Haemocytometer calculator
Turn a chamber count into cells per millilitre, with the counting error stated.
One square holds 100 nL, so a count over one square multiplies by 10000 per mL.
Four corner squares is the usual protocol.
2 if you mixed one to one with trypan blue.
Unstained, across all the squares.
Stained blue. Leave blank to skip viability.
Live cells per mL
95% interval 715000 to 893000 — about ±11% on 320 cells counted.
Total cells per mL
800000
80 cells per square · 320 counted · ±10% would need about 385 cells, ±5% about 1537
About the Neubauer improvedThe default in most laboratories. Count the four 1 mm² corner squares; each holds 0.1 µL, which is where the familiar × 10⁴ comes from.
cells/mL = n ÷ (squares × area × depth) × D; 95% CI from ½·χ²(α/2, 2n) and ½·χ²(1−α/2, 2n+2)- Exact confidence limits for a Poisson count — Biometrika, 1936
- Score interval for a proportion, used for viability — Journal of the American Statistical Association, 1927
- Trypan blue exclusion test of cell viability — Current Protocols in Immunology, 1997
- Counting accuracy in a Neubauer chamber — Journal of Clinical Laboratory Analysis, 2019
The interval covers counting error only — the randomness of how many cells happen to settle over the squares you read. It does not cover an unmixed suspension, cells lost to the coverslip edge, or clumps counted as one, all of which move the answer further than the statistics do. Mix well and load two chambers if the number matters.
When to use this
Use this to turn a chamber count into cells per millilitre, with an honest interval on it, and to work out viability from a trypan blue count. The interval is the reason it exists — counting is a Poisson process and almost nothing else reports the uncertainty.
Worked example
Four large squares counted on a Neubauer improved chamber, after a 1:1 trypan blue mix.
- Live / dead counted
- 180 / 20
- Squares counted
- 4
- Dilution factor
- 2
Result
The 95% interval spans roughly ±15%, and viability is 90% with its own interval.
What people get wrong
- Forgetting that trypan blue is itself a dilution. Mixed one to one it is a twofold dilution, and omitting it halves every number downstream.
- Counting 100 cells and reporting three significant figures. A hundred cells is worth about ±20%; the extra digits are not measurements.
- Using the familiar × 10⁴ on a chamber that is not a Neubauer. A Fuchs–Rosenthal is twice as deep, so the same multiplier doubles the answer.
Questions
+How many cells do I need to count?
For ±10% at 95% confidence, about 385. The conventional 100 gives roughly ±20%, which is fine for a passage and not fine for comparing two flasks.
+Why is the interval not symmetric at low counts?
Because it is the exact Poisson interval rather than count ± 1.96√count. The normal approximation is poor at low counts and can even go negative.
+Is trypan blue exclusion a good viability measure?
It is quick and adequate for routine passaging. It measures membrane integrity, so it will call an early-apoptotic cell alive.
Related tools
- Cell seeding density calculator — Work out the suspension and medium to seed a plate or flask at a target density.
- OD600 calculator — Convert optical density to cell density and plan culture dilutions.
- Doubling time calculator — Derive doubling time and growth rate from two cell counts.
Science last reviewed .