Nucleolar Organelles Shown by Lead Precipitation in Unfixed Cultured Cells

Abstract

Unfixed coverslip cultures of HeLa cells or of mouse ascites tumor cells are placed in a 4 mM solution of lead acetate in Michaelis buffer, pH 5.5, for 30 min at 37 C. After a thorough water wash, brief treatment with 1% aqueous ammonium sulfide exhibits multiple (1-20) dark brown granules within each nucleolus. The remainder of the nucleolus is bright yellow, and other parts of the cell are unstained. The nucleolar granules thus visualized show strict correspondence in sue and distribution to the granules of ribonucleoprotein stained by toluidine blue-molybdate, and also to the clear spaces seen in the nucleolus of living cells under phase-contrast optics. Fixation, or heating to 90 C, prevents the staining by lead but not by toluidine blue-molybdate. The reaction to the lead salt is pH dependent; optimum about 5.5. These features suggest that the retention of lead in localized areas of the nucleolus is due to enzymatic reactions which produce inorganic phosphates from endogenous substrates.