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P a g e
Step 1
Set wavelength. Default wavelength for this stored
method is 600 nm.
Press the down arrow.
Step 2
Enter the factor to compensate for different optical
configurations between this and other instruments.
Press the down arrow.
Step 3
Select whether or not to adjust the name of your
sample before each new run, using left and arrow
keys to select Yes or No. Press
.
Press the down arrow key.
Step 4
Select the units. Options are OD or cells/ml. If
cells/ml is selected two further parameters are
displayed.
OD600
Bacterial cell cultures 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 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 = 8 x 10
8
cells/ml for E. Coli.
This instrument has much smaller optics than most conventional spectrophotometers, and more light is transmitted
through to the detector resulting in lower than expected OD 600 values. Results obtained by comparing measured OD
600 with expected OD 600 (see above) indicate that a correction factor of 2.0 is required to make the data comparable to
larger instruments.
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 that changes with time, glycerol should be added to the
sample.
The procedure is as follows:
