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The ability to measure ion fluxes in bacteria depends on the
pumps they possess. A lot of the bugs will pump out your
dyes faster than you want. Staining Listeria for example
with Rhodamine, we could modulate the extrusion by sodium
chloride. The use of pump inhibitors or EDTA does also
interfere with the pumps you want to look at. So first hunt
for a suitable organism like Micrococcus luteus (no pump,
big cell). It stains beautiful in three colour staining
experiments regarding membrane potential and membrane
integrity. Otherwise you should use an activated probe like
a succinimidyl ester or a chloromethyl conjugate (see
Molecular Probes) and stain under pump inhibited conditions.
Once they are crosslinked, you can run the cells in normal
mode again.
Good Luck
Gerhard
Gerhard Nebe-v.Caron
Unilever Research, Colworth Laboratory Sharnbrook,
Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
E.mail: gerhard.nebe-von-caron@urcgb.sprint.com
______________________________ Reply Separator _________________________________
Subject: Ion fluxes in bacteria?
Author: Geoff.Osborne@anu.edu.au at INTERNET
Date: 06/09/95 18:46
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Hello,
This may sound like an unusual question but I had a request
yesterday from a user interested in detecting ion fluxes in bacteria. I
don't know of any references regarding this and wondered if someone might be
able to point me to some. I think the basic idea is to insert a sequence of
interest tagged with LacZ and detect it in those cells expressing it by
flow, and also assess the movement of things like calcium or potassium, and
hopefully see some change related to the inserted sequence.
Any and all suggestion welcome.
Thanks in advance,
Geoff
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Geoffrey Osborne | ____ __ o Ahh!
Flow Cytometry (FACS LAB) | __ `\ <,_
John Curtin School of Medical Research, | __ (*)/ (*)
Australian National University, | ==============|
CANBERRA, AUSTRALIA. | |--|
Email: Geoff.Osborne@anu.edu.au | |--|...
-----Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html------
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