Principles & Protocols

Protocols - Positive Staining

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You will need:

• Gloves
• Tweezers
• Timer
• DD H2O
• Beakers
• Uranyl Acetate
• Lead Stain
• Pipette
• Sample grids

Procedure:

• Put on gloves! (stains are very toxic)
• Place Wet filter paper in a petri dish
• Parafilm on top
• Al foil cover
• Filter each stain through a syringe filter before using
• 20 min 4% UA Stain (aqueous)
• Rinse well in DD H2O
• 3 min lead stain
• Rinse well DD H2O

Tips

• Longer times can be used if necessary.
• Resist the urge to over stain it only results in artifacts
• Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH^?

• Primarily used when samples difficult to stain (i.e. Spurr's resin)
• Penetrates more easily stain for 1-15 minutes
• Grids will sink to bottom of UA so place grid sample side up
• Shelf life is not as long.
• More risk of UA precipitating out of solution
• Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

• Penetrates more slowly stain for 1min-1 hour
• Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

• 0.01g in 10mL D20
• 3 drops of 10 N NaOH^?
• Stain samples for 30 sec to 15 minutes
• Cover sample with Petri dish when staining to prevent precipitation

• Set ALLOWTOPICCHANGE = CemfacGroup

-- DavidStokes - 18 Mar 2005