Re: [microbial cells] viability on fixed cells

Gene,

I wasn't able to find spectra for BacLight Red online, so I called MP 
and they just told me it was the same as Texas Red. From the Texas Red 
spectra, I decided that it wouldn't work with the 633 laser - am I 
mistaken here?

Carol

On Jul 24, 2006, at 9:34 AM, Pizzo,Eugene wrote:

> Carol,
>
> Have you tried BacLight Red? because I'd be surprised based on the
> excitation/emission profile that you couldn't get a decent signal from
> the 633nm diode laser of the Calibur in FL4.
>
> Gene Pizzo
>
> -----Original Message-----
> From: Carol Norris [mailto:carol.norris@uconn.edu]
> Sent: Thursday, July 20, 2006 12:24 PM
> To: Cytometry Mailing List
> Subject: 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
>
>
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