Biology and life-science lab
Cell culture calculator: seeding, counting and doubling time
Haemocytometer cell concentration and viability, seeding volumes for flasks and plates, split ratios for passaging, and population doubling time.
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Result
- Live cells per mL
- 1.00 × 10⁶
- Viability
- 95.2 %
- Total cells per mL (live + dead)
- 1.05 × 10⁶
- Live cells in the suspension
- 5.00 × 10⁶
Working
- cells/mL = (cells counted ÷ squares) × dilution × 10⁴ = (200 ÷ 4) × 2 × 10⁴
- For a Neubauer (improved) haemocytometer: each large corner square holds 1 mm × 1 mm × 0.1 mm = 0.1 µL, hence the factor 10⁴ per mL. Mixing cells 1:1 with trypan blue is a dilution factor of 2.
- Count 100–300 cells in total for a counting error near 10 %; recount if the squares differ widely.
Method and limits
Pick a calculation. Each one runs in your browser as you type.
Counting with a haemocytometer
cells/mL = (cells counted ÷ squares counted) × dilution factor × 10⁴ viability = live ÷ (live + dead) × 100 %
Each large corner square of an improved Neubauer chamber covers 1 mm × 1 mm with a 0.1 mm depth: 0.1 µL, or 10⁻⁴ mL. Trypan blue enters only cells with damaged membranes, so blue cells count as dead.
Seeding
cells per vessel = density × growth area (or × medium volume) suspension = cells ÷ (cells/mL) medium = final volume − suspension
| Vessel | Typical growth area |
|---|---|
| T-25 flask | 25 cm² |
| T-75 flask | 75 cm² |
| T-175 flask | 175 cm² |
| 100 mm dish | 55 cm² |
| 60 mm dish | 21 cm² |
| 6-well plate (per well) | 9.5 cm² |
| 12-well plate (per well) | 3.8 cm² |
| 24-well plate (per well) | 1.9 cm² |
| 96-well plate (per well) | 0.32 cm² |
Splitting and doubling time
suspension per new vessel = resuspended volume ÷ N for a 1:N split
Td = t × ln 2 ÷ ln(N ÷ N₀) doublings = log₂(N ÷ N₀) µ = ln(N ÷ N₀) ÷ t
Limits
- Count 100–300 cells in total: the counting error is roughly the square root of the number counted (about 7 % at 200).
- Doubling time assumes exponential growth between the two counts; counts in lag phase or near confluence give longer apparent times.
- Viability by dye exclusion overestimates healthy cells early in apoptosis.
Worked example
200 live and 10 dead cells over 4 squares, diluted 1:1 in trypan blue: (200 ÷ 4) × 2 × 10⁴ = 1.00 × 10⁶ live cells/mL, 95.2 % viable. Seeding 5000 cells/cm² in a T-75 (75 cm²) from that suspension needs 3.75 × 10⁵ cells: 0.375 mL of suspension and 14.6 mL of medium. Cells growing from 1.0 × 10⁵ to 8.0 × 10⁵ in 72 h doubled 3 times, so Td = 24.0 h.
References
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