FACSing Bacteria

Ray Hicks rh208 at cus.cam.ac.uk
Mon Nov 11 07:58:58 EST 1996




The settings appear to be rather improbable ( unless you've got a modified
FACScan).  FSC doesn't have a PMT on the standard model, there are two gain
stages E-1 to E03, and 1.00 to 9.99.  If you've got the coarse gain set to
E-1, youre attenuating your FSC signal (multiplying it by 0.1).  Are you
triggering on FSC? If you are, and you're getting spurious signals, I
imagine that you've got the amplifier in log, which is pretty messy.

Try switching to linear amplification, and increasing the coarse gain to
100 (E02), stepping through E00 and E01 on the way to see what sort of
background signals you get from sheath particulates.  It would be worth
your while dropping the trigger threshold to 32 so that you can see small
signals, you can rack it up to cut out noise when you see your bacteria
appearing.  You can then "centre" your bacteria using the fine gain control
(1.00-9.99).  On the FACScans that I've used it's best not to drop the
trigger threshold below 32 because the machine seems to stall.


good luck

Ray


>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.
>---------------------------------------------------------------------------
>-----
>At 2:30 pm 8/11/96, Steven Merlin wrote:
>The settings for FSC appear to be very high, resulting in large
>amplification of debris and other particulates.[snip]

                              Ray Hicks
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