bacteria by flow

Nebe-Von-Caron, G g.nebe-von-caron at unipath.com
Fri Nov 11 17:41:02 EST 2005


1: Depending on what you want to measure of the bacteria. Sheath fluids
contain preservatives such as Azide of sodium fluoride which will make
your bugs fairly unhappy to dead.
2: forget forward scatter unless you modify the instrument. Most people
trigger on side scatter as it is an intrinsic parameter of the cells.
The best thing is to use bacteria that are reproducibly fluorescent and
well dispersed like the target bacteria from the phagotest or by
staining some of your coli with a bright DNA stain such as syto9 (check
by fluorescent microscopy). You then start to set-up the instrument by
triggering on the fluorescence and raise the voltage. Once you detect a
clean population -e.g. see a nice distribution of fluorescence with a
gap to the noise- you can display it against side scatter. Once you get
the population in scatter and fluorescence away from the axis you can
try triggering on side scatter. If your system and the buffer is clean
you will still see a population separated from the noise. If not you
have to start eliminating sources for the noise. 
Ensure all your solutions are filtered as well as your sheath.
Set side scatter to log. Bacteria with a distinct rod shape might give
you a double cluster on side scatter depending on their orientation in
the beam.
3: Have concentrations the same as for cells, 10^6 to 10^7/ml. You do
not want excessive flow rates as you can not distinguish singles from
doublets.
4: For surfaces cleaning around the sample inlet we spray 75% isoprop
and wipe using mediwipes (containing chlorhexidine). For the sample
tubing we run a sample >10% bleach based domestos followed by a couple
of minutes back flush and a rinse of the sample injection rod with
plenty of 18 megohm water. This is followed by a 75% isoprop and another
distilled water rinse. 

!Ensure the bleach solution stays free off precipitates! that otherwise
clog the system.


Good luck

Gerhard

-----Original Message-----
From: Corrado Cilio [mailto:Corrado.Cilio at med.lu.se] 
Sent: 10 November 2005 22:07
To: cyto-inbox
Subject: bacteria by flow


Hi,

For the first time we are thinking to run bacteria in our FACScalibur. 
Is there someone that can give me some good advices? 
Basic questions like: 1. it is ok to resuspend in normal FACS buffer
(FCS, azide) 2. which would be the best instrument settings to get
started (mostly FSC and SSC). We want to look at cultured e-coli/eGFP
and fecal samples which should contain the same bugs. 3. cell
concentrations? 4. Special cleaning advices?

Thanks in advance

Corrado

Corrado M. Cilio, M.D., Ph.D. 
Assistant Professor 
Cellular Autoimmunity Unit 
Dept. of Clinical Sciences/Paediatrics 
Malmo University Hospital 
Lund University 
205 02 Malmo, Sweden 





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