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If you have internet access you can see an example uploaded
for the second Purdue CD for that on
ftp://ftp.cyto.purdue.edu/cdupload/gerhard
If you have freelance you can look at the whole .pre
presentation or otherwise at pages 17 and 18.gif.
The major advantage of antibodies over probes is that the
bugs remain viable, so you can sort and grow them and you
can get simultaneous viability assessment. The disadvantage
is that you need to isolate the bug first to make your
antibody.
You will find a reasonable amount of antibodies to clinical
relevant bacteria (mostly unlabelled). I have used
commercial immunofluorescence kits for counting Clamydia
elementary bodies, just adding 10ul to 50ul mashed
cellculture diluted 1:100 with PBS after 5 minutes getting
good cluster separation, but quite a number of the reagents
do not work in flow as they are against extracted antigen
like for example parts of flagella only exposed after heat
denaturation...
To set up your equipment for the first time you should use
0.5um YG-fluorescent latex with your heatfixed bacteria at
1mg/ml propidium iodide and discriminate on green
fluorescence. Once you have the beads in the middle of you
display you can switch to scatter discrimination. I prefer
to look at scatter versus red and scatter versus green (all
log) to quickly see clusters coming up.
Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com
______________________________ Reply Separator _________________________________
Subject: Questions
Author: Adrian_Moris@sratech.com at INTERNET
Date: 08/10/96 00:47
Hi folks,
One of the PI's here at SRA has asked me to start thinking about ways
to use flow to ID unknown and/or mixed bacterial cultures. In
particular, he wants to use surface markers, not DNA probes. I'm
fairly new to flow and so far have only been working with mammalian
cells. I'd appreciate any advice, especially sources of antibodies,
advice on looking at things as small as bacteria, etc. How 'bout it?
Any bacteria FLOWERS out there who can help me out?
Oh yeah. Apologies to those offended at my musical quiz. I
personally have enjoyed playing "Sort that tune" as it were, as have
many of you who replied. It helps to break up my day and let me think
more clearly. But alas, not everyone is like me, so I guess I'll keep
my notes to the list to more business-related, if mundane, arenas in
the future.
Sincerely
Adrian Moris
"If you plant ice, you're gonna harvest wind."-JG
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