OD600 calculator
Convert optical density to cell density and plan culture dilutions.
Exactly what the instrument displayed.
1 if you read it neat, 10 for a 1 in 10.
1 cm unless you are using a short-path cuvette.
Commonly reported between 2 × 10⁸ and 2 × 10⁹.
Optional, for a total cell count.
Culture density
Corrected to a 1 cm path and back to the undiluted culture.
Cells per mL
3.2 × 108
Beam saw 0.4 per cm · culture OD 0.4
About Escherichia coliThe 8 × 10⁸ figure assumes exponential growth in rich medium on a 1 cm benchtop spectrophotometer. Slow-growing cells are smaller, so the same OD represents more of them.
OD_culture = OD_read ÷ ℓ(cm) × D; cells/mL = OD_culture × F; ℓ_well = V ÷ πr²- Cells per millilitre at OD600 = 1.0 — BioNumbers, Nucleic Acids Research, 2010
- Path length and scattering in optical density measurements — BMC Biophysics, 2013
- Non-linearity of optical density in microplate readers — Scientific Reports, 2016
OD600 measures scattered light, not absorption, so the cells-per-OD factor belongs to an instrument as much as to an organism, and it drifts with cell size as growth rate changes. Treat the count as an estimate good to a factor of about two unless you have calibrated against plate counts or a haemocytometer on the instrument you are using.
When to use this
Use this to correct an optical density reading for dilution and path length, to estimate cell density from it, and to plan a dilution to a target OD. Remember it is a scattering measurement, not an absorbance one, so any conversion to cells per millilitre is a calibration rather than a constant.
Worked example
An overnight culture read after a 1 in 10 dilution.
- Reading
- 0.24
- Diluted before reading
- 10-fold
- Read on
- Cuvette, 1 cm
- Organism
- E. coli, 8 × 10⁸ cells/mL per OD
Result
About 1.9 × 10⁹ cells/mL. The reading of 0.24 is inside the linear range, so the figure is sound.
What people get wrong
- Reading a dense culture neat. Above about 0.4 on a 1 cm path the detector under-reports badly; dilute into the linear range and multiply back.
- Applying a cuvette assumption to a plate reader. A 200 µL well is roughly 0.6 cm deep, so a plate reading of 0.3 is about 0.5 per centimetre.
- Quoting a conversion factor to three significant figures. It moves with strain, growth phase and instrument; calibrate against plate counts if the number matters.
Questions
+Why do my spectrophotometer and plate reader disagree?
Different path length and different collection geometry. Cells scatter rather than absorb, so the fraction of scattered light reaching the detector depends on the instrument.
+What is the linear range?
Roughly up to 0.4 absorbance on a standard 1 cm path. The check belongs on the reading the instrument took, not on the corrected culture value.
+How do I get to a target OD for an induction?
Switch the tool to the dilution mode: give it your current OD, the target and the volume you want, and it returns the culture and medium volumes.
Related tools
- Dilution calculator — Work out how much stock and diluent to combine for a target concentration.
- Serial dilution calculator — Plan a dilution series and get the transfer volume for every step.
- Doubling time calculator — Derive doubling time and growth rate from two cell counts.
- Centrifuge RCF and RPM converter — Convert between relative centrifugal field and rotor speed, for a stated radius.
Science last reviewed .