bacteria by flow

Andy Riddell Riddell at embl.de
Thu Nov 17 06:42:30 EST 2005


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


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