Are there good ways to embed live bacteria for microscopic imaging? I tried agar(ose) which works okay, but seems a bit fussy.
Dear Zalan,
it would be of interest to know which kind of >microscopic imaging< you are going to do.
As an electron microscopist I have done several preparations from cultures done in the Bacteriology Department, and used fixed (e.g. meningococci) culture with the underlying culture substrate to process that for a) resin semithin sections (imaged by LM) as well as ultrathin sections (TEM). Due to the fact that chemical fixation always induces so called fixation artifacts it may be necessary to know also the strain you want to image (usually - with conventional fixatives and processing - the bacterial membrane is well preserved but not the bacterial body itself, nor - in most cases - the membrane-adhering glycoconjugates/glycocalyx. So always you have to consider a very special fixative - if you are not going to use e. g. HPF and Cryo-TEM for such. In case you only would like to image bacteria by LM-methods, pelleting by centrifugation in agarose (as you tell us above) might be working but perhaps needs some modifications (temperature during centrifuging, time, g-forces, etc., as well as a suited fixative and suited staining procedure). Best wishes and regards, Wolfgang MUSS, PhD, Salzburg-Austria
Dear Zalan,
To image live bacteria in native conditions, you can use Atomic force microscopy. To immobilize the bacteria, you can use filtration membrane. Using this method, we are able to image at different temperature and under dvarious buffer a lot of bacteria (GRAM + and GRAM -), yeast and viruses.
Dear David, would it be possible to tell something about the safety measurements you have in your lab doing such work? (risk of infectiosion) but perhaps you are in Bacteriological Lab where you have at least BCL-2 condition. I greatly should appreciate your kind comment on safety regulations you might have to follow with this. Thank you and best regards, Wolfgang MUSS; Salzburg, Austria
Dear Wolfgang Muss,
That depends on the bacteria. For commensal bacteria we work in normal lab but for bacteria such as Mycobacterium or yeast such as Candida we have bacteriological lab.
Best regards
David
Dear David,
your method sounds very interesting! Are the bacteria/yeasts actually viable during/after the AFM imaging? We also use porous aluminium oxide (which is also a filtration membrane) as growth surface and image microcolonies by light microscopy but the contrast is often too low to see them without additional staining. Do you use additional fixation once the microbes are on the filter?
@Wolfgang: thanks to you too for your comments! I normally use unfixed samples and mix fluorescence-stained bacteria 1:1 with 2% LMP, and was mostly wondering if people use other (simple) techniques.
Best wishes, Zalan
Dear Zalan,
thank you!
apologize for an additional question: what do you mean with “2% LMP”, guessing this could not be the instead perhaps it should be “low melting point” agarose? Sorry, I could not find an unanimous result for that abbreviation. Thank you for clarification, regards, Wolfgang
Dear Zalan,
The AFM is coupled with a flurorescence microscope and therefore we are able to do viability test. So we are sure that the cell are always viable. We can alsor perform experiment in growth medium and follow the germination of fungi for example. We don't use any chemical fixation.
To have some example you can read the following papers
Article Nanomicrobiology
Dear Wolfgang,
sorry for the sloppiness, I meant low melting point agarose.
kind regards, Zalan
(thanks David for the reference!)
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