[Cytometry] Cyanobacteria Answer-Pam Shaw

Scott Tighe scott.tighe at uvm.edu
Mon Feb 3 10:31:09 EST 2014

Dear Pam

Analysis of Cyanobacteria at 633 should be fine however I do not know 
the quantum efficiency that far in the red however seeing how 
Phycocyanin is indeed the major component of cyanobacteria, one would 
expect great emission.  We routinely excite with green 514 and catch the 
Phycoerytherin molecule and that is great.  Here is a picture of that.  
You could run some unstained yeast (sch. pombe) as a control for 
reference. It is about the same size.

1) Gloeocapsa sp. No stain. Excite 514  observed with a long BP filter 

Spectrum of PCyn:

Absorbance Spectrum of Phyco-pigments in general:

Scott Tighe
Senior Core Laboratory Research Staff
Advanced Genome Technologies Core
NextGen Sequencing/Flow Cytometry
University of Vermont and Vermont Cancer Center
149 Beaumont ave
Health Science Research Facility 303/305
Burlington Vermont 05405
802-656-2482 (AGTC)

On 2/3/2014 9:47 AM, Shaw, Pam wrote:
> Hello flow list buddies!
> I have a request a bit out of my  comfort zone.  We don’t do a lot of bacterial work, so could use some input on this request (see below).  We have a FACSAria II with 405, 488 and 633 nm lasers.  Would the 633 laser work for cyanobacteria as described below and would we be able to see a difference in size as the request indicates?
> Any input would be appreciated!
> Thanks in advance.
> --
> Pam Shaw
> Fluorescence Cytometry Core Facilitator
> Center for Environmental Health Sciences
> The University of Montana
> 32 Campus Dr, Skaggs Building 052
> Missoula, MT 59812
> Ph (406) 243-4974
> Email: pamela.shaw at umontana.edu<applewebdata://DE30ADE2-B1DF-44AF-B224-B0B84EDBD782/pamela.shaw@umontana.edu>
> www.umt.edu/cehs/Facility_Cores/Fluorescence_Cytometry/default.aspx
> From: <Miller>, Scott <Scott.Miller at mso.umt.edu<mailto:Scott.Miller at mso.umt.edu>>
> Date: Friday, January 31, 2014 at 12:51 PM
> To: "pamela.shaw at umontana.edu<mailto:pamela.shaw at umontana.edu>" <pamela.shaw at umontana.edu<mailto:pamela.shaw at umontana.edu>>
> Subject: flow cytometry question
> Hi Pam,
> By way of introduction, my name is Scott Miller, a microbiologist in DBS, with a research question that may potentially be appropriate for flow cytometry. The gist is that we are comparing wild type and mutant cyanobacteria in one of our projects. Under certain conditions, the mutant appears “clumpier” than the wild type, probably because of differences in extracellular polysaccharides, and we are exploring possible methods for quantifying this. These bacteria autofluoresce if excited with ~590 nm (peak emission at 680 nm), and so we were curious whether it might be feasible to obtain particle size distributions between wild type and mutant by flow cytometry. The bacteria are filamentous and the clumps are very small by macroscopic standards, but I don’t know how this lack of homogeneity of the sample would impact the method. Many thanks for any advice that you might have and for your thoughts on the feasibility of the approach for this question.
> Cheers,
> Scott
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