Trypan Blue Calculator
Trypan Blue Results
How to Use the Trypan Blue Calculator
Enter the number of live (clear/unstained) and dead (blue-stained) cells counted from each large square of the hemocytometer using the grid above, or enter totals directly in the fields below. Select the number of squares counted and your trypan blue dilution ratio, then click Calculate.
If you know your sample volume, enter it to get the total number of live cells in the entire sample — useful for seeding calculations and yield tracking.
Trypan Blue Exclusion Principle
Trypan blue is a membrane-impermeant dye. Live cells with intact plasma membranes actively exclude the dye and appear clear under the microscope. Dead or dying cells with compromised membranes take up trypan blue and stain dark blue. This makes it simple to distinguish live from dead cells by direct visual inspection.
Calculation Formulas
Standard Dilution Ratios
- 1:1 ratio — mix equal volumes of 0.4% trypan blue and cell suspension. Dilution factor = 2. Most common for cell culture.
- 1:4 ratio — 1 part trypan blue to 4 parts cells. Dilution factor = 5. Used when cell density is very low.
- 1:9 ratio — 1 part trypan blue to 9 parts cells. Dilution factor = 10. Use when density is very high to avoid over-counting.
Important Tips for Accurate Results
- Count cells within 3 minutes of mixing — prolonged dye exposure causes live cells to stain blue, falsely reducing viability.
- Aim for 100–300 total cells per large square for statistically reliable counts.
- Count cells touching the top and left boundary lines only; exclude bottom and right boundary cells.
- Count both sides of a double-sided hemocytometer and average the results for better accuracy.
- Use freshly prepared 0.4% trypan blue — degraded or precipitated dye gives unreliable staining.
- If viability is unexpectedly low, check trypsin exposure time and centrifugation speed — both can damage cells.
Interpreting Viability Results
- ≥ 95% — Excellent. Proceed with any application including in vivo work and cryopreservation.
- 90–94% — Very good. Suitable for transfection, assays, and routine passaging.
- 80–89% — Acceptable for most applications but investigate the cause of cell death.
- 70–79% — Marginal. Results from downstream experiments may be unreliable.
- Below 70% — Poor. Discard and revive from a frozen stock or rethink culture conditions.