bacteria by flow
umbbd at montana.edu
Mon Nov 14 12:44:06 EST 2005
We are routinely analyzing bacteria on an Aria, using all signals in log
mode. It is not easy since the bacteria are so small they are at the
noise level for the scatter signals. FSC signal is so full of noise it
is useless. SSC is useful, but if we threshold only on SSC we do
analyze some debris/noise along with the bacteria.
I would recommend that if you want to try to use the scatter signals,
that you will have to suspend the bacteria in something that is as close
to your sheath fluid as possible, keep your bacteria concentrated enough
so that you can use the lowest flow rate for your samples, so that the
bacteria are centered in the stream; if I turn up the flow rate to try
to analyze faster, then the peaks widen significantly.
Our best efforts have been with bacteria with GFP. If we can threshold
on GFP, we know we are analyzing the bacteria, not the debris/noise.
Once we have figured out where the bacteria are in the FSC vs SSC,
using the GFP to threshold, then if I go back and try to threshold on
the FSC vs SSC and look at the GFP histograms, we have always added a
substantial amount of non-GFP events.
Another issue is that of clumping of bacteria, which certainly confuses
the issue substantially and makes total counts virtually impossible. I
predict that you will be able to use your cultured GFP samples and get
decent signals, but if you try to analyze the fecal samples you will run
into many problems. Good luck!
Corrado Cilio wrote:
>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 M. Cilio, M.D., Ph.D.
>Cellular Autoimmunity Unit
>Dept. of Clinical Sciences/Paediatrics
>Malmo University Hospital
>205 02 Malmo, Sweden
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