GFP doesn't need oxygen to fluoresce, it needs oxygen to become
fluorescent.  I take it you could clearly see bacteria by some means other
than GFP fluroescence (otherwise you wouldn't have any basis for saying
that the GFP was non-fluorescent).  One problem you may have is
heterogeneity of expression within your bacterial population, particlularly
if that expression is plasmid-based.  If you simply grow your bacteria in
liquid culture, are all cells brightly fluorescent?  Is there any effect of
culture age upon fluorescence intensity?  Are antibiotics required to
stabilize expression (usually the case with plasmids)?


>Dear Steve and confocalists,
>
>I tried yesterday paraffin embedding and sectionning of plant
>tissues infected with bacteria expressing GFP. I fixed my samples
>with 3,7% formaldehyde, and then dehydrated my samples in
>increasing acetone concentration baths (30%, 50%, 70%, 100%). Then
>I observed them under confocal, and I must say that I did not see
>any green fluorescence. Doesn t this problem with GFP observation
>after paraffin embedding also comes from the fact that GFP needs
>oxygen to fluores? This is a question I asked myself, but was not
>able to answer, as some state that oxygen should be only needed
>during the maturation steps of GFP. but on the other hand I never was
>able to observe GFP after paraffin embedding.
>So if anyone would have a trick to do that, I d be as interested as
>Steve.
>So Steve, I hope this might be of some use for u,
>cheers,
>tristan
>  On  8 Mar 00 at
>10:57, Steve Barlow wrote:
>
>> Date:          Wed, 8 Mar 2000 10:57:35 -0800
>> Reply-to:      Confocal Microscopy List <[log in to unmask]>
>> From:          Steve Barlow <[log in to unmask]>
>> Subject:       GFP  and greening tissue
>> To:            [log in to unmask]
>
>> hello all
>>
>> One of my users has GFP labelled protein being expressed in mouse embryos.
>> He fixes all his tissue in buffered 4% para-formaldehyde and stores his
>> tissue in this soution for months at a time at 4 C.
>>
>> After a few months, he has noticed some of his tissue, in particular the
>> heart, turns green, making examination of tissue for green GFP fluorescence
>> impossible.  Has anyone any explanation of this effect?  I have suggested
>> he store his tissue in buffered 0.5% formaldehyde for long term storage.
>> Any suggestions for a better alternative?
>>
>> this user also wants to try paraffin embedding rather than continue his
>> cryostat sectioning.  Does anyone have a protocol that will preserve GFP
>> fluorescence during processing?  ( I thought I saw a thread recently that
>> stated alcohol or acetone dehydrations would quench GFP fluorescence.  Does
>> this mean any sectioning work  on GFP expressed protein is tissue samples
>> relies on a secondary procedure such as anti GFP antibody labelling?)
>>
>> thanks in advance for your knowledge and insights
>>
>> steve
>>
>>
>>
>>
>>
>> Dr. Steven Barlow, Associate Director
>> EM Facility/Biology Department
>> San Diego State University
>> 5500 Campanile Drive
>> San Diego CA 92182-4614
>> phone: (619)594-4523
>> fax: (619) 594-5676
>> email: [log in to unmask]
>> website:  http://www.sci.sdsu.edu/emfacility/
>>
>> Chairman, Educational Outreach subcommittee
>>   promoting access to microscopes
>> Microscopy Society of America http://www.msa.microscopy.com/
>>
>
>************************************************
>Tristan Boureau
>Biocenter Helsinki, Department of Biosciences
>Division of General Microbiology, PO Box 56
>FIN-00014 University of Helsinki, FINLAND
>E-mail [log in to unmask]
>tel: (00 358) (09)708 59 655
>     (00 358) (0)50 329 58 07
>************************************************

Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 308
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396