Dear Biofilm people,

Sorry to me so slow in responding to the post of about a month ago but,
during the recent 3D Microscopy Course on Living Cells, one of the students
was looking for GFP-labelled bacteria in biofilms when an accident caused
the bacteria to eliminate the plasmid.  This left her  with no specimen.
Rather than give up, she tried looking at the specimen with backscattered
(reflected) light.  On confocals that were set up to eliminate reflection
artifacts from the optical components, she was able to make some very
interesting 3D data sets. Each bacteria was easily visible as a small
sphere or oval.  Had the GFP still been in place, GFP-bacteria could have
been imaged in the context of all the other bacteria present.

There was general recognition that this was a very useful imaging method.
Aside from fluorescence, BSL is the only fully confocal imaging mode that
can be employed on biological specimens and bacteria seem to be the ideal
type of specimen.  (Larger objects generate specular reflections that
produce images that are less easy to understand.)  What is more, no dye is
needed and no light need be absorbed.  Presumably this should mean that no
damage is done and it should be possible to make long-term 3D time-lapse
images of biofilm development without the imaging system interfering with
the process.

Just a thought.

Jim Pawley

>Given that the principle of the kit is based not only on differential cell
>wall permeability to hex iodide as well as on the higher affinity of one
>stain for nucleic acid, maybe some more info from you on the types of
>problems you are having would be enlightening.  Are the streps labelling
>incorrectly, or is it the P ging, or maybe both?  Also, as half the kit is
>based on a "live cell" stain, it is possible that if your bugs are not
>labelling well because they are not happy.  Remember that MP designed these
>stains for application to growing planktonic cultures, not for use in
>biofilms.  Does the stain work for pure culture planktonic cells?  If not
>you are, ahem, screwed.  You must also remember that MP tested the stain on
>only a small number of pure cultures.  When last I checked, the total
>number was ten with "no plans to test further - this is an investigator
>issue."  Yeah, right.......  We have used it on biofilms that grow up from
>whole salivary inocula and see initially a vast majority of G+ that lessens
>with time - but given the complexity of the inoculum, we cannot say
>anything about how accurate that picture is other that the community
>evolution seems to resemble what goes on in the mouth.  We have generally
>seen nice results with Live/Dead (only green or red cells), but you can get
>indeterminate staining (yellow cells).  The L/D kit is much more
>straightforward in principle than is the pos/neg kit, however.
>Another factor to consider is that one component of the kit is a
>"viability" stain.  If you are working in an aerobic system, then the P
>ging are certainly not as happy as they could be, and even the facultative
>streps could be better off.  Along these lines, MP sells yet another, even
>more complicated kit that not only tells you which cells are G+ and which
>are G-, but also which ones are "viable" to boot!  The interesting point
>for you is that the Gram sign principle is completely different (and is
>easier to understand and therefore more accepted by the scientific public)
>than the kit you are currently using.  We have this kit but have not used
>it much because the only times we've really been interested in Gram sign,
>our inoculum has been so complex as to make the staining "difficult" to
>interpret (see above).
>
>
>>Hi All,
>>
>>I've been trying to label a multispecies biofilm, Streptococcus gordonii
>>and Porphrymonas gingivalis by using Molecular Probes Gram +, Gram - kit.
>>I'm having a very difficult time in the specificity of the stain, and have
>>exhausted the possibilities of manipulating the concentrations of Syto 9 to
>>hexidium iodide, per the recommendation in the instructions.
>>
>>In all fairness to MP, I haven't contacted their tech support yet, and I
>>was hoping someone else might have some other insights.
>>
>>Cheers,
>>
>>-Guy
>>
>>**************************************
>>Guy Cook
>>President
>>Bacterin Inc.
>>P.O. Box 6743
>>Bozeman, MT 59771-6743
>>(406) 582-8184
>>[log in to unmask]
>>http://www.bacterin.com
>>*************************************

                   ****************************************
Prof. James B. Pawley,                                       Phone:
604-822-6996
3D Microscopy of Living Cells: Summer Course   FAX:   604-822-6089
                                           Cabin 604-883-2095
c/o Dr. Elaine Humphrey
[log in to unmask]
Biosciences Electron Microscopy Facility
University of British Columbia, 6270 University Blvd. Vancouver, BC, V6T-1Z4

If it isn't diffraction, it is statistics:Trad. microscopist's complaint,
Anon.