NanoPhotometer
®
N120/NP80/N60/N50/C40 User Manual Version 4.3.1
97
OD600
METHOD OVERVIEW
The growth of bacteria in liquid culture media is commonly monitored by measuring the
optical density at 600 nm (OD600) in small samples taken from the cultures. OD600
measurements are typically used to determine the stage of growth of the bacterial culture,
thereby ensuring that cells are harvested at an optimum point that corresponds to an
appropriate density of live cells. Growth of bacterial cells typically progresses through a
series of consecutive phases including: lag, log, stationary and decline (see Figure 1 on
page 98). In general, cells should be harvested towards the end of the log phase using the
optical density of the samples to determine when this point has been reached. Since optical
density in the case of OD600 measurements results from light scattering rather than light
absorption, this value varies depending on the type of bacterial cells in the culture in terms of
size and shape. Cells are routinely grown until the absorbance at 600 nm (known as OD
600) reaches approximately 0.4 prior to induction or harvesting. A linear relationship exists
between cell number (density) and OD 600 up to an absorbance value of 0.6, approximately.
As mentioned above, for turbid samples such as cell cultures, the absorbance measured is
due to light scattering, and not the result of molecular absorption. Since the extent of
scattering is affected by the optics of the system (distance between the cell holder and
instrument exit slit, monochromator optics, slit geometry, etc.), different spectrophotometer
types will tend to give different OD 600 readings for the same turbid sample. Therefore, if
results from different spectrophotometers are to be compared, they must be normalized first
using appropriate calibration curves. For more information see Technical Note #8 OD 600
which can be downloaded on the Implen webpage: www.implen.de/scientific-publications/
A calibration curve can be constructed by comparing measured OD 600 to expected OD
600. Expected OD 600 is determined by counting cell number using an alternative technique
(for example microscope slide method) and converting to OD 600 using the rule of thumb
that 1 OD 600 = 5 x 10
8
cells/ml for E. coli.
The NanoPhotometer
®
comes with a correction factor of 1 as standard. To compare OD 600
values between different spectrophotometers, it is necessary to determine the constant
deviation or ratio between the absorbance values for the same sample from each instrument
and use this factor within the setting “correction factor” of your NanoPhotometer
®
Software.
Note: The use of 10 mm path length disposable cuvettes is recommended for optical density
measurements of cell culture solutions. The amount of cells is reflected in the reading and
the likelihood of fluctuating amount of cells in a drop from sample to sample can be
considered as extremely significant. It is therefore recommended to use cuvettes since the
amount of error in a bigger volume is not as significant. The cuvette measurements provide
a bigger average and therefore more reproducible readings. Also, to prevent the suspension
settling too quickly and giving an OD reading that changes with time, glycerol should be
added to the sample.
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