[RASMB] determination of phage particle concentration

[John Champagne]

Sabine et.al.,
I think that the real issue with using these batch techniques is that they are an averaged technique and may not give a very accurate determination of the concentration of bacteriophages in solution...especially if there is contamination.  Even though it may be believed that the sample is monodisperse, it is very helpful to also determine this during your characterization.  Unfortunately the initial separation of these large macromolecules is a difficult task!  I would suggest that you employ filed flow fractionation, followed by multi-angle light scattering.  The best part of this approach is that you can resolve any "other" material that may be present, and then with the 1st principle measure of the material with MALS, it is capable of actually counting the number of particles in solution and along the separation peaks.  Any other technique out there would not give you anywhere near the accuracy and precision of this approach.  I can give you the name of labs that can do the measurement for you in case you might be interested in the technique but did not want to purchase the technology.
Best,
John

> Greetings RASMB:

The scattering increases as the fourth power of the inverse wavelength.  Years ago Aviv's spectrophotometer had a function that would fit the baselines before and after an absorbance peak to subtract the scattering portion.

However, I cringe at the idea of making analytical measurements of particles via absorbance.  Absorbance of a photo can happen only once. Scattering can happen more than once, sending a photon back into the detector's view.  The detectors of different instruments view different collection angles of the sample, so the amount of scattered light is going to vary depending on the instrument design.  Reflections of scattered light off the surroundings can send photons back onto the detector. In one custom instrument Aviv mounted the detector on a rail, and apertures were mounted on both the sample's exit and detector's entrance, to control the light collection angle.  And of course the size of the particle affects the amount of light scattering.

Measure scattering with absorbance with both eyes and mind open, because the instrument is not being used as intended.

Glen


At 09:44 AM 3/5/2010, Steve Harding wrote:
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Dear Sabine
Simple turbidity measurements (making sure you are away from absorption maxima) using a good quality spectrophotometer may suffice for the sort of information you are after - particle concentration, although stricvtly speaking it is not a hydrodynamic method..  This is is the usual method for measuring concentrations (particles per ml) of very large assemblies such as spores and I think it works for smaller assemblies that are not too dilute..
Victor Bloomfield's group did quite a bit on the turbidity of phages in the late 70's and gave the relevant formulae (you may need the approx  mol. wt value) - if you look up Bahls and Bloomfield (and not Victor's QLS papers) that should give you a lead!
All best
Steve Harding <




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John Champagne
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