BECM

Negative Staining (SOP)

Negative Staining (SOP)

  

Negative staining involves the work with a radioactive heavy metal. The facility provides a 2% uranyl acetate solution for external users. Users that want to use uranyl formate or other stain have to provide it themselves. Uranyl is radioactive with a half-life of around 1 billion years and emits alpha-radiation. Alpha-radiation can travel in the air for 3 cm and is stopped by paper and human skin. The main risk therefore is the ingestion and inhalation of the substance. By Belgian law, pregnant women are not allowed to work in the room where any radioactive substance is handled. Moreover, uranyl is a heavy metal and risks occur by ingestion and inhalation.

Radioactively contaminated consumables have to be discarded properly, separating high, low and non-radioactive waste. Any small amounts of liquid should be soaked up with Whatman paper and discarded in the high radioactive dry waste with tubes and tips. Low radioactive waste can be paper tissue and gloves. Any non-contaminated waste has to be put into the regular waste bin. All radioactive work has to be performed in the designated area. Any spilling has to be cleaned immediately.

     

    Setup for negative staining: Preparation of washing solution and staining solution on Parafilm
    Setup for negative staining: Preparation of washing solution and staining solution on Parafilm

     

    Setup for negative staining: Resting position of tweezers for sample incubation and drying.
    Setup for negative staining: Resting position of tweezers for sample incubation and drying.

    We recommend following general protocol for negative staining

    1. Create a well with Parafilm and per grid optionally three spots for washing solution (20 ul water) and required three spots of staining solution (20 ul Uranyl acetate).
    2. Pick up a freshly glow discharged EM-grid (e.g. Formvar carbon film with Copper mesh) with a tweezer.
    3. Add 3 ul of your sample and incubate for 30 s. Typical concentration are in the range of 0.01 mg/ml.
    4. Remove excess liquid with Whatman paper, dip the grid into the first washing drop.
    5. Repeat step 4 two more times.
    6. For staining keep the grid in the first staining drop for 10 s, remove excess liquid, 1 s in the second drop, remove excess liquid and 1 min in the last drop.
    7. Finally, remove enough amount of liquid to leave behind a small film of uranyl.
    8. Dry the grid for 15-30 min before use.
    9. After use, clean all tweezers and other contaminated equipment to remove dried up uranyl acetate.