Difference in OD600 Readings
For turbid samples such as cell cultures, the major contributor for the absorbance measured is light scattering and not the result of molecular absorption following the Beer-Lambert Law. The measurements are therefore depending on the optical setup of the spectrophotometer (distance between the cell holder and instrument exit slit, monochromator optics, slit geometry, etc.), different instrument types will most likely 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 by either a simple correlation of the OD readings using the same sample on the two different instruments to compare

or with a more precise approach by the creation of appropriate calibration curves.
A calibration curve can be constructed by comparing measured OD 600 to expected OD 600 over a range of different concentrations. 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 108 cells/ml for E. coli. A high-quality instrument like the NanoPhotometer® or the OD600 DiluPhotometer™ comes with a “correction factor” of 1 as default, which, once adjusted will help to provide comparable results between different equipment.
The use of disposable cuvettes is recommended rather than micro/nano volume technologies (measurements in a drop) 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. |