Calculator Inputs
Use up to three replicates. Leave unused replicate rows blank.
Example Data Table
This example uses a 1:1 dye mix, three replicates, four counted squares, and a 5 mL sample.
| Replicate | Live Count | Dead Count | Squares | Live / Square | Dead / Square | Viability % |
|---|---|---|---|---|---|---|
| 1 | 86 | 14 | 4 | 21.50 | 3.50 | 86.00 |
| 2 | 91 | 11 | 4 | 22.75 | 2.75 | 89.22 |
| 3 | 88 | 12 | 4 | 22.00 | 3.00 | 88.00 |
| Average | 265 | 37 | 12 | 22.08 | 3.08 | 87.75 |
Formula Used
Mean of (live cells counted ÷ squares counted) across valid replicates.
Mean of (dead cells counted ÷ squares counted) across valid replicates.
(Average live count per square ÷ average total count per square) × 100.
(Average live count per square × dilution factor) ÷ chamber volume per square.
(Average dead count per square × dilution factor) ÷ chamber volume per square.
(Average total count per square × dilution factor) ÷ chamber volume per square.
Live concentration × available sample volume.
Target viable cells ÷ live concentration.
How to Use This Calculator
- Enter the dilution factor applied during trypan blue mixing.
- Confirm the chamber volume for each counted square.
- Add live and dead counts for one to three replicates.
- Enter how many large squares were counted for each replicate.
- Optionally add total sample volume and target viable cells.
- Click Calculate Viability to show results above the form.
- Download the summary as CSV or PDF for reporting.
Frequently Asked Questions
1. What does trypan blue viability measure?
It estimates the fraction of unstained, membrane-intact cells relative to total counted cells. Viable cells exclude the dye, while nonviable cells absorb it and appear blue under the microscope.
2. Why is the dilution factor important?
The dye mixture changes the original cell concentration. Applying the correct dilution factor scales counted cells back to the estimated concentration in the original suspension.
3. Why do I need squares counted for each replicate?
Counts are normalized per square before averaging. This keeps replicate comparisons fair, especially when one replicate uses a different number of counted grid areas.
4. What chamber volume should I enter?
A standard large hemocytometer square usually represents 0.0001 mL. If you use another chamber design or counting region, enter that specific per-square volume instead.
5. What does replicate CV tell me?
The coefficient of variation shows replicate consistency. Lower CV values suggest more repeatable counting, while high CV values may indicate clumping, pipetting variation, or uneven loading.
6. Can I use only one replicate?
Yes. One complete replicate can produce viability and concentration estimates. Multiple replicates are better because they reduce random counting error and reveal variability.
7. What happens if there are no live cells?
Viability becomes zero, dead concentration still computes, and the dead-to-live ratio or target-volume estimate may be unavailable because dividing by zero is undefined.
8. When should I export CSV or PDF?
Use CSV when you want spreadsheet analysis or batch records. Use PDF when you need a quick shareable report for lab notebooks, reviews, or experiment documentation.