Laurent wrote:
> Gerard wrote:
> > My recollection is that wavelengths in the range of 550 to 660 are used
> > to measure "scattering" or turbidity. They are appropriate for
> > microorganisms due to the refraction of light of those wavelengths
> > around the objects of about 1 um in size. One problem with this
> > technique is that it gives a representation proportional to the number
> > of "objects" in solution, i.e. it does not differenitate between live
> > and dead bacteria. To discriminate live from dead organisms, titration,
> > growth and colony counting is required.
>> It seems that 550 to 650 nm measure turbidity but it's not proportional
> to the number of "objects" in solution. It's proportional to organic
> mater in solution.
>
Indeed, I mixed up two different concepts: scattering (as in Rayleigh
scattering) and refraction. The size of the object matters for
refraction and is not appropriate here. For Rayleigh scattering, it is
actually the oscillating electron field of the molecules through which
light moves that causes the scattering, via inelastic collisions (i.e.
no energy is lost). Thus it appears that the extent of scattering is
proportional to the density of the electron nfield, i.e. the amount of
matter through which the light moves. Rayleigh found that light is
scattered proportional to L^-4 (that is the inverse of the fourth root
of the wavelength). Thus, longer wavelengths have the advantage that
they scatter less than the short wavelengths and thus are overly
sensitive. They also have the advantage of not being near the strong
absorbances cause by the double bonds in organic molecules. Of course if
there are other strong chromophores in the solution and their lambda-max
is near the wavelength used for the scattering, one again has "trouble",
in that the "loss of light transmission" used for turbidity measurements
will not be linearly proportional to the amount of material in
suspension. It can be corrected for by deterimining the absorption
spectrum and using a wavelength that is outside of the absorption of any
chromophores.
It would appear then that since scattering is due to the electron
density, the scattering would be as applicable to any material in
suspension, whether organic or not. Therefore, comments about live and
dead bacteria in solution still hold.
As a side note: for Raman spectroscopy the light that is scattered is
measured by measuring it at some acute angle to the incoming light,
whereas the above discussion deals with the loss of light transmission
due to scattering, i.e. measured at 180 degrees or in the path of the
light.
Gerard
--
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Gerard Tromp, Ph.D.
CMMG, Wayne State University vox: 313-577-8773
3116, Scott Hall fax: 313-577-5218
540 E Canfield Ave e-mail: tromp at sanger.med.wayne.edu
Detroit, MI 48201 gtromp at cmb.biosci.wayne.edu
