1.03×10⁶viable cells/mLViability 90.4%Mix 1.92 mL + 11.28 mL
Passage into a 6-well plate
Count stained cells before routine passage.
Neubauer · 4 squares · 6 wells
Hemocytometer Calculator
FigranTurn hemocytometer counts into viable cells/mL and a seeding recipe for your plates.
- Neubauer, Bürker, Thoma, Fuchs-Rosenthal
- Flags crowded or uneven counts
- Seeding volumes per well
Hemocytometer examples
Start with a scenario and adapt its counts and seeding plan.
6.20×10⁵viable cells/mLViability 91.9%Mix 532 µL + 6.07 mL
96-well plate for a CCK-8 assay
Prepare cells for 60 wells.
Neubauer · 60 wells · 5,000 cells per well
7.40×10⁶viable cells/mLViability 92.5%Mix 1.78 mL + 11.42 mL
Lymphocytes after a 1:10 pre-dilution
The first count was too crowded; load the recount after dilution.
Dilution ×20 · 12 wells
1.13×10⁵viable cells/mLViability 91.8%Mix 836 µL + 814 µL
Low-yield cells in a Fuchs-Rosenthal chamber
Count 16 large squares before seeding primary cells.
Fuchs-Rosenthal · 16 squares · 3 wells
2.00×10⁸viable cells/mLViability 94.3%Mix 500 µL + 49.50 mL
Yeast culture in a Thoma chamber
Count yeast stained with methylene blue and prepare a culture.
Thoma · methylene blue · 50 mL culture
1.28×10⁶viable cells/mLViability 94.1%Mix 1.29 mL + 64.71 mL
Mesenchymal stem cells into T-75 flasks
Plan flask expansion by surface area.
Bürker · 5,000 cells/cm² · 4 flasks
How to count cells
Use a hemocytometer to count cells and prepare a seeding mix.
1Load chamber
Mix the sample and add 10 µL to the chamber.
2Count squares
Count the top and left edges consistently.
3Enter counts
Record live and dead cells and the dilution used.
4Export results
Read the concentration and copy or download your results.
Hemocytometer formula
Turn a chamber count into cell concentration and viability, then calculate your seeding volume.
Cells per mL
C is cells/mL, n̄ is the average live or total count per square, D is the total dilution factor, and V is the volume per square in mL. For N counted live cells, relative standard error is approximately 1/√N.
Square volume and dilution
V is volume in mL; A is square area in mm² and d is chamber depth in mm. A 1 mm² square at 0.1 mm depth holds 0.0001 mL.
D is total dilution, P is the pre-dilution factor, S is sample volume and T is stain volume. A 1:1 trypan blue mix contributes a factor of 2, multiplied by any pre-dilution.
Viability
L is unstained live cells and T is all live and dead cells. Use the viable cell concentration when planning seeding.
Default example
Four large squares were counted after mixing cells and trypan blue 1:1.
| Viable concentration | 206 ÷ 4 × 2 ÷ 0.0001 = 1.03×10⁶ viable cells/mL |
|---|---|
| Suspension per well | 3.00×10⁵ ÷ 1.03×10⁶ = 291 µL |
Counting chambers and volumes
The chamber depth and counted area determine the volume represented by each square.
Improved Neubauer
Bürker
Thoma
Fuchs-Rosenthal
Malassez
| Chamber | Depth (mm) | Square counted | Area (mm²) | Volume per square (mL) | cells/mL per cell per square |
|---|---|---|---|---|---|
| Improved Neubauer | 0.1 | Large squares (1 mm²) | 1 | 1.00×10⁻⁴ | 1.00×10⁴ |
| Improved Neubauer | 0.1 | Small center squares (0.04 mm²) | 0.04 | 4.00×10⁻⁶ | 2.50×10⁵ |
| Bürker | 0.1 | Large squares (1 mm²) | 1 | 1.00×10⁻⁴ | 1.00×10⁴ |
| Bürker | 0.1 | Small squares (0.04 mm²) | 0.04 | 4.00×10⁻⁶ | 2.50×10⁵ |
| Thoma | 0.1 | Group squares (0.04 mm²) | 0.04 | 4.00×10⁻⁶ | 2.50×10⁵ |
| Thoma | 0.1 | Small squares (0.0025 mm²) | 0.0025 | 2.50×10⁻⁷ | 4.00×10⁶ |
| Fuchs-Rosenthal | 0.2 | Large squares (1 mm²) | 1 | 2.00×10⁻⁴ | 5.00×10³ |
| Fuchs-Rosenthal | 0.2 | Small squares (0.0625 mm²) | 0.0625 | 1.25×10⁻⁵ | 8.00×10⁴ |
| Malassez | 0.2 | Rectangles (0.05 mm²) | 0.05 | 1.00×10⁻⁵ | 1.00×10⁵ |
Source: Marienfeld Superior
Cell seeding density
Multiply cells per cm² by the growth area to find cells per well or vessel.
