I can tell you of my experience with the beta-galactosidase substrate
fluorescein digalactoside in Gram negative bacterial cells (E coli and a
marine bacterium Oceanospirillum).  The substrate could enter the cells
only through osmotic shock.  Fluorescence peaked five minutes after shock
and was virtually indistinguishable from back ground within twenty minutes.
The same was true for resorufin galactoside, although this fluor was easier
to distinguish from cellular autofluorescence as the excitation wavelength
is not 488.

>Dear confocalist,
>
>We are interested in using a fluorescent substrate for visualising GUS
>(beta-glucuronidase) activity in plant cells. We have been using the
>substrate sold by Molecular probes (ImaGene Green I-2908) and we encountered
>difficulties. First we got labelling in several negative controls. Second
>the pattern observed with X-gluc was sometimes very different from the
>pattern observed with ImaGene Green. We are using an Argon/Krypton laser on
>a Leica TCS-NT microscope.
>
>Does anyone has used this kind of fluorescent substrate in confocal
>microscopy with success? We would be very interested in knowing whether our
>staining conditions are appropriate or whether we should find another
>substrate.
>
>Thank you very much for your help.
>
>
>
>Olivier GRANDJEAN
>INRA GMPV Centre de Versailles
>Unité de Genetique
>route de St-Cyr
>78026 VERSAILLES CEDEX
>FRANCE
>
>
>Email: [log in to unmask]
>
>tel: 01.30.83.35.14
>fax: 01.30.83.33.19

Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 3o8
30 Convent Drive
Bethesda MD 20892
ph 301-496-2088
fax 301-402-0396