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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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