Hemocytometer calculator
Cells per mL, viability and how much to seed, from counts per square.
Cells per mL, viability and how much to seed, from counts per square.
A hemocytometer is a thick glass slide with a fine grid etched into a chamber of known depth. A drop of cell suspension is drawn under the coverslip by capillary action, so a square of known area holds a known volume. In the standard improved Neubauer chamber each of the four large corner squares is 1 mm × 1 mm under a 0.1 mm gap, a volume of 0.1 µL. Count the cells in a square and you know how many are in 0.1 µL; multiply by 10,000 and you have the number in a millilitre. For colonies on agar rather than cells in suspension, use theCFU calculator; for bacteria by turbidity, the OD600 calculator.
Worked example. Counts of 82, 90, 84 and 84 in the four corner squares give a mean of 85. The cells were mixed 1:1 with trypan blue, a dilution factor of 2. Density is 85 × 10,000 × 2 = 1.7 × 10⁶ cells/mL. In 5 mL of suspension that is 8.5 million cells. The Fuchs-Rosenthal chamber is deeper (0.2 mm) and its large squares hold 0.2 µL, so its factor is 5,000; the Bürker chamber uses the same factor as the Neubauer.
Count at least the four large corner squares, and more when counts are low. Reliable counts have 20 to 200 cells per square: fewer and the statistical error is large, more and cells overlap and are missed. The calculator flags counts outside that range. It also reports the coefficient of variation across squares; more than about 20 % means the suspension was not evenly mixed or the chamber was over- or under-filled, and the count should be repeated. Count cells touching the top and left boundary lines and ignore those touching the bottom and right, so that each boundary cell is counted once.
Trypan blue enters cells with damaged membranes and stains them blue; live cells exclude it and stay bright. Mixing cells with dye dilutes them, so the factor must be included: 10 µL of cells plus 10 µL of dye is a factor of 2, 10 µL plus 90 µL a factor of 10. Enter dead cells in their own column and the calculator reports viability as live ÷ (live + dead). Count within a few minutes of mixing, because trypan blue is itself toxic and viability falls with time. The cell viability calculator handles plate-based assays such as MTT.
Once the density is known, seeding is a dilution: the volume of suspension that holds the cells you want, made up to the working volume with medium. The table gives the growth area of common formats and the seeding densities that reach about 70–80 % confluence in two to three days for a typical adherent line; adjust for your cells' doubling time.
| Format | Area per well | Typical seeding | Working volume |
|---|---|---|---|
| 96-well | 0.32 cm² | 5,000–20,000 | 0.1–0.2 mL |
| 48-well | 0.95 cm² | 20,000–50,000 | 0.3–0.5 mL |
| 24-well | 1.9 cm² | 50,000–100,000 | 0.5–1 mL |
| 12-well | 3.8 cm² | 100,000–200,000 | 1–2 mL |
| 6-well | 9.6 cm² | 200,000–500,000 | 2–3 mL |
| T25 | 25 cm² | 0.5–1 million | 5 mL |
| T75 | 75 cm² | 1.5–3 million | 10–15 mL |
| T175 | 175 cm² | 4–7 million | 25–35 mL |
Average the number of cells in the large corner squares, multiply by 10,000 (the volume factor for a standard Neubauer chamber) and by the dilution factor. If you counted an average of 85 cells per square in a 1:1 trypan blue mix, the suspension has 85 × 10,000 × 2 = 1.7 million cells per mL.
At least four large squares, more if the counts are low. Aim for 20 to 200 cells per square; dilute the sample if you see more, concentrate it or count more squares if you see fewer. The counts should agree to within about 20 percent, otherwise the suspension was not mixed well.
Each large square of a Neubauer chamber is 1 mm by 1 mm and the coverslip sits 0.1 mm above it, so the square holds 0.1 µL. There are 10,000 lots of 0.1 µL in 1 mL, so cells per square times 10,000 is cells per mL.
The ratio of final volume to cell-suspension volume. Mixing 10 µL of cells with 10 µL of trypan blue is a 1:1 mix and a dilution factor of 2. Mixing 10 µL of cells with 90 µL of dye is a factor of 10. Cells that took up the blue dye are dead; count them separately to get viability.
Count cells touching the top and left lines of a square and ignore cells touching the bottom and right lines. Applied consistently to every square this counts each boundary cell exactly once.