Unstained cell structures, especially those which have been post-fixed with osmium tetroxide, are electron-dense and deflect electrons somewhat more than the surrounding embedding plastic.

However, this contrast is inadequate for sharp imaging of structure and sections must counterstained with heavy-metal salts which bind to components at the section surface imparting enhanced electron density and sharp, contrasty electron images.

Negative Contrast- the background area surrounding the specimen

is made electron-dense so that the specimen appears lighter than

in contrast to the darkly-stained background.

Positive Contrast- heavy metal salts attach to various organelles or

macromolecules within the section to increase their electron

density and they appear dark against a lighter backgound.

Positive Counterstains:

Uranyl Acetate (MW -422)-uranyl ions react strongely with

phosphate and amino groups so that nucleic acids and certain

proteins are highly stained.

Preparation- a 25% solution of uranyl acetate is prepared in

absolute methanol. This is a saturated solution and

must be clarified by filtering through a syringe filter

(.2 um pore size) just prior to use.

Caution:

Uranyl acetate contains trace amounts of U²³⁵ (radioactive). Powder

should not be inhaled and solutions discarded in special containers.

Reynoldís Lead Citrate (MW-1054)- lead ions bind to negatively

charged components and osmium-reacted areas (i.e. membranes)

Preparation- 1.33 gm lead nitrate, 1.76 gm sodium citrate and 30 ml

CO₂-free double distilled water (boiled for 10 mins.)

To this milky suspension add 5-7 ml 1N NaOH which

clears the suspension. Store in 50 ml volumetric flask.