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6
Measure OD600
Best practices for OD600 measurements
136
NanoDrop One User Guide
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 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
• Ensure the sample is within the instrument's
• Blank with the growth or culture media the cells of interest are suspended in.
• Run a
blanking cycle
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
• 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:
for details.
to assess the absorbance contribution of your media
absorbance detection
for more information.
Thermo Scientific
limits.
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