Electron Microscopy Negative Staining Protocol - Method 2






This protocol is used for staining virus, bacteria, microphage to show their general morphology. Virus particle count can also be performed on the images generated from the staining.


Material Needed:

  • 0.5% uranyl acetate in distilled water. Filter with 0.2um syringe filter.

  • 2% aqueous phosphotungstic acid (adjust pH to 7.3 using 1N NaOH). Filter with 0.2um syringe filter.

  • 0.01% BSA filtered with 0.2um syringe filter.

  • 1mM EDTA or EGTA washing solution.

For quantitative study, add latex spheres to samples in appropriate ratio (1:10, 1:50, 1:100, etc.)



  1. Hold the grid in forceps and wash grid briefly by applying a drop (10 ul) of 0.01% BSA for a few seconds and the draw off from the edge of the grid with filter paper.

  2. Apply 2 ul sample immediately onto the grid and leave it on for 3-5 minutes. Then draw off from the edge of the grid with filter paper.

  3. Wash grid briefly (5-10 dips) in 1mM EDTA or EGTA washing solution (or apply large drops of washing solution onto grids) if 0.5% uranyl acetate is to be used for staining. The grid does not need to be washed if using 2% phosphotungstic acid for staining in next step. Note: washing before uranyl acetate staining is to prevent participates form due to interaction between uranyl acetate and phosphate possible in samples.

  4. Stain grid immediately with 10 ul of 0.5% uranyl acetate or 2% phosphotungstic acid for 1 minute and draw off from the edge of the grid with filter paper. Place the grid directly into grid box and allow them air-dry for several hours or overnight before observation.

EM Observation:


Ten fields for each sample are randomly photographed between 10K to 80K depending on the size of virus particles.


For quantitative study, ten photographs for each sample are examined for the number of virus/phages particles and the number of beads. Beads are recognized as being much larger than virus (about 1 ½ times larger). Both beads and virus/phages particles are marked as they are counted to avoid duplicate counting. Beads are marked in black, and virus/phages in red.




For quantitative study, the washing step can not be used for accuracy.