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Halobacterium salinarum were grown on peptone agar containing 4.28 M NaCl, 0.036 M K and other salts. Stationary phase organisms were lifted onto carbon planchets, freeze-dried, carbon coated and examined in a scanning electron microscope equipped with an X-ray spectrometer. Intracellular element concentrations (mol/kg H(2)O) were determined using a bulk analysis program with appropriate standards. The cell K concentration was 110 times that of the medium. For Na this value was 0.3 and for Cl, 1.1. When Rb was present in the medium, its intracellular concentration was 77 times higher than the external value. The cation minus anion value suggests a high fixed negative charge, 0.72 equivalents. Intracellular apparent dielectric constants were calculated using cellular EMFs derived from the literature, and sodium concentration. The determined values ranged from 22-28 (vs 80 for normal water) suggesting phases of structured cell water. Ionic distributions in these extremophiles are treated according to the classical principles elucidated by Willard Gibbs and represents a heterogeneous system in thermodynamic equilibrium with the hypersaline environment. Factors to be considered are: (1) composition of Halobacterium and its immobile negative charge; (2) the physicochemical properties of the individual ions (charge, ionic radius, hydration energy, standard chemical potential); (3) the dielectric constant of the dispersion medium (water); and (4) the binding of ions, particularly potassium.

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The distribution of Calcium and Phosphorus and of Na, K, S and Cl was studied in the mineralizing matrices and strata of ameloblasts and odontoblasts in developing mouse molars (5-14 days). Sections cut in a cryostat were prepared by freeze-drying and examined in an SEM by the method of energy dispersive x-ray analysis. In enamel a gradient of mineralization was observed with respect to age and topography. Progesssive loss of sulfur was also demonstrated. Less striking mineralization gradients were found in dentin. Predentin accumulated Ca at a concentration about 2% that of dentin and the Ca/P ratio was lower than that for apatite. Significant concentrations of calcium were localized in ameloblast and odontoblast strata. The concentration increased five-fold in ameloblasts as the cells matured and enamel mineralization entered the final phases, levels in odontoblasts remained stable. With age in both cellular strata, potassium counts decreased. In maturing ameloblasts the concentrations of sodium and chloride rose.

2,4-Dinitrofluorobenzene was used without any coupling procedure as a histochemical reagent to localize some reactive groups of proteins in connective tissues and epithelial structures. Tissues were fixed by freezing-drying to avoid uncontrolled alterations of proteins and the reactions were modified in some sections by postfixation or pre-treatment with a variety of reagents, including alcohol, formalin, formaldehyde vapors, acetic anhydride, nitrous acid and N-ethylmaleimide. The staining and resolution were greatly enhanced by viewing the sections with monochromatic light at 410 mµ. When used in this way, 2,4-dinitrofluorobenzene is specific for: (1) α-amino groups of terminal amino acids; (2)ε-amino groups of lysine and hydroxylysine; (3) sulfhydryl groups of cysteine. However, the reaction seems to be dominated by the ε-amino groups of lysine in most of the tissues. In the native state not all functional groups of tissue colloids react presumably because the groups are masked by the interractions involved in forming tertiary structures or covalent bonds. This is specially notable in calcified tissues. Pyknotic nuclei, pre-dentin, carious dentin and newly formed bone are more reactive suggesting a less highly organized state of their structural proteins or some other modification in the lysine and hydroxylysine containing proteins. 2,4-Dinitrofluorobenzene is valuable as a specific cytochemical reagent and, in some instances, as an indicator of the state of organization of cellular and extracellular colloids.

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