Cell counting
Hemocytometer calculator
Enter what you counted on the chamber and get the cell concentration, viability, the total number of cells and how much suspension to take. It works for the improved Neubauer, Bürker and Fuchs-Rosenthal grids, for any square you counted, and for any dilution, including the usual 1:1 trypan blue mix.
How the hemocytometer formula works
Each square of the counting grid holds a known volume: its area times the depth of the chamber. On an improved Neubauer chamber a large square is 1 mm × 1 mm and the chamber is 0.1 mm deep, so a large square holds 0.1 mm³, which is 0.1 µL or 10⁻⁴ mL. Dividing the average count per square by that volume gives cells per mL; multiplying by the dilution undoes the dye or buffer you added.
cells/mL = (N ÷ squares) ÷ (area mm² × depth mm) × 1000 × dilution
Worked example: 200 live cells over the 4 corner squares, mixed 1:1 with trypan blue. 200 ÷ 4 = 50 cells per square; 50 × 10⁴ × 2 = 1.0 × 10⁶ live cells/mL. With 10 dead cells as well, viability is 200 ÷ 210 = 95.2 %. To seed 500,000 cells, take 500,000 ÷ 1,000,000 = 0.5 mL.
Counting rules that keep the number honest
- Edge rule: count cells touching the top and left lines of a square and skip those touching the bottom and right, so no cell is counted twice.
- Count enough cells: the counting error is Poisson, about 1/√N. 100 cells gives roughly ±10 %, 400 cells about ±5 %. Count more squares, or both chambers, when the density is low.
- Dilute crowded samples: if a large square holds more than about 100 cells, cells overlap and get missed. Dilute, recount, and enter the new dilution factor.
- Trypan blue: a 1:1 mix with 0.4 % trypan blue is a dilution factor of 2. Count within a few minutes, because live cells start taking up the dye and viability falls the longer they sit.
- Mix before loading: cells settle in the tube within seconds; pipette up and down, then fill the chamber in one smooth stroke without overfilling.
Which square did you count?
| Chamber and square | Area | Depth | Volume per square | Factor (× count per square) |
|---|---|---|---|---|
| Improved Neubauer or Bürker, large square | 1 mm² | 0.1 mm | 0.1 µL | 10⁴ |
| Improved Neubauer, one of the 25 centre squares | 0.04 mm² | 0.1 mm | 4 nL | 2.5 × 10⁵ |
| Improved Neubauer, smallest square | 0.0025 mm² | 0.1 mm | 0.25 nL | 4 × 10⁶ |
| Fuchs-Rosenthal, 1 mm square | 1 mm² | 0.2 mm | 0.2 µL | 5 × 10³ |
The factor is per cell counted per square, before the dilution. Disposable counting slides usually copy the improved Neubauer grid at 0.1 mm depth; check the slide's insert if in doubt.
Counting cells in images instead
A hemocytometer counts cells in suspension. To count nuclei or cells in a micrograph or a whole slide, use image analysis: Fiji's Analyze Particles, QuPath, CellProfiler, or SlideScope, which counts nuclei and cells on your own computer without code and reports density per mm² from the image's calibration. See how to count cells in microscopy images.
Questions
How do you calculate cells per mL from a hemocytometer?
Average the count per square, divide by the volume of one square (area × depth, for a Neubauer large square 1 mm × 1 mm × 0.1 mm = 10⁻⁴ mL) and multiply by the dilution factor. For Neubauer large squares that is average per square × 10⁴ × dilution.
What is the dilution factor with trypan blue?
Mixing equal volumes of cell suspension and trypan blue (for example 10 µL + 10 µL) halves the concentration, so the dilution factor is 2. A 1 part cells to 4 parts dye mix is a factor of 5.
How do you calculate cell viability?
Viability (%) = live cells ÷ (live + dead cells) × 100. With trypan blue, live cells stay clear and dead cells stain blue.
How many squares should I count?
Count enough squares to reach at least 100 cells, ideally 200 to 400; the error is about 1/√N. For most cultures the four corner large squares are enough; sparse samples need all nine or both chambers.
Why multiply by 10,000?
One large square of a Neubauer chamber holds 0.1 µL (1 mm × 1 mm × 0.1 mm = 0.1 mm³). There are 10,000 of those volumes in 1 mL, so cells per square × 10,000 = cells per mL.
Is this calculator free?
Yes. It runs in your browser and sends nothing anywhere. SlideScope, the desktop microscopy app made by the same team, is a paid app with a free preview.
Sources
Results are theoretical or arithmetic; check them against your instrument's documentation before relying on them for clinical or regulated work.
Measure on your real images
SlideScope opens 34 microscope and slide-scanner formats, CZI, ND2, SVS and LIF among them, with their own calibration, so lengths, areas, cell counts and scale bars come out in µm without setting anything up. Open a few of your own images with the free preview. Already decided? Subscribe now.