OD600 to Cells/mL Calculator - Cell Density and Back-Dilution
Turn an OD600 reading into a cell density, a total cell count, and the volumes for a back-dilution.
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An OD600 reading is a scattering measurement, not a cell count, and two things silently turn it into the wrong number. Above about 0.8 in a 1 cm cuvette absorbance stops being linear in cell density, so a dense culture reads LOWER than it is and every count derived from it is an underestimate. And a plate reader does not have a 1 cm path at all - it measures through the liquid column, roughly 0.58 cm for 200 uL in a 96-well plate, so the same culture reads about 40% low. This converts the reading with both corrections applied, says which of them it had to assume, and will also work out how much culture to carry into a fresh flask to start at a target OD.
A plate reader measures through the liquid column, not a 1 cm cuvette.
10 means you read a 1:10 dilution.
Fill this in to get the volumes for a fresh flask.
From E. coli convention (8.0e+8 cells/mL per OD). Factor 8.0e+8 cells/mL per OD.
- E. coli: Commonly quoted range 5×10⁸–1×10⁹ cells/mL per OD; varies with strain and growth phase.
How to use the OD600 to Cells/mL tool
- 1Enter the OD600 your spectrophotometer or plate reader showed.
- 2Pick the organism, or enter your own cells-per-OD factor if you have calibrated against plate counts.
- 3Say what you read it in - a cuvette or a plate - and any dilution you made before reading.
- 4Read the cell density, and add a culture volume for a total count or a target OD for a back-dilution plan.
Frequently asked questions
How many cells are in an E. coli culture at OD600 = 1.0?
About 8 x 10^8 cells/mL is the figure in common use, with a literature spread of roughly 5 x 10^8 to 1 x 10^9. It is a convention, not a constant: it moves with strain, growth phase, medium and the instrument's optics. Treat a cell count from OD as an order-of-magnitude answer unless you have calibrated the factor against plate counts on your own reader.
Why does my plate reader give a lower OD600 than a cuvette?
Because absorbance is proportional to the optical path, and a plate reader's path is the depth of liquid in the well rather than a cuvette's fixed 1 cm. Two hundred microlitres in a standard 96-well flat plate is roughly 0.58 cm, so the reading is about 0.58 times the cuvette value for the same culture. Set the path length here and the reading is corrected back.
Why should I dilute a culture before reading it?
Above roughly OD 0.8 in a 1 cm path, light scattered by one cell gets scattered again by another and some of it still reaches the detector, so absorbance stops rising in proportion to cell density. The culture reads lower than it is, and the error grows as it gets denser. Dilute into the linear range, read, and multiply back - which is what the dilution factor field does.
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