Re: bacteria by flow

Thanks to all the people that took their time to answer to my questions giving tips and
great suggestions. It really helped to get started! 

I have put together a summary of responses if someone is interested... or should I posted
anyway?

Thanks a lot once more

Love and peace

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 
Tel: office +46-40332395; mobil: +46-70-4330338 
"Judge a men by his questions rather than his answers" - Voltaire


----- Original Message -----
From: Andy Riddell <Riddell@embl.de>
Date: Thursday, November 17, 2005 12:42 pm
Subject: Re: bacteria by flow

> Hi Corrado,
> 
> Just to add to what what Larry has already said, I too analyse and 
> sort 
> bacteria on my MoFlo here, usually GFP. For small particles such 
> as 
> bacteria, the light scatter intensity decreases as the fourth 
> power of 
> the wavelength (Salzman Gary C. in Current Protocols in Cytometry 
> Vol 
> 1, Unit 1.13.2 - 1.13.3). I use about 1.2W of 488 light. This has 
> an 
> added benefit of near saturating the GFP on the sorter. I tested 
> saturation of the fluorochrome by increasing the light until I 
> could 
> not see any more increase in the fluorescent signal. Admittedly 
> this is 
> a rough way of doing this, however, I find it to be a reasonable 
> approximation.	A better, more accurate way of doing this is 
> described 
> by, Ger van den Engh and Collen Farmer, "Photo-bleaching and 
> Photon 
> Saturation in Flow Cytometry", Cytometry, 13:669-677 (1992). If 
> you 
> have access, have a look at the scattering unit in Current 
> Protocols in 
> Cytometry for background information and tips. The FACSCalibur	
> collection optics are much more efficient in collecting the light 
> that 
> on my MoFlo stream in air sorter, and the cells are in the 
> interrogation point longer, so you may well get a reasonable 
> discrimination signal from your bacteria. Other people have 
> reported 
> this.
> 
> 
> Anyway, Good luck.
> 
> Best Regards,
> 
> 
> Andy.
> 
> 
> On 15 Nov 2005, at 21:43, Larry Arnold wrote:
> 
> > We routinely look at bacteria on our two MoFlos.  We have looked 
> at E. 
> > coli, Pseudomonas (plant pathogen), Erwinia, and Borrelia (Lyme 
> > disease agent).  We use FSC and SSC log and trigger on the SSC.  
> > Bacteria are well resolved from noise (about 1.5 decades).	
> > Investigators have used GFP bacteria in expression cloning 
> strategies 
> > which have worked very well (see Chang et al. PNAS 102:2549) as 
> well 
> > as surface labelling with Alexa488, fluorescein and PE 
> antibodies.	We 
> > have never found it necessary to trigger on fluorescence to find 
> the 
> > bacteria.  For good FSC signals in a jet-in-air sorter it is 
> necessary 
> > to have the tip as close to the laser intercept as possible and 
> keep 
> > the drop drive amplitude as low as possible to prevent laser 
> light 
> > scatter from the stream waves getting into the FSC detector.	In 
> > general we have found it quite easy to sort bacteria with our 
> systems.>
> > Regards,
> >
> > Larry
> >
> > Larry W. Arnold, Ph.D.
> > Research Professor and Director, Flow Cytometry Facility
> > Department of Microbiology and Immunology
> > CB# 7290
> > University of North Carolina
> > Chapel Hill, NC 27599
> > Phone: 919-966-1530
> > FAX: 919-962-8103
> >
> >
> >
> Andy Riddell
> Flow Cytometry Laboratory
> EMBL Heidelberg
> Meyerhofstrasse 1, 69117 Heidelberg, Germany
> Tel: +49 [0] 6221 387-0
> Fax: +49 [0] 6221 387-8306
> 
> 
>