Implen OD600
®
User Manual Version 1.0
18
4. Implen OD600
®
Applications
The Implen OD600
®
comes with two pre-programmed applications, the OD600 application and the McFarland
application. Any application method can be selected by tapping the icon once.
OD600 Application
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). 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 OD600) reaches approximately 0.4 prior to induction or harvesting.
A linear relationship exists between cell number (density) and OD600 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 OD600 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.
A calibration curve can be constructed by comparing measured OD600 to expected OD600. Expected OD600 is
determined by counting cell number using an alternative technique (for example microscope slide method) and
converting to OD600 using the rule of thumb that 1 OD600 = 5 x 10
8
cells/ml for E. coli.
Note: The use of 10 mm path length disposable cells is recommended for optical density measurements of cell culture
solutions. To prevent the suspension settling too quickly and giving an OD reading that changes with time, glycerol should
be added to the sample.
Figure 1: Bacterial growth curve
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