The formula
viability (%) = live cells ÷ (live cells + dead cells) × 100
Count live and dead cells in the same squares, so the dilution and chamber volume cancel out; viability is a ratio and needs no conversion to cells per mL.
How trypan blue works
Trypan blue cannot cross an intact cell membrane. Live cells exclude it and stay bright under the microscope; dead cells with damaged membranes take it up and turn blue. Mix cells and 0.4% trypan blue 1:1, wait no more than a few minutes, and count: the dye is toxic, and after about 5 minutes live cells start to stain too, which makes viability look worse than it is.
A worked example
Four large squares give live counts of 58, 62, 55 and 61 (average 59) and dead counts of 6, 8, 5 and 9 (average 7).
- Viability = 59 ÷ (59 + 7) × 100 = 89%.
- Live cells per mL = 59 × 2 (trypan blue dilution) × 104 = 1.18 × 106.
Use the live count, not the total, when you work out how much to seed.
What is acceptable
Healthy cultures in log phase are above 90% viable, and most experiments want at least 85 to 90%. Freshly thawed cells can be 60 to 80% and recover after a passage. Cells detached with trypsin lose a few percent; cells left in trypsin too long, pipetted roughly or kept on ice for an hour lose more. A drop in viability between passages is an early sign of contamination, exhausted medium or a problem with the incubator.
Common mistakes
- Counting debris and stained fragments as dead cells lowers the number; counting dim, swollen cells as live raises it.
- Dead cells lyse and disappear in culture, so a viability count only shows recent death, not everything that died.
- Trypan blue misses cells in early apoptosis, which still exclude the dye; use annexin V or a dye such as propidium iodide with flow cytometry when that matters.
Calculators
The Hemocytometer Calculator takes live and dead counts and gives viability and live cells per mL. The Cell Seeding Calculator works out how much to pipette, and the Cell Doubling Time Calculator tracks growth between passages.