Culture plate and flask surface areas
| Vessel | Surface area (cm²) | Medium (mL) | Seeding (HeLa) | Cells at confluence (HeLa) |
|---|---|---|---|---|
| 6-well | 9.6 | 1–3 | 3.00×10⁵ | 1.20×10⁶ |
| 12-well | 3.5 | 1–2 | 1.00×10⁵ | 5.00×10⁵ |
| 24-well | 1.9 | 0.5–1 | 5.00×10⁴ | 2.40×10⁵ |
| 48-well | 1.1 | 0.2–0.4 | 3.00×10⁴ | 1.20×10⁵ |
| 96-well | 0.32 | 0.1–0.2 | 1.00×10⁴ | 4.00×10⁴ |
| 384-well | 0.084 | 0.08 | 1.80×10³ | 7.20×10³ |
| 35 mm dish | 8.8 | 2 | 3.00×10⁵ | 1.20×10⁶ |
| 60 mm dish | 21.5 | 5 | 8.00×10⁵ | 3.20×10⁶ |
| 100 mm dish | 56.7 | 12 | 2.20×10⁶ | 8.80×10⁶ |
| 150 mm dish | 145 | 30 | 5.00×10⁶ | 2.00×10⁷ |
| T-25 | 25 | 3–5 | 7.00×10⁵ | 2.80×10⁶ |
| T-75 | 75 | 8–15 | 2.10×10⁶ | 8.40×10⁶ |
| T-175 | 175 | 35–53 | 4.90×10⁶ | 2.33×10⁷ |
| T-225 | 225 | 45–68 | 6.30×10⁶ | 3.00×10⁷ |
Seeding and confluence values are for HeLa cells. Source: Thermo Fisher Gibco
Hemocytometer FAQ
How do you calculate cells per mL from a hemocytometer?
Divide the average cells per square by its volume in mL, then multiply by the dilution factor. A Neubauer large square has a volume of 0.0001 mL. Hemocytometer and haemocytometer are alternative spellings.
What dilution factor do I enter for trypan blue?
Equal volumes of cells and trypan blue give a factor of 2. A 1 in 10 pre-dilution followed by a 1:1 stain gives a factor of 20.
Which squares do I count, and what about cells on the lines?
For cultured cells, count the four large corner squares. Count cells touching the top and left lines and omit those touching the bottom and right. Count clumps as individual cells and apply your laboratory rule consistently to budding yeast.
How many cells should I count?
Count at least 100 cells when possible. Many protocols use approximately 20–100 cells per large square; dilute crowded samples or count more squares for sparse samples.
How do I calculate cell viability with trypan blue?
Divide unstained cells by all counted cells and multiply by 100. Count promptly after mixing, following your staining protocol.
Should I seed by viable cells or total cells?
Seed by viable cells to deliver the intended number of living cells. Use the cells per mL target to dilute a suspension to a desired concentration.
Can I use a concentration from an automated cell counter?
Yes. Select I know the concentration and enter viable cells per mL. Check whether your instrument has already accounted for dilution.
Can I use these counts for a growth curve or doubling time?
Yes. Count at each time point consistently, then calculate doubling time from the viable concentrations. Cell Doubling Time Calculator
Is my data uploaded, and what can I download?
Your inputs stay in your browser. Copy the recipe or methods text, or download PNG, SVG and CSV without signing in.
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