Bacteria sorting? Size of droplet etc

Geoffrey Osborne geoff.osborne at
Mon Mar 4 23:22:21 EST 2002

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
unless he has made some small changes since we last talked about this last
	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 at>
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 at

At 01:14 PM 3/4/02 -0800, you wrote:
>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 !

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