Bascteria sorting ?
Van Bockstaele, Dirk
Dirk.Van.Bockstaele at uza.be
Tue Mar 5 05:11:22 EST 2002
you actually also need to know your drop drive frequency and jet velocity.
Assuming a drop drive frequency of 50,000 drops/sec and a jet velocity of
say 10 m/sec, you can envisage the jet as a cylindrical column of liquid
with a circular base of surface pi.r2 with r = 25microm (nozzle radius) and
height = 10 m (for a 1 second long flow time): total volume = pi.r.r.h.
This column is broken up in 50,000 pieces (drop drive). Some quick and
dirty calculation results in 1 drop = 3.93 10-4 microl or 2545 drops will
sum up to a volume of 1microl, provided I didn't make any power of ten
mistake! Just fill in your actual drop drive and jet velocity.
If your drop drive is 50,000 Hz and to be sure that you won't have to many
coincidences I would go for an average of 1 particle passing per 10 drops
formed: i.e. total particle concentration should not exceed 5000/sec. Of
course this is just a rule of thumb: the particles will not pass equally
separated in time but at random. If you trigger on fluorescence for the
sort you first need an analytical run using scatter triggering to know the
proportion of fluorescent versus total bacterial count.
I hope this is of any help.
Prof. Dirk Van Bockstaele
Laboratory of Hematology
Head of Flow Cytometry / Molecular Diagnostics
Antwerp University Hospital
phone 32 3 821 3900 fax 32 3 825 1148
> Van: Andy Johnson[SMTP:andy at brc.ubc.ca]
> Verzonden: maandag 4 maart 2002 22:14
> Aan: Cytometry Mailing List
> Onderwerp: Bascteria sorting ?
> 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
> 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
> 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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