How to use
- Enter the cells counted in each large square. Separate them with commas, spaces or line breaks, or paste them straight out of a spreadsheet.
- Enter the dilution factor. The default is 1. Put 2 if you mixed 1:1 with trypan blue.
- The cells/mL appears large, with the mean count and the number of squares counted alongside.
- Add the dead cell count and viability is worked out. (optional)
- Add the total volume of the suspension and the total cell count is worked out. (optional)
- Add the number of cells you need and you get the volume to take. (optional)
Exponents can be entered with e — for example, 1.5×10⁻⁵ is entered as 1.5e-5.
Cell Counting Formula and practical notes
A haemocytometer is a very small chamber of precisely known volume. Count the cells inside it and you know the concentration.
A large square on a standard haemocytometer is 1 mm by 1 mm, and the coverslip sits 0.1 mm above it.
- Volume over one square
- 1 mm × 1 mm × 0.1 mm = 0.1 mm³ = 0.1 μL = 10⁻⁴ mL
So the cells counted in one large square are the cells in 10⁻⁴ mL, and turning that into a per-mL concentration means multiplying by 10⁴.
- Concentration
- cells/mL = mean count per large square × dilution factor × 10⁴
The 10⁴ comes from the dimensions of the chamber. It is not an arbitrary constant.
Everything else follows from there.
- Total cells
- total cells = cells/mL × volume(mL)
- Viability
- viability(%) = live ÷ (live + dead) × 100
- Volume to take
- volume = cells needed ÷ cells/mL
Practical notes
- The dilution factor defaults to 1. If you mixed 1:1 with trypan blue you must change it to 2. Leave it and the count comes out at half the real value. A wrong cell count does not look wrong, so this mistake tends to surface much later.
- Counting 20 to 250 cells per square is the usual practice. Too few and chance dominates; too many and they overlap and get missed. Outside that range, dilute or concentrate the sample and count again.
- Decide a rule for cells on the lines and keep to it. The common one is to count cells touching the top and left lines and not those on the bottom and right. Results diverge when people use different rules.
- Mix thoroughly before loading. Cells settle. Pipette gently a few times and load immediately.
- Trypan blue stains living cells too, given time. Count within three to five minutes of mixing. Leave it longer and viability reads lower than it is.
- Clumped cells make counting hard. Disperse to single cells before you count, and decide in advance whether a clump counts as one or as its cells.
- The reason for counting several squares is to average them. The four corner large squares are the usual choice. A large spread between squares means the sample was not mixed or not dispersed well.
FAQs
1 if you did not dilute. 2 if you mixed 1:1 with trypan blue. 10 if you added 90 μL of trypan blue to 10 μL of cell suspension. Miss this out and the result comes out low by exactly that factor.
Because one large square holds 10⁻⁴ mL. It is 1 mm by 1 mm with a depth of 0.1 mm, which is 0.1 μL, which is 10⁻⁴ mL. The number comes from the dimensions of the chamber.
The four corner large squares, usually. More squares steady the average but take longer. Values that differ a lot between squares are a sign of a mixing or dispersion problem.
Above 250 they overlap and are easily missed. Dilute the sample further, count again, and raise the dilution factor to match.
Below 20 the result is at the mercy of chance. Concentrate the sample, or count more squares to steady the average.
There is no single right answer — what matters is picking a rule and keeping to it. The common one is to count the top and left borders and not the bottom and right. That way no cell is counted twice or missed.
It may genuinely be low, but trypan blue also stains living cells if they sit in it. Check that you counted within three to five minutes of mixing.
Enter the number of cells you need and you get the volume to take. For 5 × 10⁵ cells from a suspension at 1.72 × 10⁶ cells/mL, for example, that is about 291 μL.
The counting methods differ, so some difference is expected. Automated instruments handle clumps and debris differently and focus on a different basis. Better to measure consistently by one method and record which method it was.