E. Coli. on Elite

Peter Lopez Peterl at cytomation.com
Tue Aug 4 11:28:45 EST 1998


Hi Rochelle,
	We have a number of MoFlo operators doing E.Coli work. We have
found
on our systems that log amplified side scatter triggering seems to work
better than
 forward scatter with the small bugs. What I do is first optimize the
system with drop
drive off, then turn the drop drive on (which usually adds noise at this
point), and then while allowing the noise to be part of the
picture(while running the bugs), I optimize the system to have the most
distance between the bugs and the noise. Then I run up the trigger
threshold to 
get rid of the noise. 
If you can change the trigger laser wavelength, I think you can get some
improvement 
also, although I can't speak from experience in that regard. Obviously,
if you can get
the bugs to light up, your triggering issues would go away, since you
could trigger on
fluorescence. I know Mol. Probes has some bug stains that may light up
all the bugs
for triggering purposes.

Peter


> ----------
> From: 	Rochelle A. Diamond[SMTP:diamond at its.caltech.edu]
> Sent: 	Monday, August 03, 1998 9:14 AM
> To: 	Cytometry Mailing List
> Subject: 	E. Coli. on Elite
> 
> 
> Good morning Flow-ers,
> I need a little help.  Our facility has only analyzed and sorted
> mammalian
> cells and yeast in the past.  We now have two investigators that need
> to
> analyze and sort E. Coli and other bacteria (waste water type).  
> 
> On our first attempt, I was unable to distinguish the bacteria from
> laser
> noise or sheath noise, having to crank up the forward scatter gain and
> voltage to extreme values.
> 
> Our next attempt, I first located 1 micron beads on the scatter.  The
> CV's
> are very large at the settings that I used to see them.  Then we put
> on
> the bacteria and I could not distinguish them away from buffer alone.
> 
> 
> What do people do for this?  Do I need different forward scatter
> masking?
> A different forward scatter detection system.  Smaller beads to set up
> on?
> 
> Any suggestions would be useful.  
> 
> Thanks in advance,
> Rochelle Diamond
> Caltech Biology 
> Flow Cytometry/Cell Sorting Facility
> 



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