Viability & Trypan Blue Cell Count Calculator

What is Trypan Blue Exclusion?

Trypan blue is a vital dye that cannot penetrate the intact cell membrane of living cells. When mixed with a cell suspension:

• Live cells exclude the dye and appear clear/unstained
• Dead cells take up the dye and appear blue

This makes it easy to distinguish viable from non-viable cells under a microscope.

Calculating Viability Percentage

Viability is expressed as the percentage of live cells relative to total cells:

Where Total Cells = Live + Dead (trypan blue–positive) cells counted.

Hemocytometer Cell Counting

A hemocytometer is a specialized counting chamber with a defined geometry:

• Chamber depth: 0.1 mm
• Large square area: 1 mm × 1 mm = 1 mm²
• Volume per large square: 0.1 µL (0.0001 mL)

This gives the standard conversion factor of 10⁴:

Understanding Dilution Factor

The dilution factor accounts for how much you diluted your original sample before loading the hemocytometer. For example:

• 1:1 mix of cells:trypan blue → dilution factor = 2
• 1:4 dilution (1 part cells + 3 parts diluent) → dilution factor = 4

Include all dilutions between the original sample and the counting chamber.

Typical Applications

• Assessing cell culture health before experiments
• Determining cell concentration for seeding or passaging
• Quality control after thawing frozen cell stocks
• Monitoring cell viability during drug treatments

Limitations

• Counting accuracy depends on technique and sample preparation
• Cell clumps can lead to underestimation
• Early apoptotic cells may still exclude dye temporarily
• Extended trypan blue exposure can cause false positives
• Pipetting variability affects reproducibility