Lots of chatter on the phage quantitation. So, the essential question
is one of exactly what is required and the accuracy of the measurement
(first approximation or +/- 1%).
Bottom line, absorbance measurements will be fast and easy get you
into the ballpark without going through all the hoopla - all the
caveats notwithstanding.
Peter provided the best approach overall, however; titer for
infectious phage and microscopy for particles. Both would be
quantitative and accurate if done appropriately.
carlos
On Mar 9, 2010, at 5:52 AM, Glen Ramsay wrote:
Dear RASMB:
I should have realized that my comments might cause concern,
especially given the many successful prior publications. I'll try to
be more helpful.
What absorbance range worked you, Peter? "Highly" turbid solutions
will cause nonlinearity in the signal-to-concentration relationship.
You can see this effect by shining a beam of visible light through
slightly turbid, and highly turbid, solutions. The slight solution
will clearly show the beam path. The highly turbid solution will have
a diffuse beam path, and show light outside of the beam path due to
more than one scattering event. Unless the detector has special
optics to see only the transmitted light path and nothing else then
there is a chance that the light signal will over-state the
concentration. My feeling is that if you keep ~0.1 OD or less that
the problem is negligible, while at 1 OD the problem is significant.
If the sample is highly scattering I would expect problems in the
diffusion / shape fitted parameters, but not necessarily the size.
My other concern is that the same scattering sample can produce
different absorbance signals on different instruments architectures,
due to different optical layouts and susceptibilities to scattered
light. Fortunately, that is not the goal of AUC experiments. Rather
the AUC measurements are relative to each other (much like
fluorescence) with the same sample and hardware. The analysis
software is not as sensitive to absolute amplitudes, but rather the
relative shape(s) of the radial scan(s). Therefore, for slightly
turbid solutions I see no problem for a single species. If there is a
large range of particle sizes, then I would expect the sizes to be
correct, but the relative concentrations to be off due to larger
particles scattering light more than small particles.
My $0.02 for people who haven't already coped with these issues.
Glen
At 06:38 AM 3/9/2010, Peter Edward Prevelige Jr wrote:
> We have determined (in control experiments for our experiments using
> turbidity to monitor phage assembly) that the turbidity increases
> linearly with the concentration of phage capsids over the useful
> working range of a spectrophotometer regardless of wavelength. As
> Borries mentioned you just need a standard to get absolute number.
> We use phage with a particle/pfu ratio of very nearly 1 and obtain
> absolute concentration by titer.
>
> Peter
>
>
> Peter E. Prevelige Jr.
> Professor
> Dept. of Microbiology, BBRB 416/6
> Univ. of Alabama @ Birmingham
> 845 19th St. South
> Birmingham AL. 35294-2170
> Phone 205 975-5327
> FAX 205 975-5479
> prevelig at uab.edu
> http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm
>
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org [ mailto:rasmb-bounces at rasmb.bbri.org
> ] On Behalf Of Borries Demeler
> Sent: Tuesday, March 09, 2010 5:17 AM
> To: Steve Harding
> Cc: rasmb at server1.bbri.org
> Subject: Re: [RASMB] determination of phage particle concentration
>
> I wonder if one could mitgate the problems with light scattering by
> preparing a known, significant dilution, staining with sybrgreen
> and looking at fluorescence intensity in a velocity experiment.
> Sybrgreen bound to DNA is very sensitive and this can be done at very
> low concentration.
>
> We have done this with good success using the Aviv fluorescence
> machine
> to measure phage aggregation behavior. Dilutions provided linear
> changes
> in fluorescence intensity. Sybrgreen seems to have no problem
> leaking into
> the phages we investigated and lights them up like a christmas tree.
> One
> issue though is that one would need some standard, and I am not sure
> how
> to get this. At the very least one could use this to determine if
> there
> is leaked DNA in the prep or other contaminating DNA, and sybrgreen
> has
> a very low background.
>
> Serwer P, Hayes SJ, Thomas JA, Demeler B, Hardies SC. Isolation of
> novel
> large and aggregating bacteriophages. Methods Mol Biol.
> 2009;501:55-66.
>
> Regards,
> -Borries
> _______________________________________________
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> _______________________________________________
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> RASMB at rasmb.bbri.org
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Glen Ramsay, Ph.D.
Chief Scientist
Aviv Biomedical, Inc.
750 Vassar Avenue, Suite #2
Lakewood, NJ 08701
(732) 370-1300, Ext. 25
(732) 370-1303, FAX
glen at avivbiomedical.com
www.avivbiomedical.com
Skype: GlenAtAviv
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