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I had some very useful replies and several requests for a summary of
the answers, so here they are!
1) Several people felt that we already have the technology necessary
to make a range of useful measurements, and that the real problem
lies in educating the microbiologists - making them aware of the
method, the advantages of the method and getting them to accept it.
To aid this process some people suggested ways that this could be
done e.g.
a) We need more simple cytometers with a better resolution.
b) Foolproof, robust, intuitive, icon driven, windows/Mac
c) Costs little but looks great!?!
As one respondent pointed out, something that has improved acceptance
quite a bit is the use of the Autoclone on the Elite to sort single
cells on agar plates. "This makes it easier for the microbiologists to relate
to the fancy coloured dots and clusters on the computer screen."
2) There were however calls for other developments. Bacteria have
a much lower e.g. DNA content than mamallian cells and several people
sugeested the need for more specific dyes with higher quantum yield.
While some stated that present instruments are sensitive enough,
others require higher sensitivity, e.g. for measuring
autofluorescence of marine photosynthetic prokaryotes.
3) In environmental monitoring there may be many different cell types
present and so to distinguish between different organisms the need
for 5 or more colours in addittion to 2 light scattering channels was
expressed. This would require efficient narrow-band optical filters
and v. sensitive PMTs.
4) Fluorescently-labelled antibodies for microorganisms are not
readily available "off the shelf". This is seen as a big problem as
regards identification of bacteria in mixed environmental samples.
There are further complications here, relating to microbiology rather
than flow cytometry, in that the antigens expressed may vary
according to growth conditions - "laboratory bacteria" grown on rich
media can be very different to those found in some natural
environmental samples.
5) For some potential applications the low throughput of flow
cytometry is seen as a problem. This may seem odd as we are all used
to describing flow cytometry as a rapid method, but the problem is
that it can only process samples sequentially, rather than in
parallel. For example, flow cytometric sorting *could* be used to
isolate a mutant that produces a high amount of X from a cell
population, but you could use a 96-well plate reader to screen many
isolates simultaneously.
6) ACCURATE total counts and viable counts are very important to many
microbiological procedures. The extent to which a flow cytometer will
allow you to obtain reliable numbers varies between models /
manufacturers.
7) The development of low-cost light sources such as the laser diode
may be expected to make the technique of flow cytometry much cheaper
and thus more accessible. These systems may be expected to be much
smaller and more robust, and thus portable for field analysis.
And just to give fellow microbiologists out there a bit of
encouragement - we are in a growing field!
Hope that some of you find this useful and interesting. I'd be happy
to hear peoples opinions on any of the above.
Many thanks to those who sent responses to my original question.
Haze.
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|Sefydliad y Gwyddorau Biolegol, Prifysgol Cymru, ABERYSTWYTH, Dyfed, CYMRU |
|Inst. Biological Sciences, University of Wales, ABERYSTWYTH, Dyfed, WALES |
|hlr@aber.ac.uk, http://144.124.112.37/index.htm (FCM | Welsh | Brewing) |
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