OD600 DiluPhotometerTM User Manual
Version 1.3 Page 9 / 14
4. OD600TM - DILUCELLTM APPLICATION
4.1 Bacterial Cell Culture Measurement
4.1.1 Description
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 = 8 x 108cells/ml for E. coli. Clumping together of cells
will also affect readings, so the medium they are suspended in will also make a difference.
4.1.2 Operation Instructions
Making a measurement:
Step 1 Switch the instrument on by pressing the ON/OFF button.
Step 2 Place a reference into the cuvette compartment.
Step 3 Press the blank button. The display will show 0.00.
Step 4 Remove the reference and replace with the sample solution in a cuvette.
Step 5 Press the sample button. The display will show the OD of the sample in absorbance units.
Important Information:
If using DiluCellTM remember to multiply the reading from the OD600TM after the measurement with the
proper virtual dilution factor:
Virtual dilution factor are: 10 for DiluCell™ 10
20 for DiluCell™ 20
Multiple samples can be compared with the same reference by placing different samples in the cuvette chamber and
making measurements for each one. It is recommended to re-reference with the reference solution every 10 to 15
minutes to avoid any slow instrument drift. Please re-reference always if in doubt.
