Came across a problem I never had before: when I use transmission electron microscope to visualize some tobacco mosaic virus particles, I can see the rod-shaped particles as indicated by blue arrow, buy they seem to be buried under tons of black dot-like things (red arrow). Thus I cannot get a good picture of them (Fig 2 will be a big aggregation of TMV with tons of those black dots). Are those black dots negative stain? why would it be like this? How can I get rid of this?
Here is a brief method how I stained.
Formvar coated on 300 grids
2uL undiluted TMV sample (of low concentration), for 1min, then absorb excess
2uL 1% phosphotungstic acid (newly prepared) for 25 sec. then absorb excess
I also tried using 5% stain +1% trehalose, but that seems always darken the background.
Are you filtering your stain with micropore syringe filters? If you are not you may want to try it. We do routinely but if the stain has "gone off" the particles will pass through even syringe filters.
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