Silver Staining for Nucleolar Organizer Regions
Trude Schwarzacher and Molecular Cytogenetics Research Group


Borate buffer:0.01M Na2B4O7, pH=9.22

Aqueous silver nitrate solution:-

                1 g AgN03 (reasonably high grade, clean, and not too old)

                1 ml good distilled water

                10 ul formalin (= .37% formaldehyde, optional, increases staining in old slides)

                Mix well and put in a plastic syringe with a millipore filter.

                Do not bring in contact with metal objects.


Silver nitrate stains clothes lab benches and fingers black, normally after about 10-1 6h. It is corrosive, but not really dangerous to human beings, and is used occasionally as a disinfectant. The skin sheds and the black spots disappear after a few days. It is advisable to wear gloves as the black spots are not very attractive. Cloths and lab benches are normally soiled for life.


  1. Incubate slides in borate buffer for 10-20 min (to wash slides from remaining acetic acid, and to adjust for a high pH which is favourable for silver staining).

  2. Rinse in distilled water.

  3. Air dry.

  4. Apply 200-300 /ul freshly made silver nitrate solution directly from the millipore filter on the slide

  5. Cover with a 25x40 mm clean nylon mesh (200-300 um pore size, we use Swiss Silk Bolting Cloth-Fabrik, Zurich, Switzerland, Nybolt 68GG-243; but I think others do as well). Make sure nylon mesh is soaked thoroughly. If necessary apply another drop of silver nitrate solution on top.

  6. Incubate in a moist chamber at 370C until golden brown (about 1-3h). For quick results incubate at 600C for 5-20 min.

  7. Rinse in plenty of distilled water.

  8. Air dry.

  9. Examine under the through light microscope.

Put a few drops of xylene on the preparation and cover with a large No.0 cover slip. Re-apply xylene if it dries out. For storage, leave the slide on the bench until all the xylene has evaporated, remove the coverslip carefully and put slide in a box. This method gives excellent contrast, but you have to cope with a little bit of xylene smell. Alternatively, you can examine the slides directly with immersion oil. This will not destroy the silver staining. Oil can be removed with xylene.


Nuclei should be visible without phase contrast. They should appear yellow to light brown and nucleoli brown. Too long staining results in black nucleoli and eventually black nuclei. If black precipitate occurs, slides were not clean or the silver nitrate solution was contaminated.

At metaphase, silver nitrate stains nucleolus organizer regions which have been active in the preceding interphase. Although amount of silver nitrate is in some relation to the amount of activity, or number of active, rDNA copies; it cannot be used for an accurate quantitative study, partly because of differences of silver staining strengths within and between slides depending on fixation procedures, density of nuclei, preparation quality and age of slides. Sometimes, e.g. with rye, heterochromatic regions also stain a pale brown color.


Kodama et al. 1980: An improved silver staining technique for nucleolus organizer regions by using nylon cloth. Jpn J Human Genet 25, 229-233

Schwarzacher T, Kraemer PM and Cram LS 1988: Spontaneous in vitro neoplastic evolution of cultures Chinese hamster cells. Nucleolus organizing region activity. Cancer Genet Cytogenet 35, 119-128.