bacteria by flow

Dan Rosson Ph.D. Dan.Rosson at mail.jci.tju.edu
Fri Nov 11 15:38:08 EST 2005


I have a user analyzing and sorting bacterial on a Calibur.

1 Sheath fluid is certainly OK
2 You should trigger on SSC. We have the Mode on Lin, Voltage at 443, amp 8.
The threshold value is 25
     With FSC at Lin, V= E01, amp =1
3 Concentration of bacteria should be about 1-5 x 10(6)/ml as with
eukaryotic cells.
   That's a dilution of about 1/500 for an overnight culture of E. coli.
   Your instrument settings will, of course, need tweaking. Check your
settings (SSC voltage and threshold) by finding the point at which PBS just
shows a about 10 events/ second.
   Use the flow rate adjustments to get about 2000 events/ second with
bacteria. If you can't get in this range with anywhere between Low and High,
make another dilution.
4 As for cleaning up fecal samples, I'm not going to touch that line!


Happy Flowing
Dan Rosson PhD
Thomas University Flow Jock



----- Original Message ----- 
From: "Corrado Cilio" <Corrado.Cilio at med.lu.se>
To: cyto-inbox
Sent: Thursday, November 10, 2005 5:07 PM
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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