How should I inactivate my adenoviruses for transmission electron microscopy (negative staining)? Which methods affect their visible structure the least?
Dear Satu,
In case have to do regular TEM it is fairly simple. In the regular procedure you start with a fixation in glutaraldehyde and/or formaldehyde. This first step already counts as an inactivation. In the past an incubator of culture hood with persisting fungal infections could be sterilised by using a glutaraldehyde- or formaldehydebomb. The aldehydes crosslink proteins very well and that serves as a sterilisationmethod.
Most procedure do a second fixation with OsO4. Also that counts as a protein crosslinking and hence inactivation of organisms including virusses.
So I would advise you to follow a regular EM fixation and embedding procedure. You can find procedures on the web.
Never feel so save that you forget your gloves during the procedure. Isn't it to avoid contact with a escaping viral particle, then do it to prevent the aldehyde, oxidizing agents, organic substances to touch your hands.
Good luck
Duco Kramer
We use 4% paraformaldehyde.
The best stain for NS is 1% uranyl acetate water solution.
Very good article about virus inactivation for TEM:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353909/
Good luck!
Hi dear, you can read this article
My best wishes
Article Single-use technology for solvent/detergent virus inactivati...
Thank you for your answers and links! In the end, I was able to image the virus without inactivation. I got some decent adenovirus images using 1 % PTA stain (90 sec - 120 sec staining time).
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