Dear Stefan
In principle you can use any flow cytometer to measure bacteria to various
degrees of perfection. Indeed a flowcell is of big advantage as (if coupled
correctly) it improves the numerical aperture of a system. Another important
feature is the slower flow speed (m/sec) inside a flow cell which helps to
increase sensitivity for small particles and the good hydrodynamic focusing
achieved by long flow cells as in the old Ortho systems.
Light scatter discrimination is limited. We measure routinely particles of
320nm on the Coulter XL analyzer and the Elite by light scatter analysis. With
the XL we discriminate on the side scatter diode (peak signal) for that purpose.
Unfortunately you can find bigger bacteria that show less light scatter signals
than those beads and bacteria growing under extreme conditions seem to fall
under this category.
Regarding multiple lasers I enjoy your enthusiastic approach. The adjustment of
different laserbeams is more a question of skill and practice. The choice of
excitation wavelength depends more on your budget and in what dyes you believe.
I get away with 488nm and 632nm which is also less pricy which might also prove
better for viable sorting.
I attached the abstract for the ISAC tutorial on microbial cytometry in 1998. I
am certain there will be a number of people that use different equipment for
different purposes you can talk to. Otherwise you are very welcome to visit our
laboratory for some practical demonstration or contact myself for further
off-line discussion.
Regards
Gerhard Nebe-v.Caron
Unilever Research, Colworth Laboratory
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel.: +44-(0)1234-222066
FAX: +44-(0)1234-222344
E.mail: gerhard.nebe-von-caron@unilever.com
Abstract for the tutorial on Microbiology:
Despite 23 years of microbial flow cytometry the technique
is still not so widely used amongst microbiologists as it
could be. Thus the aim of the tutorial is to encourage
people to take microbial cytometry on board as an additional
service that a cytometry facility can offer.
The tutorial will contain three short presentations
discussing biotechnological, environmental and medical
applications and address the fundamental question of
microbial viability and cell injury. This will be followed
by a technical discussion about instrument setup and other
useful tips and tricks. It is aimed at people that have
already a basic understanding of flow cytometry. As most
cytometers currently on the market can theoretically be used
for a number of microbial applications, the exchange of
technical information should allow operators to gain
confidence in microbial analysis with their particular
equipment.
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______________________________ Reply Separator _________________________________
Subject: right FCM for natural bacteria
Author: Stefan.Andreatta@uibk.ac.at at INTERNET
Date: 20/11/97 20:30
Dear listmembers,
We are going to purchase a flow cytometer with sorting capabilities
for use in aquatic microbial ecology. Our main task is to measure
bacteria from natural samples (DAPI, SYTO13, CY3,...).
Unfortunately these organisms are very small (down to 0.2 microns)
and they do not live in the water all by themselves - there will
be lots of algae, flagellates, detritus etc.
So we need to have good sorting capabilities to check what we are
actually measuring in the microscope, but the big problem (I feel)
is the instrument's sensitivity.
I would like to ask experienced users out there:
1- Which features, do you think, are of particular importance to our
application. (PMT for forward scatter is certainly one). Other
important features?
2- What do you think of jet in air vs. cuvette sensing. Flat surfaces
should enhance scatter measurement, but seemingly only Coulter is
ready to provide a sorting(!) cuvette. Other important features?
3- We need three excitation wavelengths (UV, 488, 514 or higher) for
various experiments, not all three at the same time but all possible
combinations of two of them. This will involve a lot of laser
adjustment. How complicated is it on different instruments e.g. to
shift a laser beam from second to first sensing position?
4- I would highly appreciate hearing about any relevant experience on
different instruments. What we have in mind is to bye one of the
following (in alphabetic order):
-Becton Dickinson FACS Vantage
-Coulter Epics Elite ESP
-Cytomation MoFlo MLS.
Thanks for any hints
Stefan
----------------------------------------------------
Stefan Andreatta
University of Innsbruck, Austria
Institute of Zoology and Limnology
Technikerstrasse 25, A-6020 Innsbruck
phone: +43-(0)512-507-6122
fax: +43-(0)512-507-2930
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