NanoPhotometer
®
P-Class User Manual
Version 2.1 Page 36 / 70
4.3 Bacterial Cell Culture Measurement (OD600)
4.3.1 General Information
The stage of growth of a bacterial culture needs to be monitored to ensure that the cells are harvested at the
optimum point for the greatest density of live cells. An exemplary growth curve is given below. Cells should be
harvested towards the end of the log phase. The optical density of the sample indicates when this point has been
reached. This value varies dependent on the cells being grown. Routinely the cells are 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 approx. 0.6
It is important to note that for turbid samples such as cell cultures, the absorbance measured is due to light
scattering, and not the result of molecular absorption. The amount of scatter is affected by the optics of the system
(distance between the cell holder and instrument exit slit, geometry of this slit and the monochromator optics).
Different spectrophotometer types therefore give different responses for the same turbid sample; to compare
results, they must be normalized using calibration curves.
A calibration curve can be determined 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.
Additionally your NanoPhotometer
®
P-Class is coming with a correction factor of 1 as default. To compare OD
values between different spectrophotometer, you have to determine the constant deviation between the
Absorbance values for the same sample within those instruments and use this factor within the setting “correction
factor” of your NanoPhotometer
®
P-Class Software.
The use of 10 mm pathlength disposable cells is recommended for optical density measurements of cell culture
solutions; to prevent the suspension settling too quickly and giving an OD that changes with time, glycerol should
be added to the sample.
The Submicroliter Cell is not recommended for optical density measurements of cell culture solutions.
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