The overview
Cell counting measures how many cells are in a millilitre of suspension, and usually how many of them are alive. Labs count before they seed, passage, transfect, freeze or dose. The common methods are a hemocytometer under a microscope, an image-based automated counter, flow cytometry and electrical impedance; for microbes, optical density and plate counts.
Updated
Almost every cell experiment starts from a number of cells: so many per well, per flask, per vial frozen or per dose. If the count is off, everything downstream is off by the same factor, and an experiment repeated next month starts from a different place without anyone noticing. The count is usually two numbers, the concentration in cells per mL and the viability, the share of cells that are alive, and a seeding calculation uses the live concentration.
| Method | What it reads | Live and dead | The catch |
|---|---|---|---|
| Hemocytometer, counted by eye | Cells in a known volume of a ruled glass chamber, counted under a microscope. | Yes, with trypan blue. | Slow, and two people counting the same sample get two numbers. |
| Image-based automated counter | A camera images a slide or chamber and software counts the cells. | Yes, with trypan blue or fluorescent stains. | A dedicated instrument, often with its own disposable slides. |
| Flow cytometry | Cells pass one at a time through a laser; the count comes from a known volume or reference beads. | Yes, with a viability dye. | An expensive instrument and a trained operator; worth it when you need to know what the cells are. |
| Electrical impedance (the Coulter principle) | Each cell passing through a small aperture changes the electrical resistance, which gives its count and size. | Not on its own. | Debris and clumps of a similar size count as cells. |
| Optical density | How cloudy a culture is, usually at 600 nm, converted to cells with a calibration curve. | No. | An estimate for bacteria and yeast growth curves, not a count. |
| Plate count | Colonies grown from a diluted sample, reported as colony-forming units. | Only cells that grow are counted, so it is a viable count by design. | A day or more before there is a number. |
The hemocytometer is the reference most protocols are written for, and the method teaching labs start with. How to count cells with a hemocytometer, step by step.
For a hemocytometer, every count becomes a concentration the same way: cells per mL = the average count per 1 mm square × 10,000 × the dilution factor. A 1 mm square under a cover glass 0.1 mm above the floor of the chamber holds 0.1 µL, a ten-thousandth of a millilitre, which is where the 10,000 comes from. For the 0.2 mm squares of the centre grid, each holds 0.004 µL and the factor is 250,000.
The dilution factor is the total volume divided by the volume of sample in it, so equal parts sample and trypan blue is a factor of 2. The formula with worked examples, and a calculator for your own count.
Viability is the share of cells that are alive: live ÷ (live + dead) × 100. With trypan blue, a live cell keeps the dye out and looks clear, and a cell whose membrane has failed takes it up and looks blue. Count soon after mixing, because the dye slowly gets into live cells too, and the viability you read falls the longer the sample sits. Trypan blue exclusion, defined.
Cells settle in a chamber at random, so a count of N cells carries an error of about one in √N before anyone makes a mistake: about ±10% for 100 cells, about ±5% for 400. Counting more cells is the only way to shrink it, which is why protocols ask for several squares and a dilution that puts roughly 100 cells in each.
On latex beads of a known concentration, the cleanest possible case, one lab re-counting the same sample spreads by ~5%, and different labs by 14.6%. Two blinded experts counting the same cell cultures have been measured to differ by roughly 20%. The loading, the choice of squares, the rule for cells on a line, the dilution arithmetic and the judgement of what is a cell all add to it. The studies are cited on the validation page.
CellCount is a web app with a free plan that counts cells in hemocytometer images. Photograph the chamber with a phone at the eyepiece or a microscope camera; CellCount finds the grid by itself (if the rulings cannot be found, you can place the crossings by hand), counts live and dead cells by trypan blue exclusion, and gives viability and cells per mL with the dilution you set.
Every detected cell is marked on your image, you correct the ones you disagree with, and the lab shares one protocol for the squares, the dilution and the sensitivity, so the count is done the same way every time.