Yes, I heard this from others who responded to my (inadvertent - oops -
hope that's the last time I make that mistake!) post to the list.  In fact,
this was the major complanint - that they just couldn't see the forest for
the trees.  Now, what about antibodies?  Here, the major problem is
specimen preparation (like usual) so if you know what you are looking for,
can make a decent antibody to it, and aren't overly concerned about the
time involved to process the specimen (say 8-10 hours), then you can get
the critters labeled rather brightly.  Also, you have the luxury of
choosing your excitation wavelength to get away from the "bad" UV and blue
excitation that causes a lot of your autofluorescence.

>Rob,
>
>In my case the problem is not just distinguishing bacteria from eukaryotes but
>finding the needle in the haystack.  With so much background fluorescence from
>the huge amounts of eukaryotic tissue (not just cells but collagen, ECM, etc.
>etc.) the bacteria just get lost.  Also, our applications usually involve
>situations where there are very few bacteria to begin with.  We really need
>something that will light them up like stars in the night sky in order to find
>them (and to be able to take advantage of the "contrast effect" so we
>don't have
>to do all our hunting at highest mag.).  It's a real challenge, and my meager
>efforts at seeking an appropriate dye have been disappointing, except for a
>couple slight possibilities as I mentioned previously.  I've tried two of the
>Molecular Probes Live/Dead combo's but had poor results.  Running more
>exhaustive
>trials of all their bacterial stains is one item on my long To-Do list.   If
>anyone else has already done this, it would save me a lot of time and
>frustration!
>
>Karen
>
>
>Robert Palmer wrote:
>
>> Hi Guy - I'm having a hard time envisioning a microscope with the
>> resolution necessary to positively identify bacteria but that doesn't
>> provide the sort of image necessary to distinguish bacteria from eukaryotic
>> cells, particlularly from epithelial cells which are rather big and
>> regularly shaped.  I suspect part of the problem is that the device will be
>> "automated" and some computer program will tell the doctor how many
>> bacteria are present.  In other words, humans are not interpreting the
>> image, and that usually results in discrimination problems.
>>
>> So, on another tack, how about B2K?  I think I got a positive response from
>> you when I asked if Bacterin would care to take part in the "microscopy
>> workshop" this August.  What would you folks like to show and how much
>> space might you need?  It's getting time for me to start writing things
>> down now, so I wanted to check in again with you.
>>
>> So far, we have commitments from BitPlane and from Leica.  Zeiss has
>> declined for B2K but will send a machine to the workshop at ASM in May.
>> Have yet to hear from Vaytek and BioRad.  Nikon will also likely show up
>> again.  Know of any other companies that might be interested in some cheap
>> publicity?
>> Rob
>>
>> >Rob,
>> >
>> >This isn't my project, but I was asked to checked into the possibility of
>> >such a stain.
>> >
>> >The application is to image vaginal bacterial flora in-vivo.  A minimally
>> >invasive confocal microscope, about 1mm in diameter, would be used to image
>> >the bacteria.  Yes, it does exist, I've seen it, and it works.
>> >
>> >The problem (among others) now seems to be differentiation of the bacterial
>> >cells from the epithelial tissue.  When I left the CBE a few years ago,
>>this
>> >group was playing around with conjugating the vitamin B12 to various
>> >bacterial cell membrane targets.
>> >
>> >I was curious if the listserve had any similar needs/experiences.
>> >
>> >-Guy
>> >
>> >Guy Cook
>> >President
>> >Bacterin
>> >910 Technology Blvd.
>> >P.O. Box 6743
>> >Bozeman, MT 59715
>> >406-582-8184
>> >Fax 406-586-0396
>> >http://www.bacterin.com
>> >ftp.bacterin.com
>> >
>> >
>> >
>> >
>> >> -----Original Message-----
>> >> From: Confocal Microscopy List
>> >> [mailto:[log in to unmask]]On Behalf Of Robert Palmer
>> >> Sent: Friday, February 11, 2000 5:33 AM
>> >> To: [log in to unmask]
>> >> Subject: Re: Vital Dyes
>> >>
>> >>
>> >> I suppose this is a naive question and one that, if you were to
>>answer it,
>> >> would require that I be shot afterwards, but here goes - why is it so
>> >> critical that the dye NOT stain eukaryotic cells?  I wasn't aware of
>> >> significant problems distinguishing eukaryotic cells from bacterial
>>cells.
>> >>
>
>--
>Karen S. Zaruba  [log in to unmask]
>3M Company, St. Paul, MN