[Cytometry] FW: flow cytometry question
g.nebe-von-caron at alere.com
Tue Feb 4 10:14:30 EST 2014
There is no way you can obtain any real size information you can get from light scatter. Please read my open access article Nebe-von-Caron G (2009) Standardization in microbial cytometry. Cytom Part A 75A: 86–89 were I demonstrate the discrepancy of light scatter of bugs and beads regarding size.
You might want to look down a microscope for independent assessment of aggregation. Peak/area measurement may be unfavourable with the small particles but plotting log side scatter vs log fluorescence would be a good indication of aggregation I if not already delivering discrete clusters from their natural fluorescence, then possibly a DNA stain might reveal the aggregation level in a better way.
Considering that your forward scatter is unlikely to detect the bacteria you should trigger on side scatter. However you also should be aware that rods or filamentous bacteria my result in double cluster due to preferential orientation in the beam. This effect however gets lost in higher aggregates.
You can make sonicated control samples (usually ~20 sec in a hot spot of a bath) to see if they break apart albeit this could affect autofluorescence but please do not confuse scatter with size. In particular if the bacteria use gas bubbles for buoyancy control or have inclusion bodies from things they accumulate or produce you can get a lot of strange signals.
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of christopher.fogarty at helsinki.fi
Sent: 04 February 2014 07:18
To: Shaw, Pam; Cytometry Mailing List
Subject: Re: [Cytometry] FW: flow cytometry question
I have previously worked with bacteria in the FACS machine and have found a pretty significant change in scatter properties if I don't vortex the samples just before the run. Given that it's relatively straight forward to see the different scatter properties before and after vortex, it very well may be possible to see differences in clumping between a population that clumps and one that doesn't as much using FSC-A vs -H and SSC.
It seems that testing the hypothesis would be pretty straight forward, as least. But keep in mind that vortexing affects the scatter properties.
Hope this helps,
Quoting "Shaw, Pam" <pam.shaw at mso.umt.edu>:
> 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
> pamela.shaw at umontana.edu<applewebdata://DE30ADE2-B1DF-44AF-B224-B0B84E
> DBD782/pamela.shaw at umontana.edu>
> 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
> Cytometry mailing list
> Cytometry at lists.purdue.edu
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