[RASMB] determination of phage particle concentration

[Timothy Dafforn]

Hi Glen,
You are absolutely correct.
Mathematical fitting of wavelength dependent scattering and then subtracting it from the data still remains an essential method in CD. Particularly as in the Far UV the scattering distortion is very bad (if only we could all use infrared!)
We have published such a method as have others.

It is also clear that it doesn't always obey the 4th power rule.

This method does have the potential to produce very accurate estimates of macromolecular concentration and certainly shouldn't be discounted..
Cheers
Tim

PS how is Jack?
________________________________________
From: rasmb-bounces at rasmb.bbri.org [rasmb-bounces at rasmb.bbri.org] On Behalf Of Glen Ramsay [glen at avivbiomedical.com]
Sent: 08 March 2010 14:08
To: Steve Harding; rasmb at server1.bbri.org
Subject: Re: [RASMB] determination of phage particle concentration

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

http://www.nottingham.ac.uk/ncmh

> Dear all,
>
> we are trying to obtain the concentration of bacteriophages in a
> solution, concentration meaning the number of particles per ml. The
> solution is supposed to be monodisperse and the MW is (roughly) known
> but could be determined exactly. Is the any idea of how to do this
> with a hydrodynamic method (staining is difficult to compare between
> phage mutants and counting infected cells is tedious). I`ve got the
> "feeling" that one could use the number-averaged MW, but maybe this is
> totally wrong.
>
> Thank you for any hints, literature is also welcome.
>
> Cheers,
>
> Sabine
>
>
>
>
> Sabine Kaltofen
> PhD student
>
> Universität Potsdam
> Department of Physical Biochemistry
> Institute of Biochemistry and Biology
> Karl-Liebknecht-Str. 24-25, Haus 25, Raum B/0.05
> D-14476 Potsdam-Golm
> Telefon: +49-(331)-977-5245
> Email: kaltofen at uni-potsdam.de
>
>

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