Cell culture
Cell counting
Turn a haemocytometer count into a concentration and a total, and work out how much suspension to take for a given number of cells. If your counter already reports a concentration, switch to the second mode and skip the grid arithmetic.
Total and volume to take
The total is the concentration times the volume you enter. The last figure is how much of this suspension to pipette to obtain the target number of cells.
The 10⁴ factor is fixed by the standard Neubauer chamber, where each large corner square holds 0.1 microlitre. The dilution factor is the fold dilution of your sample before loading — 2 for a 1:1 mix with trypan blue. Count several squares and average; the more cells counted, the smaller the counting error.
Formula
concentration = (cells counted ÷ squares counted) × dilution factor × 10⁴
- cells counted
- the total counted across the large squares you scored
- squares counted
- how many large corner squares you counted, usually four
- dilution factor
- the fold dilution before loading; 2 for a 1:1 mix with trypan blue
- 10⁴
- fixed by the chamber: each large square holds 0.1 microlitre, and there are 10⁴ of those in a millilitre
Worked example
A four-square count with a 1:1 trypan mix
- 180 cells over 4 squares
- dilution factor 2
- suspension volume 10 ml
- Average per square: 180 divided by 4 is 45.
- Concentration: 45 times 2 times 10⁴ is 9 × 10⁵ cells per ml.
- Total: 9 × 10⁵ times 10 ml is 9 × 10⁶ cells.
Answer 9 × 10⁵ cells per ml, 9 × 10⁶ in total
Common mistakes
- Forgetting the dilution factor. If you mixed one part cells with one part trypan blue, the sample is twice diluted; leaving the factor at 1 halves your count.
- Counting too few cells. Below about ten per square the count is dominated by chance; count more squares or a denser sample so the total counted runs into the hundreds.
- Counting clumps as one. Trypsinise to a single-cell suspension first, or the concentration reads low and the viability reads wrong.
Frequently asked
Which cells on the boundary do I count?
Pick two sides of each square, say top and left, count cells touching those, and ignore the other two. Applied consistently this avoids counting the same cell twice.
Does the 10⁴ factor ever change?
Only if your chamber is not the standard 0.1 mm depth. For an ordinary improved Neubauer it is fixed. If you use a different chamber, its own factor applies.
Why average several squares?
Cells settle unevenly. Averaging four corners and, ideally, repeating the fill evens out both pipetting and distribution error.