Calculator
The cell count formula: cells per mL = the average count per 1 mm square × 10,000 × the dilution factor, and viability is the live cells divided by all the cells counted. Enter what you counted and the calculator works it out: live and total cells per mL, viability, the cells in your suspension and the volume to seed, with every step shown.
Updated
Cells per mL = (cells counted ÷ squares counted) × 10,000 × dilution factor
Viability (%) = live cells ÷ (live + dead cells) × 100
For the 0.2 mm squares of the centre grid, use × 250,000 in place of × 10,000.
A large square of an improved Neubauer chamber is 1 mm by 1 mm, and the chamber is 0.1 mm deep, so the sample over one square is 0.1 mm³, which is 0.1 µL. Cells per µL is the average count per square divided by 0.1, and cells per mL is a thousand times that: the average per square times 10,000. Multiply by the dilution factor to get back to the sample before it was diluted. The word is also spelled haemocytometer, in British English, or hemacytometer, and the device is also called a counting chamber or a Neubauer chamber.
Viability is the clear cells divided by all the cells counted. Trypan blue enters cells whose membrane has failed, so blue cells count as dead. Count within a few minutes of staining, because the dye eventually enters live cells too. The terms are defined in the glossary.
Live cells per mL
1.88 × 10⁶
1,880,000 cells/mL
The working
Average per square: (376 + 35) ÷ 4 = 102.75
All cells per mL: 102.75 × 10,000 × 2 = 2,055,000
Viability: 376 ÷ 411 = 91.5%
Live cells in your suspension: 1,880,000 × 10 mL = 18,800,000
Opens on the worked example from the hemocytometer guide: four corner squares, 376 clear and 35 blue, equal parts trypan blue. Free, no account.
376 clear and 35 blue cells in the four 1 mm corner squares; sample mixed one to one with trypan blue (×2).
186 clear and 14 blue cells in five 0.2 mm centre squares; one to one with trypan blue (×2).
112 clear and 28 blue cells in four 1 mm squares; diluted 1 in 10 in medium, then one to one with trypan blue (×10 × 2 = ×20).
The dilution factor is the total volume divided by the volume of sample in it: 10 µL of sample in 20 µL altogether is a factor of 2, and 10 µL in 100 µL is a factor of 10.
Equal parts sample and trypan blue is written 1:1 but multiplies the count by 2, and confusing the two halves or doubles every concentration. The calculator always shows both. For two steps, multiply them.
| What you mixed | Multiply by |
|---|---|
| Nothing added | ×1 |
| 10 µL sample + 10 µL trypan blue | ×2 |
| 10 µL sample + 40 µL medium | ×5 |
| 10 µL sample + 90 µL medium, then equal parts trypan blue | ×10 × 2 = ×20 |
Even a perfect counter is limited by sampling. 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. Counting more cells is the only way to shrink it; aim for about 100 cells per large square over four squares.
| Cells counted | Counting error alone |
|---|---|
| 25 | about ±20.0% |
| 100 | about ±10.0% |
| 400 | about ±5.0% |
| 1,000 | about ±3.2% |
If a large square holds more than about 200 cells they touch and get missed; dilute further. For small cells at high density, count squares of the 0.2 mm centre grid instead: each holds 0.004 µL, so the factor is 250,000 rather than 10,000. Two people counting the same sample by hand still differ by much more than this, which is the subject of the validation page.
CellCount does this count from a photo of the chamber, taken with a phone at the eyepiece or a camera on the microscope. It finds the grid in the image by itself, or lets you place the crossings by hand if the rulings cannot be found, then counts the clear and the blue cells, applies the dilution you set and does exactly the arithmetic above, with every cell marked on your image so you can check it. There is no tally to keep, and you can start on the Free plan with no card.