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Ray-
        over the years, we have learned that before we use and interpret
BacLight as a live/dead indicator, we have to do lots of controls. Some
cultures don't seem to stain with PI at all, in others, every cell is red.
We see a lot of yellow too. The killing agent we use prior to staining seems
to interfere pretty seriously with staining - for example we may from
plating, know that most of the cells are viable, but they stain red, not
green. If you want to test biocidal agents like we often do, there is a ton
of preliminary work to do first. We also find that we can get separate red
and green cells in some Gram positive cultures, but not in some Gram
negatives - there seem to be a million variables, which render the assay
anything but cookbook. So, yes, we see what you do too, and in such
situations, it appears to be the staining not the microscope filters and
settings. So we generally hesitate to interpret the green/red as live/dead
without doing a lot of preliminary work first.

Betsey Pitts

-----Original Message-----
From: ray hester [mailto:[log in to unmask]]
Sent: Tuesday, June 17, 2003 8:50 AM
To: [log in to unmask]
Subject: BacLight


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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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