How to count cells with a hemocytometer
A hemocytometer is a thick glass slide with a counting grid of known volume. Counting the cells over part of that grid tells you how many cells are in each millilitre of your suspension. With trypan blue, the same count also gives viability.
Step 1: Prepare the sample
- Resuspend the cells thoroughly so there are no clumps. Clumped cells make counts unreliable.
- Mix the cell suspension 1:1 with 0.4% trypan blue (for example 10 µL cells + 10 µL dye). This is a dilution factor of 2.
- Count within a few minutes. Trypan blue slowly becomes toxic and starts staining live cells too.
Step 2: Load the chamber
Place the coverslip on the hemocytometer, then touch the pipette tip to the edge of the coverslip and let about 10 µL flow under by capillary action. The liquid should fill the chamber without overflowing into the moats.
Step 3: Count
A Neubauer grid has nine large squares of 1 mm × 1 mm. With the 0.1 mm chamber depth, each large square holds 0.1 µL. Count the four corner squares under a 10× objective.
- Count unstained (bright) cells as live, and blue cells as dead.
- For cells touching the border lines, count those on the top and left lines but not those on the bottom and right, so no cell is counted twice.
- Aim for about 20–100 cells per square. Dilute further if there are more, concentrate if there are fewer.
Step 4: Calculate
cells/mL = average cells per large square × dilution factor × 10⁴
viability (%) = live cells ÷ (live + dead cells) × 100
Example: live counts of 52, 48, 45 and 51 in the four corner squares, plus 9 blue cells in total, after a 1:1 trypan blue dilution.
- Average live cells per square: 196 ÷ 4 = 49
- Live cells/mL: 49 × 2 × 10⁴ = 9.8 × 10⁵ cells/mL
- Viability: 196 ÷ 205 × 100 = 95.6%
The factor 10⁴ converts the 0.1 µL square volume to 1 mL (1 mL = 1000 µL = 10,000 × 0.1 µL).
Common sources of error
- Not mixing the suspension right before taking the sample. Cells settle within a minute.
- Overfilling or underfilling the chamber, which changes the volume over the grid.
- Counting too few cells. Counting both sides of the chamber and averaging improves precision.
Frequently asked questions
Why multiply by 10,000?
Each large square of a standard Neubauer chamber holds 0.1 µL. Multiplying the count per square by 10,000 converts it to cells per millilitre.
What is the dilution factor with trypan blue?
Mixing cells and trypan blue in equal volumes is a 1:1 mix, so the dilution factor is 2. If you also diluted the cells beforehand, multiply the two factors.
What viability is acceptable?
Healthy, actively growing cultures are usually above 90% viable. Many protocols require at least 80–90% before seeding or transfection.