Calculate cell concentration, viability, and seeding volumes from hemocytometer counts.
Count live (clear/bright) and dead (blue-stained) cells in the large corner squares of your hemocytometer after mixing with Trypan blue (typically 1:1, giving a dilution factor of 2). Enter the counts and the calculator gives you concentration, viability, and total cell number. Use the seeding section to calculate how much suspension to add per well.
Concentration: Each large square on a hemocytometer holds 0.1 µL (0.0001 mL). So: cells/mL = (cells counted / squares counted) × dilution factor × 10⁴.
Viability: % viability = (live cells / total cells) × 100. Healthy cultures typically show >90% viability. Below 80% suggests problems with culture conditions, passage number, or handling.
Worked example: You count 185 live and 15 dead cells across 4 squares with a 1:1 Trypan blue dilution (DF = 2). Live cell concentration = (185/4) × 2 × 10⁴ = 9.25 × 10⁵ cells/mL. Viability = 185/(185+15) × 100 = 92.5%.
Accuracy tips: Count cells touching the top and left borders of each square but not the bottom and right borders (to avoid double-counting). Aim for 100–200 total cells across 4 squares for statistical reliability — if you see fewer than 50, reduce your dilution; if more than 300, increase it.