From: Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
Date: Wed Apr 08 1998 - 06:37:14 EST
Dear Steve
Assuming you run plain PBS as a sheath and run your laser
power at about 15 mW you will not do the cells any harm with
the cytometer. Regarding the sorter adjustment I would
recommend to use the 0.5um yellow green beads, as indeed
there seems to be some difference in the whole system
alignment compared to big particles. I always sort drop
triplets as the single drop recovery always lies between 90
and 95 percent (with fluorescent beads) which was not good
enough for our viability studies. For completely unstressed
cells we have 98% to 100% recovery on single cell sorts.
As I reported on the ISAC meeting, the major problem for
recovery of stressed cells is that they dye of intrinsic or
extrinsic oxidative damage when exposed to growth stress.
The recovery in particular on agar plates can drop for
several decades. If you sort facultative anaerobes you
should sort into "SPRINT" medium (Oxoid,Basingstoke UK)
which provides an anaerobe, detoxified environment that
allows the highest possible recovery of injured cells. You
can aerate the wells after 6 hours of repair.
Good luck
Gerhard.Nebe-von-Caron@unilever.com
______________________________ Reply Separator _________________________________
Subject: E.coli and plate sorting
Author: steve@habanero.cb.uga.edu at INTERNET
Date: 07/04/1998 23:31
Hi folks,
I'm getting frustrated. You may remember past queries from me about
working with the EPICS 753 autoclone. Well, I have it working, and I've
been very happy with it's performance with beads. We have used it to
sort sperm for PCR and been happy with the results. So I bragged about
it.....
Now I have a user who wants to clone E.coli expressing GFP. We had
previously modified this 753 by adding a PMT for FALS, and we have
one of the "sort-sense" flow cells, so we can detect the bacteria (by
scatter and fluor.), but I've sorted into plates twice and nothing
grows up! I have sorted their bacteria into tubes, and upon
re-analysis we certainly had separated GFP- and GFP+ bacteria, but it's
not working w/ plates.
I make up plain PBS, so there are no preservatives to suppress growth.
Using 500mW definitely bleached the GFP in earlier tests, so I dropped
down to 50mW and still got great signal. In the latest experiments we
went down to 10 (probably grossly inaccurate w/ a 90-5) but it made no
difference. Sheath pressure is 13, but if I lower it I can't get good
enough deflection to hit my target. Besides, I would have thought that
they could have handled 13 psi through a 100um orifice.
Am I killing them, or just missing them? I can imagine that the
sorting dynamics might be different for 10um DNAchecks vs E. coli, but
it worked when sorting into tubes. Maybe my recovery was lower than I
thought, but it only made a difference w/ single cell deposition? They
got a few colonies when we sorted 10/well, so I'm leaning toward this
idea.
Has anyone done this? Any ideas on how to make this work would be
greatfully appreciated--my head's on the block!
-------------------------------------------------------------
Steve G. Hilliard flowman@uga.edu
University of Georgia Cell Analysis Facility
http://floweb.cb.uga.edu/Floweb/
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