| Chamber and squares | Volume per square | Factor |
|---|---|---|
| Neubauer improved or Bürker, large 1 mm² squares, 0.1 mm deep | 0.1 µL | 10,000 |
| Neubauer improved, small centre squares (0.2 × 0.2 mm) | 0.004 µL | 250,000 |
| Fuchs-Rosenthal, 1 mm² squares, 0.2 mm deep | 0.2 µL | 5,000 |
The dilution is the trypan blue dilution times any dilution you made before counting. Equal volumes of cells and trypan blue is a 1:2 dilution.
Worked example
You mix 10 µL of cells with 10 µL of trypan blue and count the four large corner squares of a Neubauer chamber: 248 unstained (live) cells and 17 blue (dead) cells in total.
- Live cells per square = 248 ÷ 4 = 62
- Live cells per mL = 62 × 2 × 10,000 = 1.24 × 10⁶
- Viability = 248 ÷ (248 + 17) = 93.58%
- In 10 mL of suspension: 1.24 × 10⁷ live cells
The Use in cell seeding button carries the live concentration straight into the cell seeding calculator.
How precise is a count?
Cells land in the chamber at random, so even a perfect count varies. The spread follows a Poisson distribution: counting N cells gives a 95% uncertainty of about ±1.96 ÷ √N. Counting 100 cells gives about ±20%, 400 cells about ±10%. The calculator shows this for your count and suggests counting more when fewer than 100 cells were counted. Pipetting, mixing and how the chamber was loaded add to this, so treat it as the best case.
Tips for a reliable count
- Mix the suspension well just before taking the sample, as cells settle quickly.
- Aim for roughly 20 to 200 cells per large square. Dilute more if it is crowded.
- Count cells touching the top and left lines of a square but not the bottom and right, so none are counted twice.
- Count within a few minutes of adding trypan blue. Over time live cells start to take up the dye, so viability reads low.
- Load both sides of the chamber and average them if the count matters.
Accuracy
The calculator is checked against independently worked-out answers for each chamber type, earlier dilutions and missing inputs.
This calculator is checked against independent calculations before every release. How the calculators are checked.
References
- Strober W (2015). Trypan blue exclusion test of cell viability. Current Protocols in Immunology 111: A3.B.1–A3.B.3.