Measure OD600
130 NanoDrop One User Guide Thermo Scientific
In the case of living cells, most of the incident light is transmitted through the sample rather
than scattered, reflected or absorbed. The amount of scattered light is low and can vary from
instrument to instrument. As a result, calculated absorbance readings are typically very low.
The calculated absorbance values are used to determine the density of cells in solution in
cells/mL. The physical concepts and formulas that relate optical properties of living cells to
concentration include:
• Cells, which have a different index of refraction from the surrounding medium, randomly
reflect and scatter light out of the incident light path. The amount of scattering is
proportional to the density of cells in the sample.
• The Beer’s Law equation is used to relate absorbance to concentration. See Calculations
for OD600 Measurements for details.
• For cuvette reading with the NanoDrop One instrument, accurate absorbance readings
are typically in the range between 0.04 A and 1.5 A. Serial dilutions of the sample are
usually needed to bring the absorbance readings within this range.
• All measurements should be made on the same type of spectrophotometer and method
(i.e., pedestal vs. cuvette) as the amount of scattered light captured varies based on the
optical configuration. When using a different spectrophotometer or method, calculate
and apply a conversion factor to the reported results. For example, to compare OD
readings using the pedestal vs. a cuvette, a conversion factor can be calculated as follows:
Conversion factor = Cuvette OD/Pedestal OD
Best practices for OD600 measurements
• Ensure the sample is within the instrument’s absorbance detection limits.
• Blank with the growth or culture media the cells of interest are suspended in.
•Run a blanking cycle to assess the absorbance contribution of your media solution. If the
media solution exhibits strong absorbance at or near the analysis wavelength (600 nm),
you may need to choose a different media solution or application. See Choosing and
Measuring a Blank for more information.
• Make dilutions as necessary to ensure sample cultures do not exceed the linear dynamic
range of the assay before the culture reaches the stationary phase. The linear range
depends largely on optical configuration and, therefore, differs for pedestal and cuvette
measurements. To determine the linear range:
– Measure a series of dilutions using a young overnight culture (~16 hrs) of the
microbial strain
– Graph the OD600 measurements against the dilution factor
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