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This is not unusual.  Your suggestion that you need to "fine tune"
the ratio of Syto to PI is exactly right - you want the confocal
image to approximate that which you see through the oculars using a
LP FITC filter.  You should see (means  through the oculars) a
mixture of green and red cells.  If the culture is "viable", then the
vast majority of cells will be green.  Sometimes you may see yellow
cells - not to worry!  Just get the scanned image to approximate what
you see in the oculars.  Bleaching may be a problem (Hg illum AND
laser illum) so move to new fields frequently.  Once you get things
set up pretty well, save these setting and use them as the starting
point for other samples.
Some hints on "controls":
viable cells: fresh culture (exponential phase growth) - these are
the cells on which you would try your different mixes of Syto/PI
"dead" cells: make culture 50% with respect to ethanol

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Has anyone using BacLight from Molecular Probes (PN L-7012) to assess
>bacterial viability had an experience similar to the following?
>
>An investigator is using it with Vibrio vulnificus and even with log phase
>cultures (which they are fairly certain contain a large majority of viable
>bacteria), we see, on our Leica TCS SP2 confocal, mostly doubly stained
>cells.  We are exciting with 488 line and detecting green fluorescence at
>500 to 535nm and red fluorescence at 555-620nm in a sequential scan mode.
>
>The kit employs SYTO 9 green fluorescence nucleic acid stain and propidium
>iodide.  SYTO 9 will apparently stain all bacteria (those with intact as
>well as those with damaged membranes) whereas the PI will stain only those
>with damaged membranes.  In dead cells, PI, with its higher affinity for
>nucleic acid, displaces the SYTO 9 from dead cells resulting in dead/red
>cells and leaving the viable cells green.
>
>In our samples, all of the cells are red - either singly red or red/green -
>and these are cultures in which we would expect a high percentage of
>green-only, viable cells.  In fact it would seem that the percentage of
>red/green cells correlates closely with the percentage of viable cells.
>
>This probably means we need to fine tune the SYTO9 and/or PI concentrations,
>or maybe the staining procedure, but we also wondered if perhaps others
>might have had a similar experience and can offer suggestions.
>
>Thanks.
>
>Ray Hester
>Univ. of South Alabama
>Mobile, AL
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Robert J. Palmer Jr., Ph.D.
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