i am working on Scanning Electron Microscope, i cultured S.aureus on agar, and then took cells and washed them in PBS few times and then fixed over night in 2% Gluteraldehyde and then took a 10 micrometer on carbon tape to visualize, but i wasnt able to visualize the bacterial cells.
The end of your protocol is not clear to me. What do you do after fixation? did you dehydrate and dry your sample before the carbon tape step? Or did you put directly your liquid sample on carbon tape?
Without coating = without metal sputtering?
the best is to put small pieces of aluminum foil in wells (24 well-box for example) and then put a liquid solution of your living bacteria in the wells. You let them attach then fix, dehydrate and dry the aluminum foil. You can now put your aluminum foil on the stub with carbon tape.
After over night fixation in 2% Gluteraldehyde, i centrifuge sample and discard supernatant and then again wash cells twice with PBS, and then i pour 10 microlitre of sample on carbon tape already affixed on stub, and then i put stub in incubator in 50 degree celcius for 15-20 min to dry.
For now I cannot understand why you don't see your sample. By drying your drop, your sample should deposit on the stub.
I see that you dry your sample in PBS. You should wash your sample with water before drying because you get PBS salt crystal if not.
Thank you very much Nicolas for your valuable guidance, after fixation i will wash cells with de-ionised water and then to drop on carbon tape
You should read a bit about biological specimen preparation (google it).
And, by the way, carbon tape for bacteria is not a good idea.
Hello Abdul,
I totally agree with Vladimir.
Please, what information do you expect to get from air-dried bacteria samples? I think that with your preparation procedure you may get a lot of artifacts (cell aggregation, cell flattering, etc.) caused by surface tension forces.
I would not recommend to use aluminum foil in living bacteria culture. Aluminum is not inert enough. The better way is to use 12 mm sterile circular glass coveslips coated with poly-L-lysine (the best way is to use silicon wafers instead of glass coverslips).
We have done a lot of work on Streptococcus pneumoniae. If you want to get our protocol I can send it to you. However, we usually use critical-point drying and sputter-coating steps in our sample preparation procedure.
Regards
Oldrich
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