Re: [microbial cells] viability on fixed cells

Hi Julie,

I have been helping a user set up EMA staining of Pseudomonas putida. 
We are still in preliminary trials, but it looks like 10ug/mL is 
working pretty well to distinguish mixtures of live and heat-killed 
bacteria, and this discrimination is maintained after fixation with 
buffered formalin (stain, expose to light, wash, fix).	It's a little 
easier to see in FL3 than FL2 (FACS Calibur). We haven't tried storing 
the stained samples for days and then re-analyzing.  Actually, the 
BacLight kit seems like it would be the easier option. We can't use it 
here because eventually we will be using bugs that express GFP, and we 
don't have a laser that will excite BacLight Red.  Regardless of stain, 
I think I would try the fixation and storage to make sure things are 
stable over 3 days.  The EMA stain should be OK even if there are 
changes in membrane permeability over time in fix  (I've seen this with 
mammalian cells), as long as any unbound EMA is washed out before 
storage.

About the staying up for three days - I can't think of any way to make 
that easier!

Carol

(On Jul 17, 2006, at 1:48 PM, Julie Nelson wrote:

> Hi ya'll,
>
> I have a researcher interested in performing a time course experiment 
> measuring the
> viability of E.coli.	He wishes to sample every two hours for three 
> days (or some such
> madness) and we were wondering if anyone out there has ever stained 
> bacteria with EMA,
> fixed them and then measured them at a later time for viability.  I 
> know this can be done
> with mammalian cells but bacteria?  Or, are there other kits that 
> would work?  Also, if
> anyone has any other suggestions, we would welcome them.  At this 
> point, he plans to use
> the Baclight kit from Invitrogen and stay up for 3 days.  Surely there 
> is a better way?
>
> Thanks for any and all advice,
>
> Julie
> Julie Nelson
> Laboratory Manager
> Flow Cytometry Facility
> Center for Tropical and Emerging Global Diseases
> University of Georgia
>
Carol E. Norris, Ph.D
Facility Scientist
Flow Cytometry/Confocal Microscopy Facility
Biotechnology/Bioservices Center
University of Connecticut Unit 3149
91 N. Eagleville Rd
Storrs, CT 06269-3149

Phone (860) 486-3080
Fax (860) 486-5005