Before you start
- Mix the suspension well just before sampling — cells settle quickly.
Reagents and materials
Totals are for 4 counts (+10% extra). Per-count amounts are in the middle column.
| Reagent | Per count | Total |
|---|---|---|
| Cell suspension | 10 µL | 10 µL |
| 0.4% trypan blue | 10 µL | 10 µL |
Buffers and recipes
Calculation per count
| Cells per mL | (cells counted ÷ squares counted) × dilution factor × 10⁴ |
| Dilution factor (1:1 with trypan blue) | 2 |
| Viability (%) | live (unstained) ÷ total × 100 |
| Total cells | cells per mL × suspension volume (mL) |
Equipment
- Hemocytometer and coverslip (or automated counter)
- Microscope
- Tally counter
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Mix with dye
Mix 10 µL cell suspension with 10 µL 0.4% trypan blue.
Load
Load 10 µL under the coverslip by capillary action without overfilling.
Count
Count live (bright) and dead (blue) cells in the four large corner squares; count cells touching the top and left lines but not the bottom and right.
Calculate
Cells/mL = (total live ÷ 4) × 2 × 10⁴. Aim for 50–200 cells per square for accuracy; dilute more if needed.
Critical points
- Count within 3–5 min of adding trypan blue; it becomes toxic and stains live cells over time.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Counts vary a lot | Mix better, count more squares, use the right dilution. |
| Clumps | Pipette or filter the suspension before counting. |
Safety
Trypan blue is a possible carcinogen: wear gloves.
Further reading
- Strober W. Trypan blue exclusion test of cell viability. Current Protocols in Immunology (2015).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.