[RASMB] determination of phage particle concentration

[John Correia]

Berkowitz and Day Biochemistry 1980  19, 2696-2702.  Turbidity
Measurements in an Analytical Ultracentrifuge:  Determinations of Mass
per Length for Filamentous Viruses fd, Xf and Pf3.  Combines scattering
and interference measurements in a Model E.  Can it be done in an XLI is
the question.

>>> On 3/8/2010 at 8:08 AM, in message
<20100308141543.BE7051742AF at rasmb.bbri.org>, Glen Ramsay
<glen at avivbiomedical.com> wrote:
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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