<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">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%).<div>Bottom line, absorbance measurements will be fast and easy get you into the ballpark without going through all the hoopla - all the caveats notwithstanding. </div><div>Peter provided the best approach overall, however; titer for infectious phage and microscopy for particles. Both would be quantitative and accurate if done appropriately. </div><div>carlos</div><div><br></div><div><br><div><div>On Mar 9, 2010, at 5:52 AM, Glen Ramsay wrote:</div><br class="Apple-interchange-newline"><div> Dear RASMB:<br><br> I should have realized that my comments might cause concern, especially given the many successful prior publications. I'll try to be more helpful. <br><br> 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.<br><br> 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. <br><br> My $0.02 for people who haven't already coped with these issues.<br><br> Glen<br><br> <br> At 06:38 AM 3/9/2010, Peter Edward Prevelige Jr wrote:<br> <blockquote type="cite" class="cite" cite="">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.<br> <br> Peter<br><br> <br> Peter E. Prevelige Jr.<br> Professor<br> Dept. of Microbiology, BBRB 416/6<br> Univ. of Alabama @ Birmingham<br> 845 19th St. South<br> Birmingham AL. 35294-2170<br> Phone 205 975-5327<br> FAX 205 975-5479<br> <a href="mailto:prevelig@uab.edu">prevelig@uab.edu</a><br> <a href="http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm" eudora="autourl"> http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm</a><br><br> -----Original Message-----<br> From: <a href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [<a href="mailto:rasmb-bounces@rasmb.bbri.org" eudora="autourl"> mailto:rasmb-bounces@rasmb.bbri.org</a>] On Behalf Of Borries Demeler<br> Sent: Tuesday, March 09, 2010 5:17 AM<br> To: Steve Harding<br> Cc: <a href="mailto:rasmb@server1.bbri.org">rasmb@server1.bbri.org</a><br> Subject: Re: [RASMB] determination of phage particle concentration<br><br> I wonder if one could mitgate the problems with light scattering by<br> preparing a known, significant dilution, staining with sybrgreen<br> and looking at fluorescence intensity in a velocity experiment.<br> Sybrgreen bound to DNA is very sensitive and this can be done at very<br> low concentration.<br><br> We have done this with good success using the Aviv fluorescence machine<br> to measure phage aggregation behavior. Dilutions provided linear changes<br> in fluorescence intensity. Sybrgreen seems to have no problem leaking into<br> the phages we investigated and lights them up like a christmas tree. One<br> issue though is that one would need some standard, and I am not sure how<br> to get this. At the very least one could use this to determine if there<br> is leaked DNA in the prep or other contaminating DNA, and sybrgreen has<br> a very low background.<br><br> Serwer P, Hayes SJ, Thomas JA, Demeler B, Hardies SC. Isolation of novel<br> large and aggregating bacteriophages. Methods Mol Biol. 2009;501:55-66.<br><br> Regards,<br> -Borries<br> _______________________________________________<br> RASMB mailing list<br> <a href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a><br> <a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb" eudora="autourl"> http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><br> _______________________________________________<br> RASMB mailing list<br> <a href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a><br> <a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb" eudora="autourl"> http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a></blockquote> <x-sigsep><p> <br> Glen Ramsay, Ph.D.<br> Chief Scientist<br> Aviv Biomedical, Inc.<br> 750 Vassar Avenue, Suite #2<br> Lakewood, NJ 08701<br> (732) 370-1300, Ext. 25<br> (732) 370-1303, FAX<br> <a href="mailto:glen@avivbiomedical.com">glen@avivbiomedical.com</a><br> <a href="http://www.avivbiomedical.com/" eudora="autourl"> www.avivbiomedical.com<br> </a> Skype: GlenAtAviv<br><br> <font size="1">The information transmitted is intended only for the person or entity to which it is addressed and may contain confidential and/or privileged material, the disclosure of which is governed by applicable law. 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