OD600 to Cell Count Calculator
Convert an OD600 absorbance reading into cells per millilitre and total cells using a calibration factor.
How it works
cells/mL = OD600 × calibration factor · total cells = cells/mL × volume
Optical density at 600 nm measures light scattered by cells, and over the linear range (roughly OD 0.1–0.8 for E. coli) it is proportional to cell concentration. The calibration factor — cells per mL at OD 1.0 — must be measured for your own strain, instrument and path length, because published values range from about 5 × 10⁸ to 1 × 10⁹. Above the linear range the reading saturates and underestimates the true count, so dense cultures should be diluted before reading.
Worked example
An OD600 of 0.5 with a factor of 8 × 10⁸ cells/mL per OD gives 0.5 × 8 × 10⁸ = 4 × 10⁸ cells/mL. In a 10 mL culture that is 4 × 10⁸ × 10 = 4 × 10⁹ cells.
Frequently asked questions
Is the OD-to-cell factor universal?
No. It depends on the organism, cell size and shape, the spectrophotometer and the cuvette path length. Calibrate against colony counts or a counting chamber for your own setup.
Why dilute a dense culture before reading?
Above about OD 0.8 the linear relationship breaks down as scattered light is re-scattered, so the reading underestimates cell number. Diluting back into the linear range and multiplying by the dilution restores accuracy.
Does OD600 count dead cells?
Yes — scattering does not distinguish live from dead cells, so OD600 reports total biomass. For viable counts use CFU plating instead.
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This calculator is for educational purposes. Double-check important results before acting on them.