E. Coli. on Elite

Hazel Davey hlr at aber.ac.uk
Tue Aug 4 04:28:54 EST 1998


Rochelle A. Diamond wrote:

> We now have two investigators that need to
> analyze and sort E. Coli and other bacteria (waste water type).  

Here are several thoughts based on our analysis of bacteria 
including E. coli :

1) Make sure that all buffers etc are filtered. With our bacterial 
samples I use a 0.1um filter rather than a 0.22 to prepare sheath 
fluid / dilution buffers etc.

2) Is it possible to use a fluorescent stain for your E. coli that 
would allow you to discriminate them from the other particles?

3) If you have the gated amp electronics on your Elite use them 
even if your only using one laser - we find it really does improve 
detection.

4) I doubt that settings are directly transferrable between 
machines, but just to show that you don't need to push the voltage 
etc on the forward scatter detector to extreme levels, here are our 
starting "cytosettings" for bacteria using the Argon ion 488 nm 
laser (All detectors are set to logarithmic):

FS 400 Volts, IGAIN=5, PGAIN=1, DELAY=UP
PMT1 (<440nm) 630 Volts, IGAIN=3, DELAY=LOW
PMT2 (Side Sc) 400 Volts, IGAIN=5, PGAIN=5 DELAY=UP
PMT3 (525nm) 570 Volts, IGAIN=5, PGAIN=10 DELAY=UP
PMT4 (575nm) 700 Volts, IGAIN=5, PGAIN=10 DELAY=UP
PMT3 (>600nm) 500 Volts, IGAIN=3, DELAY=UP

With this setup "noise" is all below channel 50, and roughly 
speaking, bacteria are in the 200-500 channel number region and 
yeast appear around channel 700.

I hope some of this helps,

Haze
----------------------------------------------------------
|            Hazel Marie Davey   hlr at aber.ac.uk          |
|Sefydliad y Gwyddorau Biolegol*Inst. Biological Sciences|
|Prifysgol Cymru               *      University of Wales|
|     ABERYSTWYTH, Ceredigion, CYMRU / WALES SY23 3DD    |
|            http://pcfcij.dbs.aber.ac.uk/index.htm      |



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