The manual method
Mix the sample one to one with trypan blue, load about 10 microlitres under the cover glass of an improved Neubauer chamber, count the clear and the blue cells in a few 1 mm squares at 10x, and multiply the average per square by 10,000 and by the dilution factor to get cells per mL. Viability is live divided by all.
An improved Neubauer hemocytometer with its cover glass, 0.4% trypan blue, a pipette that delivers 10 microlitres, a microscope with a 10x objective, a tally counter or a pen, and a written protocol that says which squares are counted and at what dilution. The chamber is glass and reusable; the only thing you use up is the dye.
Resuspend the cells well and mix one part sample with one part 0.4% trypan blue. Count within a few minutes, because the dye eventually enters live cells too. The one-to-one mix is a dilution factor of 2; multiply in any dilution you made before it.
Place the cover glass on a clean improved Neubauer hemocytometer, touch about 10 microlitres of the mix to the edge of the cover glass and let the chamber fill by capillary action without overflowing into the moat. Give the cells a moment to settle.
Under the 10x objective, count one 1 mm square at a time; the four corner squares, or the four corners and the centre, is the usual pattern. Clear cells are live, blue cells are dead. Pick one rule for cells on a line, for example count those touching the top and left lines and not the bottom and right, and keep it.
Cells per mL = the average number of cells in one 1 mm square × 10,000 × the dilution factor, because a square over the chamber’s 0.1 mm depth holds 0.1 microlitre. Viability = live ÷ (live + dead). Aim for roughly 100 cells per square, and dilute or concentrate the sample if you are far from it.
Record the dilution, which squares, who counted and when, next to the number. Two people who follow the same written protocol get comparable counts; two blinded experts with no shared protocol have been measured to differ by about 20% on the same cultures.
Four corner squares, sample mixed one to one with trypan blue, so the dilution factor is 2.
| Square | Clear (live) | Blue (dead) | All |
|---|---|---|---|
| Square 1 | 88 | 9 | 97 |
| Square 2 | 95 | 7 | 102 |
| Square 3 | 91 | 11 | 102 |
| Square 4 | 102 | 8 | 110 |
| Total | 376 | 35 | 411 |
About 2.06 million cells per mL, of which 1.88 million are live. A seeding calculation uses the live concentration, so keep both numbers.
The method is simple and the spread is not. 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, with a stain and clumps and a live-or-dead call to make, have been measured to differ by roughly 20%. The differences come from the loading, the choice of squares, the rule for cells on a line, the dilution arithmetic and the judgement of what is a cell, and a written protocol removes most of them. The studies are cited on the validation page and the terms are defined in the glossary.
CellCount does this count from a photo of the chamber: a microscope camera on the port, a phone held to the eyepiece, or a file you already have. It measures the rulings in the image to get the scale, counts every cell inside the square, calls each one live or dead, and applies the dilution and the counting pattern from a protocol the whole lab shares, so the arithmetic above happens the same way every time. Every detected cell is marked on your image; you add, delete or flip the ones you disagree with and accept the count, and the record keeps who accepted it and when.