Hi Andy, I'd candidly say most of us don't know the finer details of flow and fluid dynamics but I can tell you what I've established. Most calculations about droplet volume in flow over the years are nearly always based upon the mathamatical approach outlined by Daniel Pinkel and Richard Stovel in their excellent chapter "Flow Chambers and Sample Handling" which appeared in "Flow Cytometry: Instrumentation and Data Analysis", 1985 ISBN 0-12-712150-1. Get your hands on that and have a read if you can. They state, that a jet vibrating at optimal frequency for droplet formation .i.e. lambda = 4.5d will produce droplets 3.53d(power 3). These are the basis of the calculations that Joe Trotter applies here http://facs.scripps.edu/optimize3.html unless he has made some small changes since we last talked about this last (Joe?). This will give you an indication of droplet diameter from which you can work out the volume. I tested this bit of theory at one point in a moment of weakness and it seemed quite different from the a magic number of 0.0017ul volume per drop for a 70um nozzle on the Vantage, which was emperically derived. As to whether there is are other non fluorescent bacteria in a single droplet, the only way I know of to test that is single drop/single cell sort onto plates and grow them up. Given the size of the particles relative to beam diameter I think it is too tough a call for normal pulse processing to deal with, but then I've been wrong before... Lastly have a look at a posting Howard made a while ago From: Howard Shapiro <hms@shapirolab.com> Subject: Microbial Analysis at the Single Cell Level - Available On-Line The Journal of Microbiological Methods - Special Issue Microbial Analysis at the Single-Cell Level Volume 42, Number 1 (September, 2000): Available On-Line Hope this helps Geoffrey Osborne Specialist, Flow Cytometry, John Curtin School of Medical Research, The Australian National University, Canberra, 0200, ACT. AUSTRALIA email: geoff.osborne@anu.edu.au http://jcsmr.anu.edu.au/facslab/facshome.html At 01:14 PM 3/4/02 -0800, you wrote: > >Hello > >For those of you who know the true finer details about flow and fluid >dynamics ... can somebody tell me the calculation to work out the volume >size of droplets. The mathamatical approach rather than collecting and >counting the volume. > >We are using a 50 µm nozzle at 30psi (BD vantage) and trying to single cell >sort bacteria onto 96 well plate. > >The first question is how large are the droplets (volume) ? > >Can you determine the dilution factor of your samples (bacteria) ? > >We are sorting bacteria and triggering on the fluorescence signal to reduce >background noise. The questions is : at what rate of sample can we assume >that the instrument is detecting a single cell in a droplet ? > >Looking at the numbers it would appear that non-fluorescent bacteria must >be in the same droplets, but I may be assuming something incorrectly. > >Any help appreciated and further information available if it will help you >with the solution ! > >Andy > > >
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