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Lead acetate (3-10%, pH between 4.3 and 7.0, alone or containing 2% glutaraldehyde), when used as fixative, has been demonstrated to produce an intracellular microcrystalline precipitate of lead orthophosphate, Pb(5)(PO(4))(3)OH (lead hydroxyapatite). This confirms earlier work with the light microscope (6). In interphase cells the nucleoli are sharply delimited by the massive lead phosphate precipitate. Some diffuse precipitate is found in the nucleoplasm; it is always delimited by the nuclear membrane. Nucleolar localization of this orthophosphate pool is not a diffusion artifact; the pool is probably in a loosely bound state and is not retained by conventional fixatives. In maize root cells in advanced mitotic stages the lead phosphate crystals are seen distributed throughout the cytoplasm and also relatively concentrated on the late anaphase-early telophase chromosomes. This pool of inorganic phosphate anions may be involved in the mitotic cycle of chromatin condensation, and it may be partially responsible for the absence of mature ribosomes in the nucleolus through the chelation of divalent cations. It is evident that the siver-reducing component detected in the nucleoli of fixed cells (6) is a completely different substance.

Potassium pyroantimonate, when used as fixative (saturated or half-saturated, without addition of any conventional fixative) has been demonstrated to produce intracellular precipitates of the insoluble salts of calcium, magnesium, and sodium and to preserve the general cell morphology. In both animal and plant tissues, the electron-opaque antimonate precipitates were found deposited in the nucleus-as well as within the nucleolus-and in the cytoplasm, largely at the site of the ribonucleoprotein particles; the condensed chromatin appeared relatively free of precipitates. The inorganic cations are probably in a loosely bound state since they are not retained by conventional fixatives. The implications of this inorganic cation distribution in the intact cell are discussed in connection with their anionic counterparts, i.e., complexing of cations by fixed anionic charges and the coexistence of a large pool of inorganic orthophosphate anions in the nucleus and nucleolus.

For localization of pyroantimonate-precipitable cations, mouse testes were fixed with a saturated aqueous solution of potassium pyroantimonate (pH about 9.2, without addition of any conventional fixative), hardened with formaldehyde, and postosmicated. A good preservation of the cell membranes and over-all cell morphology is obtained as well as a consistent and reproducible localization of the insoluble antimonate salts of magnesium, calcium, and sodium. Four sites of prominent antimonate deposits are revealed, besides a more or less uniform distribution of the precipitates. These sites are: (a) In the walls of the seminiferous tubules, localized in two concentric layers corresponding to the inner and outer layers of the tubular wall; (b) Around the blood vessels and adjacent connective tissue; (c) At the area of contact between the Sertoli cell and spermatids, where a double line of precipitate surrounds the head of the mature spermatids; and (d) In the cell nuclei, disposed between regions of the condensed chromatin. The nucleus of mature spermatids did not show any sign of antimonate precipitation. The implications of this inorganic cation distribution are discussed with relation to their anionic counterparts, their localization in other animal and plant tissues, and the possibility that those sites may represent barriers to the free passage of ions.

Lead acetate treatment of unfixed cells immobilizes the intracellular water-soluble, inorganic orthophosphate ions as microcrystalline lead hydroxyapatite precipitates (see reference 1). These precipitates have been analyzed with the electron microprobe. A much higher concentration of phosphorus has been found in the nucleoli of maize root tip cells fixed in lead acetate-glutaraldehyde (organic phosphorus plus inorganic orthophosphate), as compared to the nucleoli of roots fixed in glutaraldehyde alone (organic phosphorus). The concentration of the inorganic orthophosphate pool in these nucleoli is three to five times as high as the concentration of the macromolecular organic phosphate. Since nearly all of the latter is in RNA, the concentration of inorganic phosphate in the nucleolus is calculated to be roughly 0.5-0.8 M. About 30%-and up to 50%-of the total cellular inorganic phosphate is accumulated in the nucleolus since the mean concentration per cell is about 10(-2)M. In the extranucleolar part of the nucleus the mean concentration was estimated by densitometry to be roughly six times less than in the nucleolus ( 0.1 M), and appears more concentrated in the nucleoplasm than in the condensed chromatin. While there is no direct evidence for the concentration in the cytoplasm, it certainly must be much lower than the mean cellular level (i.e., < 10(-2)M) since the nucleus is about 10% of the total cell volume. The implications of this compartmentation in the intact cell are discussed in connection with (A) the availability of orthophosphate ions for the cytoplasm in those processes in which these ions affect the rate of enzymatic reactions, and (B) protein nucleic acid interactions within the nucleus and nucleolus.