Hemocytometer Calculator: Cells per mL, Viability and Seeding

A hemocytometer is a thick glass slide with a grid etched into a chamber of known depth. Count the cells over part of the grid and you know how many sit in a known volume, and so in your whole tube. The calculator does that arithmetic and then plans the seeding.

How to count cells on a hemocytometer

These steps assume the common improved Neubauer chamber and trypan blue.

1. Clean the chamber and coverslip with 70 percent ethanol, dry them and seat the coverslip over both counting areas.
2. Mix the cell suspension well, since cells settle within minutes. Take a sample.
3. Mix the sample with an equal volume of trypan blue (a 0.4 percent solution is the usual stock). Equal volumes make a dilution factor of 2.
4. Touch the tip to the edge of the coverslip and let about 10 µL run under it. Stop before liquid spills into the side channels; overfilling changes the depth.
5. Under a 10× objective, find the grid. It has nine large squares, each 1 mm × 1 mm. Count the four corner squares, each split into 16 smaller squares.
6. For cells touching the boundary lines, count those on the top and left lines and skip those on the bottom and right lines. Any consistent two-sides rule works; it stops edge cells being counted twice or missed.
7. Count clear, bright cells as live and blue cells as dead, keeping two tallies.
8. Count within a few minutes. Trypan blue slowly enters healthy cells too, so a slide left sitting reads low viability.

If the squares are nearly empty or crowded, dilute or concentrate and reload (see the FAQ for a target).

Why the formula multiplies by 10^4

Each large square covers 1 mm² and the chamber is 0.1 mm deep, so the liquid above one square is 0.1 mm³. That is 0.1 µL, or 10⁻⁴ mL. Cells per square divided by 10⁻⁴ mL is the same as cells per square times 10,000, which gives cells per mL.

• cells/mL = (cells counted ÷ large squares counted) × dilution factor × 10^4
• viability (%) = live ÷ (live + dead) × 100
• cells needed = seeding density (cells/cm²) × growth area per well (cm²) × number of wells
• volume to seed = cells needed ÷ live cells per mL

The fields and their defaults

The growth areas are typical for standard plasticware; brands differ slightly.

Worked example with the default values

• Live cells per mL: (180 ÷ 4) × 2 × 10^4 = 45 × 2 × 10,000 = 900000
• Total cells per mL, live plus dead: (200 ÷ 4) × 2 × 10^4 = 1000000
• Viability: 180 ÷ 200 × 100 = 90 %
• Live cells in the 5 mL tube: 900000 × 5 = 4500000
• Cells needed: 20000 × 1.9 cm² = 38,000 per well, × 24 wells = 912000
• Volume to seed: 912000 ÷ 900000 = 1.013 mL, which the tool shows as 1010 µL (it rounds to three significant figures)
• Per well: 1013.3 µL ÷ 24 = 42.2 µL, shown as "Per well or vessel: 38000 cells in 42.2 µL"

Each well gets 42.2 µL of suspension, topped up with medium. Diluting the whole amount first is easier: at 0.5 mL per well, bring about 1.01 mL of suspension to 12 mL with medium and dispense 0.5 mL per well. If the volume to seed is larger than the suspension you have, the calculator warns you.

Common mistakes

• Counting nine squares but entering 4, or counting the small central squares as if they were large ones. The 10^4 factor holds only for 1 mm² squares.
• Forgetting the trypan blue dilution, which halves every result.
• Sampling an unmixed suspension, or overfilling the chamber.
• Seeding from the total count. The calculator seeds from live cells per mL.

Limits

A manual count carries chance error: about 10 percent with 100 cells counted (the square root of 100, over 100) and about 5 percent with 400. Trypan blue only marks cells whose membranes have already failed, so early apoptotic cells still look alive and viability can read high. Manufacturing labs that release cell therapy doses add viability dyes, automated counters and marker counts, outlined on our stem cell technology page. To follow how fast a culture grows between counts, take two counts into the doubling time calculator. If you pellet and resuspend cells before counting, the rpm to × g converter sets a gentle spin.

FAQ

Which squares do I count on a hemocytometer?

For most cultured cells, the four large corner squares of the improved Neubauer grid, each 1 mm × 1 mm. Very dense or very small cells are often counted in the central large square instead. Enter however many large squares you counted, and the calculator divides by that number.

How do I count cells that touch the lines?

Count the cells touching the top and left boundary lines of a square and leave out those touching the bottom and right lines. The exact pair of sides does not matter as long as you use the same rule every time, so no cell is counted twice.

Why multiply by 10,000?

The liquid above one large square is 1 mm × 1 mm × 0.1 mm, which is 0.1 µL or 0.0001 mL. Cells per square times 10,000 converts that tiny volume to cells per mL.

How do I calculate viability with trypan blue?

Divide the unstained (live) count by the total of live plus blue (dead) cells and multiply by 100. With 180 live and 20 dead cells, viability is 90 percent. Count within a few minutes of adding the dye, as live cells slowly take it up.

How many cells should I count?

Aim for roughly 20 to 50 cells per large square and about 100 or more in total. Fewer cells make the count noisy, and very crowded squares are easy to miscount.