bacteria by flow

Mark Lunzer mlunzer at
Tue Nov 15 14:02:27 EST 2005


I run E. coli in a FACScalibur all the time. The trick is to have the 
machine clean and run on the lowest setting. To clean the machine I run 
straight bleach for 5 min followed by 5 tubes of 0.2 um filtered PBS. 
This is all done in the carousel. When you get to the last tube you 
should see very few counts (15 events per second every now and then).

I use YO-PRO-1-iodide to stain cells that have been treated with T5 
bacteriophage, chloramphenicol, and EDTA. You could probably use my 
settings as a guide, so here they are,

FSC  E01  Log
SSC  415  Log
FL1  505  Log
FL2  477  Log
FL3  568  Log

Trigger on both FSC & SSC
Thresholds are
FSC   90
SSC   320


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

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