The OD is measured in a (spectro)photometer (Fig. 3). The underlying principle is that
most of the light scattered by the cells no longer reaches the photoelectric cell, so that
the electric signal is weaker than with a cell-free cuvette. The OD of a bacterial culture is
thus primarily not an absorbance, as in the case of a dissolved dye. Cells of many
bacteria are almost colorless and real light absorption is marginal. It is therefore not
correct (even though unfortunately common) to designate the OD of a culture an
absorption; the most appropriate term would indeed be turbidity. Only strongly
pigmented cells such as phototrophic (purple and green) bacteria cause a significant
absorption of light in addition to scattering. One should be aware that the photoelectric
cell does not “know” what causes the decrease in light intensity. It just notices “less light”
which is displayed as a number, usually as extinction value. But even if the display has
the label “absorption”, you know that you are dealing with turbidity.
The practical use of the photometer requires consideration of four points:
(a) The intensity of light scattering and thus the OD of a culture depend on the
wavelength. Scattering of short wavelengths (such as of blue light) is stronger than that
of long wavelengths (such as of red light). You can choose any wavelength for
measurement, but once you have chosen you must keep this wavelength for all
following measurements. This wavelength (e.g. 600 nm) should be indicated as
subscript (e.g. OD600).
(b) The OD measured by a photometer depends on the geometry of the light beam,
the position of the cuvette, and the photoelectric cell, i.e. on the photometer model and
manufacturer. Hence, you cannot change the model of the photometer within an
experiment. One should not even change between two photometers of the same model
because there can be slight differences