Here is an image I tried to take using TEM ( JEM-1400S) for human adenovirus. I used uranyl acetate to negatively stain the sample. However, this image I have indicates the sample is positively stained. I wonder if the center dark spot of the virus indicates the virus particle membrane is breaking apart. The Uranyl Acetate enters the particle and interacts with the DNA phosphorus group.
Yep. Something is not good here. It would be easier to answer your question if you provide the protocol which you used for sample preparation. In general, there are a few possible causes:
1. The film on the grids is too thick. What kind of grids did you use?
2. The concentration of UA was too low or staining time was too short. The most common parameters for viruses: 1% water solution of UA, 20-30 sec staining time;
3. The surface of the film is too hydrophobic and the stain was just ran away, when you lifted the grid from the droplet. In such a case some weird "semi-stained" virions can be found. Sometimes it can be done intentionally to reach positive staining - just wash the grid on the droplet of water or weak buffer after the staining. To avoid this effect, you can use a standard glow discharge treatment of the grids or just a fresh thin carbon coating (a few nanometer) right before the sample preparation.
Typically I use a simple UA negative staining protocol with ultrathin formvar-carbon grids: https://www.mdpi.com/1999-4915/11/10/955
Good luck!
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