wavelength to read cultures

George W. Chang changlab at nature.berkeley.edu
Tue Oct 4 21:14:07 EST 1994

In article <36r3o8$cna at hermes.uni-konstanz.de>,
Andreas.Brune at uni-konstanz.de (Andreas Brune) wrote:

> In article <36ptvg$gd at mserv1.dl.ac.uk>, "RICHARD A. MURPHY 312-996-8630"
<U14663 at UICVM.UIC.EDU> says:
> >
> >When reading ODs of bacterial cultures with a spectrophotometer, what
> >wavelength should be used and is it the same for all bacteria?.
> The sensitivity of the measurement is WL-dependent (decreasing with
> increasing WL), so it is advisable to calibrate with dry weight or 
> protein per OD unit.
> If you have small turbidity readings (little growth, or tiny cells), 
> you can use this phenomenon to significantly increase your instrument's 
> response by switching to, e.g., 450 nm. 

        As Andreas points out, it is a trade off.  Shorter wavelengths
give more light scattering and therefore higher sensitivity.  But
yellowish culture medium absorbs very strongly at shorter wavelengths. 
This will interfere with turbidity readings.
        Some workers would use wavelengths well above 600 nm in order to
be free of interference from even fairly dark culture medium.  But they
have to live with lower sensitivity to cell mass.
        In addition, it helps to have a well-collimated exit beam from
your sample cell.  You can get this by choosing a machine with a long
cell-to-detector distance, moving the components of your instrument around
a bit, and/or collimating the exit beam with something as simple as a
darkened piece of cardboard.

Good luck!

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