Estimate the number of bacterial cells per millilitre from an OD600 reading, or work backwards from a target cell density to the OD you need. Enter a value below; the conversion factor is adjustable so you can match your strain and spectrophotometer.
Edit OD600 or cells/mL. Default factor 8×108 cells/mL per OD 1.0 is a common value for E. coli; it varies by strain, growth phase and spectrophotometer — calibrate for accurate work. Reliable only in the linear range (roughly OD < 0.8; dilute and re-read above that).
OD600 and cell number
Optical density at 600 nm (OD600) measures how much light a culture scatters, which is proportional to cell density within a limited range. A widely used rule of thumb for E. coli is that OD600 of 1.0 ≈ 8 × 108 cells/mL, but published factors range from about 5 × 108 to 1 × 109. The exact value depends on the strain, growth phase, cell size and — importantly — the optics of your specific instrument, so the factor in the tool is editable.
Stay in the linear range
The OD-to-cell relationship is only linear at low densities, roughly OD600 < 0.8. Above that, cells shadow one another and the reading underestimates the true density. For dense cultures, dilute (e.g. 1:10), read again, and multiply the result by the dilution factor.
Cells vs CFU
OD measures total cells (live and dead). Colony-forming units (CFU/mL) count only viable, culturable cells and are determined by plating. The two are correlated but not identical; for healthy exponential-phase cultures they are close, while in stationary or stressed cultures CFU can be substantially lower than the OD-based total. For viable counts, a plate assay is the reference method.
Calibrating your own factor
For accurate work, calibrate once: measure OD600 of several dilutions, plate to count CFU/mL, and fit the slope (cells or CFU per OD unit). Enter that slope as the factor here and your conversions will match your lab’s conditions.
Frequently asked questions
How many cells is OD600 = 1?
For E. coli, commonly about 8 × 108 cells/mL, though values from 5 × 108 to 1 × 109 are reported. Adjust the factor for your strain and instrument.
How do I convert OD600 to CFU/mL?
Use a CFU-based factor (from plating) instead of a total-cell factor. CFU counts only viable cells and is best determined empirically for your culture.
My culture reads OD600 = 2. Is that accurate?
Probably not — that is above the linear range. Dilute (e.g. 1:10), re-read, and multiply by the dilution factor.
Does the factor depend on the spectrophotometer?
Yes. Path length and optics differ between instruments, so calibrate the factor on the machine you use.
Related guide: DNA & RNA quantification →
