Cell counting (counting chamber)
A chamber count (Neubauer, Malassez) in cells/mL, with trypan blue viability and the Poisson uncertainty.
Result
1 × 10⁶ /mL
- Calculation
- C = 200 / 0.4 µL × 2 = 1000 /µL
- Counting uncertainty
- ±7.0711 %
- Concentration per microlitre
- 1000 /µL
- Counted volume
- 0.4 µL
Note
The original suspension contains 1 × 10⁶ cells/mL.
Calculation, assumptions and sources
OverviewConcentration in cells/mL of the original suspension, from a chamber count and the dilution factor.
Concentration in cells/mL of the original suspension, from a chamber count and the dilution factor. Preset chambers: improved Neubauer large square (0.1 µL) and Malassez rectangle (0.01 µL); any other geometry can be entered. Trypan blue viability is computed when dead cells are counted. The Poisson uncertainty is 1/√N, that is ±10% for a hundred cells.
Method & formulasC (cells/µL) = counted / (units × unit volume) × dilution
The surveyed volume is units × unit volume; the dilution factor scales the concentration back to the original sample.
- Counting chamber (hemocytometer)
- a gridded slide hollowed to a calibrated depth; each grid pattern bounds a known volume under the coverslip.
- Improved Neubauer
- the most widespread chamber: nine large squares of 1 mm², 0.1 mm depth, that is 0.1 µL per large square.
- Malassez
- one hundred rectangles of 0.01 µL, one microlitre for the whole grid; the usual chamber in French-speaking labs.
- Trypan blue
- exclusion dye that only enters dead cells: live cells refractile, dead cells blue. It kills cells within minutes; count quickly after mixing.
Validity domainThe calculation assumes a homogeneous suspension (vortex, pipette without letting it settle), a coverslip seated at the right depth (interference fringes visible) and a constant edge convention (cells straddling two sides only).
The calculation assumes a homogeneous suspension (vortex, pipette without letting it settle), a coverslip seated at the right depth (interference fringes visible) and a constant edge convention (cells straddling two sides only). Clumps counted as one cell underestimate the concentration; dissociate them before counting. The Poisson uncertainty is a minimum, to which pipetting and homogeneity biases add.
Counting uncertaintyCells are distributed at random over the grid: the count follows a Poisson law with standard deviation √N, and the relative uncertainty 1/√N depends neither on the operator nor on the microscope.
Cells are distributed at random over the grid: the count follows a Poisson law with standard deviation √N, and the relative uncertainty 1/√N depends neither on the operator nor on the microscope. It is ±32% for 10 cells, ±20% for 25, ±10% for 100, ±5% for 400. Aim for about a hundred cells in total, dilute if the grid overflows, count more units if it is sparse.
Reading the resultAfter thawing, viability below 90% is normal; in routine culture, below 70% it signals a problem.
After thawing, viability below 90% is normal; in routine culture, below 70% it signals a problem.