Biology / Cell culture
Hemocytometer Calculator (Cells per mL and Viability)
Turn hemocytometer counts into cells per millilitre: enter the live and dead cells in each large square and the dilution, and get the live and total concentration, viability, and the cells in your suspension.
Hemocytometer Calculator (Cells per mL and Viability): On an improved Neubauer chamber, each large square holds 0.1 µL, so cells per mL = average count per square × dilution × 10⁴. Counts of 52, 47, 55, and 49 live cells with a 2× trypan blue dilution give 50.75 × 2 × 10⁴ = 1.015 × 10⁶ live cells per mL; with 18 dead cells among them, viability is 203 ÷ 221 = 91.9%. Runs 100% locally in your browser with zero server file uploads.
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On an improved Neubauer chamber each large corner square holds 0.1 µL (1 mm × 1 mm × 0.1 mm), so cells per mL = average count per square × dilution factor × 10,000. Count the four corner squares, including cells on two of the edges but not the other two, and aim for 20–200 cells per square; dilute more if there are more. Trypan blue enters dead cells and stains them blue; mixing equal volumes doubles the dilution.
Seeding a plate
To seed 2 × 10⁵ cells per well from a suspension at 1.015 × 10⁶ live cells per mL, add 0.197 mL, about 197 µL, to each well.
To dilute the suspension to a target density, use the dilution calculator.
Improving accuracy
Mix the suspension well before sampling, load the chamber in one smooth flow, and count both chambers: the counting error falls as the square root of the number of cells counted, so 400 cells give about ±5%.
The average calculator also shows the spread between squares, which flags uneven loading.
How to use it
- Count the cells in each large corner square, live and dead (stained) separately.
- Enter the counts and the dilution factor, 2 for an equal mix with trypan blue.
- Read the live cells per mL, the viability, and the total.
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Frequently asked questions
Which squares should I count?
The four large corner squares (or the centre square's 25 small squares for small cells, in which case enter the total as one square and adjust); count cells touching the top and left lines but not the bottom and right.
How many cells should a square have?
About 20–200 for an accurate count; dilute more if there are more, or count more squares if there are fewer.
What viability is acceptable?
It depends on the cells and the experiment; above 90% is common for healthy cultures and below 80% suggests a problem.
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