OD600: What It Measures and Why It Is Not a Cell Count

OD600 is the optical density (absorbance) of a culture measured at a wavelength of 600 nm. It is a relative turbidity reading, not a cell count. It tracks cell density only within an instrument’s linear range, and converting it to cells/mL requires a calibration for your own instrument, organism and conditions. For laboratory use only; this page gives no medical advice.

Definition and scope

OD600 is a spectrophotometer or plate-reader measurement at 600 nm. A bacterial or yeast culture scatters light, so the reading rises as the culture gets denser, even though little light is truly absorbed. The general relationship between absorbance and transmitted light is covered in absorbance and optical density, and the instrument itself in UV-Vis spectrophotometer basics; this page is about what the 600 nm number does and does not tell you.

“Original cell density”

The phrase usually means the density of the undiluted culture. If a sample is diluted so the reading falls in the instrument’s linear range, the reading describes the diluted sample, and the dilution factor has to be applied to get back to the original culture. A cell dilution calculator covers that arithmetic.

How OD600 is used in practice

OD600 is a quick, non-destructive way to follow culture growth and to decide when to sample or dilute. Its value as a number depends on the instrument: Beal et al. (2020) report that readings are “relative to the configuration of a particular instrument”, and Stevenson et al. (2016) state that different spectrophotometers need to be cross-calibrated even in the single-scattering regime. A 0.4 on one instrument is therefore not automatically a 0.4 on another.

OD600 to cells/mL: why there is no single factor

Two peer-reviewed studies agree on the core points:

• Linearity is limited. Beal et al. found OD is not even linearly related to cell count except within a limited range. Stevenson et al. describe the Beer-Lambert relationship as applying, with some considerations, only to low-density cultures, roughly up to OD600 of about 0.2 (single scattering). Above that, OD is expected to depend on cell number in a parabolic rather than linear way.
• The factor depends on the cells. Stevenson et al. conclude that the most practical approach is to calibrate, once for each cell size and refractive index. If cell size changes (their example is antibiotic-induced filamentation), OD is unreliable and cells should be counted directly.
• The factor depends on the instrument. See above: instrument configuration changes the reading.

So a rule of thumb such as “OD600 of 1 equals a fixed number of cells/mL” cannot be applied to every strain and instrument. A conversion is only defensible when it comes from a calibration done with your own setup.

Calibration methods compared

Beal et al. (2020) compared three ways of calibrating OD600 across 244 laboratories using E. coli strains. The table summarises what that paper reports.

Method	What it gives	Findings reported by Beal et al.
Silica microsphere serial dilution (0.961 µm beads)	Particles per OD600 unit	Recommended by the authors; 95.5% of results within 1.2-fold using their pipetting-error model; lab-to-lab geometric SD about 2.2-fold.
LUDOX in water	A single reference point	Cheap and precise, but its particles (under 50 nm) mean it cannot estimate cell number; lab-to-lab geometric SD about 2.2-fold.
Colony-forming units (CFU)	Viable cells per mL	Counts viable cells only and cells-per-colony is uncertain; per-cell fluorescence was 28.4-fold higher than by flow cytometry, indicating CFU underestimates cell number; only 7.3% of replicate sets within 1.1-fold; geometric SD about 2.4-fold.

The authors also note that calibrated results are reliable only within the instrument’s linear range: dilution levels where the signal saturates show insufficient slope and should not be used.

Where it matters: what to report with an OD600 value

This checklist is derived from the findings above (editorial synthesis of the two papers, not a published standard):

1. The instrument and whether the reading was taken in a cuvette or a plate reader (Stevenson et al. show that plate-reader readings need instrument- and plate-specific corrections).
2. Whether the reading was inside the linear range, and the dilution factor if the sample was diluted.
3. The calibration reference used (microspheres, LUDOX or CFU) and the unit it yields (particles per OD600 unit, or CFU/mL).
4. The organism and growth conditions, since cell size and refractive index change the conversion.

For related turbidity standards, see the 0.5 McFarland standard; for direct counting, see what a hemocytometer is. Growth readings are often paired with kill-curve work such as time-kill assays.

Common misunderstandings

• “OD600 counts cells.” It measures light scattering, which only approximates cell number within a limited range.
• “A higher reading is a more informative result.” Past the linear range the relationship is no longer linear, so dilute and re-read.
• “CFU is the ground truth.” CFU counts only viable cells and, in Beal et al., underestimated cell number relative to flow cytometry.
• “One conversion works for every lab.” The calibration depends on instrument and cell properties.

FAQ

What does OD600 mean?

Optical density at 600 nm: the absorbance reading of a culture at that wavelength, used as a relative measure of turbidity and therefore of cell density.

How do I convert OD600 to cells/mL?

There is no universal factor. Stevenson et al. recommend calibrating for your own instrument and cell type; Beal et al. compare microsphere, LUDOX and CFU calibrations (table above).

What is original cell density?

The density of the undiluted culture, recovered by multiplying a diluted sample’s result by its dilution factor.

What is the linear range of OD600?

Stevenson et al. place the single-scattering, roughly linear regime at about OD600 of 0.2 or below; your own instrument’s range should be checked with a dilution series.

Sources

• Beal J et al. Robust estimation of bacterial cell count from optical density. Communications Biology 2020;3:512. doi:10.1038/s42003-020-01127-5 (read 2026-11-07).
• Stevenson K et al. General calibration of microbial growth in microplate readers. Scientific Reports 2016;6:38828. doi:10.1038/srep38828 (read 2026-11-07).