Viable cell count (Trypan Blue) → % viability and viable cells/mL
The Trypan Blue exclusion assay distinguishes live cells (unstained) from dead cells
(blue-stained). You count live and dead cells in a hemocytometer grid and then compute:
viability percentage and viable concentration (live cells/mL). If the sample was diluted
(commonly 1:1 with Trypan Blue), you apply a dilution factor.
What this calculator computes
- % viability from live and dead counts.
- Live (viable) cells/mL using hemocytometer square volume rules and dilution factor.
- Dead cells/mL (optional but useful for QC).
- Seeding helper (optional): volume needed to pipette to seed a target number of cells.
Inputs and counting modes
-
Per-square mode (preferred): enter live and dead counts for each of N squares.
This shows variability across squares.
-
Totals mode: enter total live and total dead across N squares (faster if you already summed).
Internally, the calculator converts totals to averages by dividing by N.
-
Square type: live/dead cells per mL depends on the volume of the counted square
(area × chamber depth). Choose the same square type you actually counted.
-
Dilution factor (DF): for a 1:1 mix with Trypan Blue, DF = 2.
If undiluted, DF = 1.
Core formulas
1) Viability percentage
\[
\%\text{viability} = 100\cdot\frac{\text{live}}{\text{live}+\text{dead}}
\]
Here, “live” and “dead” can be totals across all counted squares (recommended), or means per square.
The ratio is the same either way.
2) Average counts per square
If you counted N squares:
\[
\overline{n}_{\text{live}}=\frac{\text{live total}}{N}
\qquad
\overline{n}_{\text{dead}}=\frac{\text{dead total}}{N}
\]
3) Volume of one counted square
For a square with dimensions \(L \times W\) and chamber depth \(D\) (in mm):
\[
V_{\text{sq}}=L\cdot W\cdot D\ \ (\text{in mm}^3)
\]
Convert to mL using:
\[
1\ \text{mm}^3=10^{-3}\ \text{mL}
\]
So:
\[
V_{\text{sq}}(\text{mL})=L\cdot W\cdot D\cdot 10^{-3}
\]
4) Cells/mL conversion (with dilution)
Concentration is “average count per square divided by square volume”. Apply dilution factor DF:
\[
C_{\text{live}}=\frac{\overline{n}_{\text{live}}}{V_{\text{sq}}(\text{mL})}\cdot DF
\]
\[
C_{\text{dead}}=\frac{\overline{n}_{\text{dead}}}{V_{\text{sq}}(\text{mL})}\cdot DF
\]
Common hemocytometer preset (large square)
A common counting area is the large square:
\(1\ \text{mm} \times 1\ \text{mm}\) with chamber depth \(0.1\ \text{mm}\).
\[
V_{\text{sq}}=1\cdot 1\cdot 0.1=0.1\ \text{mm}^3
\]
\[
0.1\ \text{mm}^3 = 0.1\cdot 10^{-3}=1\times 10^{-4}\ \text{mL}
\]
Therefore, for the large square:
\[
\frac{1}{V_{\text{sq}}}=10^4\ \text{mL}^{-1}
\]
So a convenient shortcut is:
\[
C_{\text{live}}=\overline{n}_{\text{live}}\cdot 10^4\cdot DF
\qquad
C_{\text{dead}}=\overline{n}_{\text{dead}}\cdot 10^4\cdot DF
\]
Optional seeding calculation
If you want to seed a target number of viable cells per well, use the viable concentration:
\[
V_{\text{per well}}=\frac{\text{target cells per well}}{C_{\text{live}}}
\]
If you seed W wells:
\[
V_{\text{total}}=\frac{\text{target cells per well}\cdot W}{C_{\text{live}}}
\]
Convert mL to µL using:
\[
1\ \text{mL}=1000\ \mu\text{L}
\]
Boundary-line rule (avoid double counting)
Use a consistent rule for cells touching boundary lines. A common convention is:
count cells touching the top and left lines, and do not count cells touching
the bottom or right lines. The key is consistency across all squares.
Worked example
You count 4 large squares (1 × 1 × 0.1 mm). Live counts: 120, 110, 125, 118.
Dead counts: 10, 12, 8, 9. Trypan Blue mix is 1:1 → DF = 2.
Step 1 — Totals and viability
\[
\text{live total}=120+110+125+118=473
\qquad
\text{dead total}=10+12+8+9=39
\]
\[
\%\text{viability}=100\cdot\frac{473}{473+39}
=100\cdot\frac{473}{512}=92.38\%
\]
Step 2 — Averages per square
\[
\overline{n}_{\text{live}}=\frac{473}{4}=118.25
\qquad
\overline{n}_{\text{dead}}=\frac{39}{4}=9.75
\]
Step 3 — Volume and cells/mL
\[
V_{\text{sq}}=1\times 10^{-4}\ \text{mL}
\]
\[
C_{\text{live}}=\frac{118.25}{1\times 10^{-4}}\cdot 2
=2.37\times 10^{6}\ \text{cells/mL}
\]
\[
C_{\text{dead}}=\frac{9.75}{1\times 10^{-4}}\cdot 2
=1.95\times 10^{5}\ \text{cells/mL}
\]
Common pitfalls
- Wrong dilution factor: DF = 2 for 1:1 dilution, not 0.5. You multiply by DF to recover original concentration.
- Square mismatch: using the wrong square type changes the volume and can shift cells/mL by large factors.
- Cell clumps: poor mixing increases square-to-square variability and can bias results.
- Inconsistent boundary rule: changing inclusion/exclusion rules between squares biases totals.
- Very high density: if squares are overcrowded, consider a higher dilution for more reliable counting.
