Monday, April 16, 2012

FAQ: For cell growth assay, why would I use light-scattering nephelometeric measurements in replace of turbidity measurements?

Answer: For turbidity measurements, the amount of light that passes directly through a sample (unscattered light) is measured by a spectrometer or spectrophotometer. The reciprocal of this transmitted light is then used to obtain absorbance optical density values (O.D.). In contrast, nephelometery measures the amount of light that is scattered by a sample using a detector (i.e. photodiode) which is usually at an angle to the incident beam.

This difference in principal is apparent when measuring samples that have higher concentration of particles, i.e. more cells.  For turbidity measurements, a plateau is eventually reached at a certain O.D. because very little light can pass through the sample anymore. For instance an O.D. of 4.0 means that only one photon of light out of 10,000 passes through to the spectrometer, which is usually no higher than the background noise of the detector. This is the reason why most absorbance assays are considered not reliable above an O.D. of 3.5. Nephelometry on the other hand measures the scattered light that is reflected (not absorbed) by the particles in solution. This allows for a larger dynamic range of cells to be measured (more than 5 decades), especially at higher concentrations.

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