FACSing Bacteria

Steven Merlin merlin at patho.unibe.ch
Fri Nov 8 09:30:06 EST 1996


On Nov 6, Scott Simpson wrote:
>
>        I'm trying to quantify the amount of complement factor H deposited on
>Neisseria gonorrhoeae by FACS. I'm using a BD FACScan machine. When
>acquiring the data I have the FSC set at E-1 (PMT is at 868) and I'm
>noticing that I'm getting non-specific background noise that will not go
>away even when water is running thru the machine.  Do you have any
>suggestions about what settings (FSC/SSC) I should be using to analyze
>bugs with this machine and any thoughts on the background noise.
--------------------------------------------------------------------------------

The settings for FSC appear to be very high, resulting in large
amplification of debris and other particulates. This is most likely the
reason why high background counts are being obtained when water is running
on the instrument.

We start off with an FSC detector level of E01, Amp gain of 5.70 and Log
Mode. These settings will cover particles from approx 0.3 to 2.5 microns in
size. Switch over to linear if needed and readjust the amplifier gain. A
number of companies sell various size beads which can be used to set an
upper and lower size gate. Polysciences in Warrington, PA is one such
company offering bead sizing kits.

If the in-line 0.45 micron saline filter has not been changed in the past 6
months, it is recommended to replace it. I prefer using a Millipore 0.22
micron Milli-Fil GS Filter Unit. It can be easily accomodated on the
FACScan with luer fittings and CPC Quick disconnect couplers. The
commercially prepared sheath fluid is fine. If you prepare your own, be
sure to adequately filter it before running it through the cytometer.




Steven Merlin
University of Bern
FACS Core Facility
Murtenstraase 31
CH-3010 Bern, Switzerland
Lab:     ++41-31-632-8876
Office: ++41-31-632-9960
FAX:     ++41-31-381-8764
e-mail: merlin at patho.unibe.ch





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