How to use the Coulter Epics Elite
Flow Cytometers at Aberystwyth
We used to have a Skatron Argus but this has been retired to Cardiff. Information on this flow cytometer is still available here.
There is more information on the features of these machines below. First of all though there follows a brief description of what is meant by the term "flow cytometer", and what it is that they do.
Introduction
In a typical microbial flow cytometer individual particles pass through an illumination zone,
typically at a rate of some 1000 cells.s-1 (although much higher rates are possible
in specialised instruments, and appropriate detectors, gated electronically, measure the magnitude
of a pulse representing the extent of light scattered. The magnitudes of these pulses are sorted
electronically into "bins" or "channels", permitting the display of histograms of the number of cells
possessing a certain quantitative property vs channel number. Although many of the applications of
flow cytometry, such as microbial discrimination, require only a qualitative output,
the flow cytometric approach also offers quantitative information.
The angular-dependence of scattered light provides information on the nature of the scattering particles but more importantly, appropriate fluorophores may be added to the cell suspension. These may be stains which bind to (or react with) particular molecules such as DNA, RNA or protein, fluorogenic substrates which reveal distributions in enzymatic activity, indicators which change their property as a function of pHinor which are taken up in response to membrane energisation, or, increasingly, antibodies or oligonucleotides tagged with a fluorescent probe. 2- or 3-variable histograms or contour plots of, for example, light scattering vs fluorescence from a DNA stain (vs fluorescence from a protein stain) etc. may also be generated, and thus an impression gained of the distribution of a variety of properties of interest amongst the cells in the population as a whole.
The Skatron Argus 100 Flow Cytometer
Given that both the cell volume and the DNA content of bacteria is some 1000-fold less than that of higher eukaryotic cells, other flow cytometers have until recently proved unsuitable for the study of unstained microorganisms. The Skatron has a specially designed open flow chamber resulting in a high signal-to-noise ratio and is therefore ideal for detecting light scattered by microorganisms. Some notes on using the Skatron are provided here.
Coulter Epics Elite
Like most flow cytometers the Elite
uses laser light, infact our model has 4 lasers thus giving us a range of excitation
possibilities, allowing a wide range of fluorochromes to be studied. The Elite was
designed primarily for use with mamalian cells, but its sensitivity is just about sufficient
for the study of light scattering by bacteria, and where they have been stained with
fluorescent compounds they can be detected and analysed with ease.The Elite has the advantage over the Skatron that it is able to sort the cells of interest based on the light-scattering and/or fluorescence characteristics measured. Typically cells are sorted onto a microscope slide for further analysis or live cells maybe sorted into test tubes or into wells of a microtiter plate to be grown up for further study.
Aber Instruments Microcyte
The Microcyte was launched at
the ISAC 1996 conference in Rimini. It exploits a red (635 nm) laser diode as the light
source and has an internal battery pack allowing it to be used as a fully portable flow
cytometer. The instrument has been designed primarily for the analysis of
microorganisms and can detect and provide accurate absolute counts of (i.e. without an
internal standard) particles as small as 0.4 micrometres. For more information on this flow
cytometer you can visit my Microcyte pages,
Aber Instruments or
Optoflow
Flow Cytometry Home Page