I can't find a reference that explains what, for example, OD600 = 0.1 means.

For example, Wikipedia states

OD600 is an abbreviation indicating the absorbance, or optical density, of a sample measured at a wavelength of 600 nm.

In other words, the OD600 value reports (a function of) what percent of light goes through a sample. But that depends on the thickness of the sample. More light will go through 1 millimeter of fluid than through 1 meter of the same fluid. So what is the thickness of the sample involved when reporting OD600? Or is the OD600 independent of that, so that it actually has units that people simply omit? In that case, what are the units? What is a precise physical interpretation of what is meant by the measurement OD600 = 0.1?

Suppose OD600 = 0.1 for some sample. I shine 1 mW of 600nm laser light through a 1 cm path length of the sample. What is the transmitted power?


For most laboratory spectrophotometers the path length in cuvette that holds your sample is 1 cm, and unless another path length for the light is specified you can always assume that the Beer-Lambert Law for absorption of light is using 1 cm.

When growing a bacterial liquid culture in an incubator, the OD (wavelength is usually 550 nm or 600 nm) is a approximation of the number of cells, or colony forming units, or cfu, per mL of culture.

The recipe, or protocol, will normally specify the bacterial strain to use, and the desired OD (at which point one either harvests the growing cells, or possibly adds something else to the culture, like a drug, or a viral stock).

To actually know the number of cfu/mL for a specific bacterial strain one needs to construct a growth curve by taking aliquots at defined time points, and reading the OD, and also plating out some serial dilutions. After incubating the plates at the correct temperature one can count the number of cells per mL and plot cfu/mL vs. OD600 (for example).

  • $\begingroup$ Nitpick: I believe that OD600 correlates better with total biomass than with the number of cells (e.g. growing but non-dividing cells do show continued increase in OD). This also makes more sense intuitively (at least to me). $\endgroup$ – Victor Chubukov Jul 18 '16 at 2:59

OD600 is usually used in molecular biology as a measurement of the density of bacteria in a sample. That frequency of light typically has little other scattering and the bacterial bodies

The interpretation of a specific absorbance is by Beer's law:

A = abc

a = specific absorbance of the light at that frequency by the specimin b = the concentration of the specimen c = the path length; the length of sample along the path of light shining through the sample

In practice for OD600 an absorbance of 0.1 is usually not converted but is understood as a low concentration of bacteria in the sample. Typically E coli and most bacteria will start growing in logarithmic fashion, dividing regularly so the OD600 doubles in a period ranging from 10 minutes on up, depending on the bacteria.

Around OD600 = 1.0 the bacteria will stop dividing, which is called 'stationary phase'. In practice most measurements of OD600 are not accurate above this level in most instruments since Beer's Law is only accurate in linear form in dilute samples.

  • $\begingroup$ Suppose OD600 = 0.1 for some sample. I shine 1 mW of 600nm laser light through a 1 cm path length of the sample. What is the transmitted power? $\endgroup$ – Mark Eichenlaub Jun 16 '16 at 15:26

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