Cell culture · Counting
Hemocytometer cell count calculator.
From a chamber count to cells per mL, total cells, percent viable and the volume to seed.
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How a hemocytometer count becomes cells per mL
Each large square of the grid is 1 mm x 1 mm and the coverslip holds it 0.1 mm above the grid, so one square holds 0.1 mm3 = 100 nL = 10-4 mL. Therefore cells/mL = (cells counted x dilution factor / squares counted) x 10,000. The dilution factor is the final volume divided by the cell volume, so mixing equal parts cells and trypan blue gives 2.
Worked example. 325 cells over 5 squares with a 1:1 trypan blue mix: 325 / 5 = 65 per square, x 2 = 130, x 10,000 = 1.3 x 106 cells/mL. In 10 mL that is 1.3 x 107 cells. To seed 500,000 cells you need 0.385 mL of suspension.
Viability is unstained cells / (unstained + stained) x 100. If your chamber has a different depth or square size, change the last two boxes. To prepare a lower concentration afterwards, see the dilution calculator.
Frequently asked questions
Why multiply by 10,000?
One large square holds 10^-4 mL (1 mm x 1 mm x 0.1 mm), so multiplying the per-square average by 10^4 gives cells per mL.
How do I handle trypan blue dilution?
Include it in the dilution factor. Equal parts cell suspension and trypan blue is a factor of 2.
Which squares should I count?
Follow your lab's protocol and count the same pattern each time. The calculator just needs the total and the number of squares.
Does it work for other chambers?
Yes. Enter the square area and chamber depth for your chamber, since the multiplier depends on the volume per square.
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Research use and scope
This is arithmetic for research-bench planning, not clinical, diagnostic or safety advice. Conversion factors are standard approximations; strain, instrument, reagent and protocol differences matter. Check results against your own protocol, kit manual and lab supervisor, and follow your institution's biosafety rules.