I agree with Rob, for monolayer or very thin films, reflected light DIC
works very well. It also helps to have a highly reflective, or well
polished substratum. For thicker biofilms, I have not been able to
distinguish individual cells within a biofilm colony, and resort to fluor
labeling for the best images.
Ace Baty at the Center for Biofilm Engineering (CBE) has published a few
papers using the reflected light DIC technique. Also, Andy Rice, also of
the CBE, has just finished his thesis tracking a mixed population of
GFP-plasmid labeled Pseudomonas aeruginosa PAO1 and unlabeled Pseudomonas
aeruginosa PAO1. He switched between fluor excitation and reflected light
DIC to track motility of the labeled cells inside of a biofilm colony, but
it was always a very thin biofilm.
-Guy Cook
Bacterin
----------
> From: robert palmer <[log in to unmask]>
> To: [log in to unmask]
> Subject: Re: Gram +/- Stain
> Date: Friday, July 16, 1999 1:44 PM
>
> Reflection is a very under-utilized technique in biology. It works just
> fine on monolayers and thin biofilms - but in my experience becomes less
> satisfactory with films over about 5 um. We use it just as Jim has
> described: to show the presence of non-fluorescent cells, e.g., cells
with
> no GFP signal in a biofilm containing GFP-expressing cells. The damage
> issue is another ball of wax. The laser is running and light is
certainly
> absorbed, but the damage must be less than when a fluor is involved.
> Fortunately for us prokaryotic people, bacteria are generally amazingly
> hardy and continue to divide even after having been imaged with Syto
stains
> hanging all over their nucleic acids...
> Rob Palmer
> CEB/UT
>
> >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.
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