Negative staining

Negative staining is a widely used method in microscopy for examining optically opaque specimens in fluid. It involves the use of heavy metal salts to enhance the contrast between the background and small biological specimens such as viruses, bacteria, bacteriophages, mycoplasmas, protein complexes, macromolecules, subcellular fragments, as well as nucleic acids and protein filaments.

One of the advantages of negative staining is its simplicity and directness. To perform negative staining, a suspension of the biological specimen (centrifugation and pelleting may be required depending on the sample type) is placed on a surface such as a staining tray, parafilm, or petri dish. A copper grid coated with Formvar is then carefully placed, Formvar side down, on top of the suspension for approximately 1-3 minutes. Afterward, the grid is lifted, excess liquid is absorbed using filter paper, and the grid is transferred onto a drop of phosphotungstic acid (PTA) for one minute. The excess PTA is removed, and once the grid is completely dry, it is ready to be introduced into the electron beam.