Negative staining using 1% uranyl acetate

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A 1% solution of uranyl acetate has been prepared for your use in the laboratory exercise. We have included instructions for preparing the stock 4% as well as 1 and 2% solutions in the event you have to do this in you own laboratory as a later date. Please note that uranyl acetate is toxic and radioactive. Use the solutions on the bench set aside for negative staining. In the event of a spill, notify a staff member.

Note: Although not as dangerous as handeling P32, you should exercise caution in handeling the stock chemical of solutions. Breathing particles of the salt from dried spills, for example, is very hazardous to your lungs.

1 Materials needed:

  1. fine forceps

  2. P20 pipetteman

  3. pre-filtered 1% solution of uranyl acetate (UA)

  4. Pasteur pipette and bulb

  5. small plastic containment cup

  6. 400 mesh Gilder grids coated with a formvar and carbon supporting film or only carbon supporting only film.

  7. UV light source or glow-discharge unit to treat grids before staining

  8. specimen

  9. vortex mixer

  10. solid and liquid waste containers

2 The staining procedure:

Prepare 2 negative stained specimen using 1% UA solution (we will use baselbody from bacterial flagella preparation from a mutant strain of Salmendella typhyimurium, SJW-1660).

  1. making the grids bydrophilic by putting them carbon side up under the UV lamp for about 5-10 minutes or by glow discharging in air for approximately 1-2 minutes (this is done by using the EmiTech Glow DIschage unit or the Edwards high vacuum coating unit).

  2. secure a grid (carbon side up) using a pair of fine forceps.

  3. mix specimen on vortex mixer(setting 5-6) for a few seconds.

  4. apply 3-5 micro-liter of specimen to carbon side of grid (glow discharged side of grid).

  5. wait approximately 30 seconds to 1 minute to allow the specimen to absorb to the carbon support film.

  6. using a Pasteur pipette, gently rinse the surface of the grid with several drops of 1% UA solution making sure each rinsing drop stays only on the carbon side of the grid - avoid getting the solution on the opposite side of the grid.

    Alternatively, you can float the grid (specimen side down) on the drop of negative stain that has been placed onto a pieace of parafilm.

  7. gently bot away the last drop of UA solution by holding a small wedge of Whatman #1 filter paper near the edge of the grid.

  8. let the grid air dry for about 5 minutes

  9. the grid is now ready for viewing on the microscope.