# Hemocytometer cell count and cell seeding calculator

> Free hemocytometer calculator: cells/mL and viability from trypan blue counts, then how much cell suspension and medium to seed any number of wells.

URL: https://www.labcanvas.com/tools/cell-count-calculator

Cells per mL = the mean count per large corner square × 10,000 × the dilution factor; with equal volumes of cells and trypan blue the dilution is 2. Viability is live cells ÷ (live + dead) × 100. The calculator then gives the volume of suspension and medium to seed a set number of cells in each well.

## Quick answer

- **What it does:** Calculates cells per mL and viability from hemocytometer counts with trypan blue, then the volumes of cell suspension and medium to seed your wells.
- **Formula:** cells/mL = mean count per large square × 10,000 × dilution; viability = live ÷ (live + dead) × 100.
- **Example:** A mean of 49 clear cells per large square, mixed 1:1 with trypan blue: 49 × 10,000 × 2 = 980,000 cells/mL; with 3 blue cells, viability = 49 ÷ 52 = 94.2 %.

Free, in the browser, no account needed. Updated 2026-11-08.

## Features

- Cells per mL from the live (unstained) count in each large square, with the dilution from the volumes of cells and trypan blue you mixed
- Viability and total cells per mL from the blue (dead) counts
- Any earlier dilution and the total number of cells in your tube
- Seeding by cells per well or by density (cells/cm²) and growth area
- Volume of cell suspension and medium for any number of wells, with optional extra for pipetting losses
- Warns when the suspension is too dilute to reach the seeding density
- Copy the result or download a CSV; inputs are remembered in your browser

## How to use it

1. **Mix and load**: Mix your cells with trypan blue (for example 10 µL + 10 µL), load the hemocytometer and count within a few minutes.
2. **Enter the counts**: Type the live (clear) cell count of each large corner square, and the blue (dead) counts in the same order for the viability.
3. **Enter the dilution**: Type the volumes of cells and trypan blue you mixed, and any dilution made before that.
4. **Plan the seeding**: Enter cells per well (or cells/cm² and the growth area), the number of wells and the volume per well.
5. **Read and pipette**: Read the volume of cell suspension and medium to mix, then add the stated volume to each well.

## At a glance

| | |
| --- | --- |
| Formula | \(\text{cells/mL} = \text{mean per large square}\) \({}\times 10{,}000 \times \text{dilution}\) |
| Viability | \(\text{live} \div (\text{live} + \text{dead}) \times 100\) |
| Dilution | \((\text{cells} + \text{trypan blue}) \div \text{cells}\), times any earlier dilution |
| Seeding | By cells per well or by cells/cm² and growth area |
| Output | Cells/mL, viability, suspension and medium volumes; CSV |

## How it works

$$\begin{aligned} \text{cells/mL} &= \text{mean count per large square} \\ &\quad {}\times 10{,}000 \times \text{dilution} \end{aligned}$$

$$\begin{aligned} \text{dilution} &= \frac{\text{cell volume} + \text{dye volume}}{\text{cell volume}} \\ &\quad {}\times \text{earlier dilution} \end{aligned}$$

$$\text{viability (\%)} = \frac{\text{live}}{\text{live} + \text{dead}} \times 100$$

In the hemocytometer protocol of Phan et al. (2022) the unstained cells in each large corner square (16 small squares) are counted, the counts are averaged, and the mean is multiplied by 10,000 and by the trypan blue dilution to give viable cells per mL of the original suspension. A cell is counted when it lies inside the square or on its right-hand or bottom line, so cells on the edges are not counted twice. The factor 10,000 means each large square holds \(10^{-4}\) mL.

Trypan blue enters cells whose membrane is damaged, so live cells stay clear and dead cells turn blue (Strober 2015). In Strober's protocol equal parts of cell suspension and 0.4% trypan blue are mixed, a 2-fold dilution; Phan et al. mix 0.1 mL of cells with 0.4 mL of dye, a 5-fold dilution. Strober gives viability as \(\text{viable cells per mL} \div \text{total cells per mL}\) \({}\times 100\), asks for counting within 3 to 5 minutes of mixing (longer incubation kills cells) and for serum-free solution, because serum proteins take up the dye.

For seeding, the cells needed are \(\text{cells per well} \times \text{number of wells}\) (plus any extra), the volume of suspension is \(\text{cells needed} \div \text{viable cells per mL}\), and the rest of the final volume is medium.

Sources: Phan A, Romeis E, Tantalo L, Giacani L. In vitro transformation and selection of *Treponema pallidum* subsp. *pallidum*. *Curr Protoc* 2022;2:e507 (Support Protocol 2), [doi:10.1002/cpz1.507](https://doi.org/10.1002/cpz1.507). Strober W. Trypan blue exclusion test of cell viability. *Curr Protoc Immunol* 2015;111:A3.B.1–A3.B.3, [doi:10.1002/0471142735.ima03bs111](https://doi.org/10.1002/0471142735.ima03bs111).

## Worked example

Cells mixed 10 µL + 10 µL with trypan blue (dilution 2). The four large corner squares hold 48, 52, 45 and 51 clear cells (mean 49) and 3, 2, 4 and 3 blue cells (mean 3).

Viable cells: \(49 \times 10{,}000 \times 2 = \mathbf{980{,}000\ cells/mL}\). Total cells: \(52 \times 10{,}000 \times 2 = 1.04\times10^{6}\ \mathrm{cells/mL}\). Viability: \(49 / 52 \times 100 = \mathbf{94.2\%}\).

To seed 20,000 cells in each of 24 wells in 0.5 mL: 480,000 cells are needed, which is \(480{,}000 / 980{,}000 = \mathbf{490\ \upmu L}\) of the suspension, made up with **11.51 mL** of medium to 12 mL (40,000 cells/mL); add 0.5 mL to each well.

## Questions

### How do you calculate cells per mL from a hemocytometer?

Average the counts of the large corner squares, multiply by 10,000 and by the dilution factor. With a mean of 49 cells per square and cells mixed 1:1 with trypan blue, that is \(49 \times 10{,}000 \times 2 = 980{,}000\) cells/mL (Phan et al. 2022).

### Why multiply by 10,000?

Each large corner square of the hemocytometer holds \(10^{-4}\) mL, so the number of cells in one square times 10,000 is the number per mL of the mix you loaded.

### What is the dilution factor with trypan blue?

The total volume divided by the volume of cells: equal volumes (for example 10 µL + 10 µL) give 2, and 0.1 mL of cells with 0.4 mL of dye gives 5. Multiply by any dilution you made before adding the dye.

### How is cell viability calculated?

Viability (%) \[ = \frac{\text{live (unstained) cells}}{\text{total (unstained + blue) cells}} \times 100 \] (Strober 2015). With 49 live and 3 dead cells per square, it is 94.2%.

### Which cells on the lines should I count?

Count cells inside the square and those touching its right-hand or bottom line, and leave out those on the top and left lines, so no cell is counted twice (Phan et al. 2022).

### How long can cells sit in trypan blue?

Count within 3 to 5 minutes of mixing; longer incubation kills cells and lowers the viability. Mix in serum-free solution, because serum proteins stain with trypan blue (Strober 2015).

### How do I work out how much cell suspension to seed?

Multiply cells per well by the number of wells, divide by the viable cells per mL to get the volume of suspension, and make it up to the final volume (wells × volume per well) with medium.