Re: Cytek info and bacteria
Hector Nolla (hector@soest.hawaii.edu)
Mon, 30 Jan 1995 15:46:07 -1000 (HST)
On Thu, 26 Jan 1995 hilliard@zookeeper.zoo.uga.edu wrote:>
> On a related note, can anyone give me some tips on analyzing bacteria with
> a 753 or Elite Analyzer? We haven't attempted anything yet, but the idea
> is to label the strain of interest with an antibody, and I assume I might
> have to gate on SSC or discriminate on the FITC fluorescence. Anyone
> having any luck with these sub-micron particles?
>
> Thanks in advance,
> Steve
> ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
> Steve G. Hilliard, Cell Analysis Facility |
> University of Georgia | "Be good and you will
> hilliard@zookeeper.zoo.uga.edu | be lonesome..."
> ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
>
> We routinenly analyse bacteria in sea water in a EPICS 753. We fix the
samples with PFA and store them frozen until analysis. Instead of FITC
conjugated antibodies we label the DNA with Hochst 33342 (UV excitation,
450nm emmision) and measured the fluorescence using a "Biosense" flow cell
tip. This approach has worked pretty well for us, however, alignment can
be a pain for the following reasons. You have to find the "sweet" spot in
the flow cell tip( this is the position where the fluorescence
magnification lens in the flow cell tip is at optimum alignment with the
"pick up" fluorescence lens that transmits the signals to the PMTs in the
back. You can do this using alignment beads such as DNA check 10 um beads.).
Since you will probably be working close to the edge of detection, it is
best not to use the gated amplifier( this can result in the amplification
of "noise" and give you overstimates of your populations) We also use a
mixture of submicron beads from polysciences(0.46um UV fluoresbrite beads
Cat#18339 and 0.98um fluoresbrite beads cat#18860 as well as another
visible excitation 0.6um bead). Since we also collect red fluorescence
from chlorophyll we excite with the 488nm line of a coherent 90-5 laser
colinear with our UV laser. If you are going to use FITC labelled
antibodies, you may want your "green" fluorescence to be your trigger
signal(this will clean up a lot of the "noise" as well as smaller size
listmode files). One final point, dont be afraid of boosting the power
output of your 488nm laser, we routinly hit our samples with 1 watt of
power. I hope this helps, good luck.
Hector Nolla
Dept of Oceanography
University of Hawaii.> >
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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