To all:
Is there a definitive EM method of determining whether an unknown bacterium (in this case a plant pathogen) is G+ or G-? This one gives ambiguous results at the LM level.
TIA.
Doug Davis
doug_davis@maillink.berkeley.edu

Doug:
I don't think there is such a thing as a definitive TEM method. :-) let alone for G+/G-... But having said that, if you look at a thin-section TEM of your plant pathogen and see both inner and outer membranes, it's probably G-. G+ will have a denser peptidoglycan layer (electron dense). But EM is fraught with artifacts, etc. so your best choice would be to find a local EM expert to help you out. (if G+/G- is truely important for you).
Molecular probes also sells a fluorescently conjugated lectin which is specific for peptidoglycan. They have a nice protocol for staining with it, and this may give you a cleaner, cheaper result than traditional Gram staining, using only a fluorescence microscope. Although the lectin is slightly expensive, it's a drop in the bucket compared to time/materials/ microscope usage for the TEM stuff.
Regards
Dave Graham
dgraham@life.uiuc.edu


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