Cytochemistry of epinephrine and norepinephrine adrenomedullary cells

Summary Rat adrenomedullary tissue was fixed in 3% glutaraldehyde in a variety of buffers at different osmolarities. Incubations were performed in potassium dichromate (DC) at pH 4.1 to identify norepinephrine (NE) specifically and in osmium tetroxide to enhance contrast and to define membrane structures. Different buffer systems were also used for the DC and the osmium processes. It is evident from the results that both the osmolarity of the initial fixative and the incubation process itself are critical for adrenomedullary catecholamine (CAM) localization. Furthermore, the Schiff monobase formed by the amine and the aldehyde in the initial reaction was found to be reactive before and even after the tissue had been epoxy-embedded. Thus the technique of on-grid staining of tissue with DC has now been made possible and works sufficiently well to show a high degree of promise for future investigations. In vitro experiments were performed using purified CAMs. Reactions were conducted using the purified CAMs in solution with glutaraldehyde. The resulting reaction products then were incubated with DC and collected by centrifugation. The amount of chromium (Cr) present in the products was determined by the use of an electron probe as well as by chemical analyses by a commercial laboratory. In all cases it was evident that indeed Cr was in the reaction product, indicating this to be the electron-dense product seen microscopically in the tissue. In essence, as a result of this investigation, the specificity of the glutaraldehyde-DC pH 4.1 reaction has been made more sensitive, more utilizable cytochemically, and further substantiated by the chemical analyses of in vitro reaction products. Moreover, the on-grid staining technique leads to a new concept, not only in the electron microscopy of CAMs but in other heavy metal reactions as well.This investigation was supported by HEW Grant NS06980 and the Nathanael Henry Asten Fund for Research in Paralysis Agitans.     

Effect of High Oxygen Tensions on the Growth of Selected, Aerobic, Gram-negative, Pathogenic Bacteria

The in vitro effects of high O(2) tensions (P(O2)) on aerobic, enteric pathogens were examined at pressures of up to 3 atm absolute. Organisms from the genera Salmonella, Shigella, and Vibrio were usually subjected to 24-hr exposures. Tensions of 0.87, 1.87, and 2.87 atm absolute of O(2) (plus traces of CO(2) and N(2)) became progressively inhibitory for Salmonella and Shigella growth, but were bactericidal only for V. comma strains at tensions greater than 0.87 atm absolute of O(2). Growth inhibition of enteric organisms resulted from increased P(O2), rather than pressure per se, and could be mitigated nutritionally; an appropriate carbohydrate source is at least partially involved. Further studies with vibrios indicated that such mitigation was independent of medium pH. In addition, a synergistic relationship existed between O(2) and sulfisoxazole when tensions from 0.87 to 2.87 atm absolute of O(2) were maintained for 3 to 24 hr. Synergism occurred even under nutritional conditions which negated growth inhibition by O(2) alone. Bactericidal concentrations of sulfisoxazole, in the presence of increased P(O2), were reducible up to 4,000-fold. The combined procedure employed in this investigation, by use of an antimicrobial drug of known action, which also synergizes with O(2), plus nutritional studies, suggests a means for establishing a site of O(2) toxicity. These data support the concept that O(2) inhibition of growth represents a metabolic disturbance and that metabolic pathways involving p-aminobenzoic acid may be O(2)-labile. Such an approach could also guide development of antimicrobial agents as O(2) substitutes for promoting synergism.     

HALE STAIN FOR SIALIC ACID-CONTAINING MUCINS : Adaptation to Electron Microscopy

The feasibility of using the Hale stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Three kinds of mouse ascites tumor cells were fixed in neutral buffered formalin, exposed to fresh colloidal ferric oxide, treated with potassium ferrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acetate and potassium permanganate was done after sectioning in order to increase contrast. Those cells known to be coated with sialomucin showed deposits of electron-opaque ferric ferrocyanide crystals in the areas where sialomucin concentrations were expected. When these cells were treated with neuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with electron microscopy to identify sialomucin.     

Clinical Evaluation of a New Sequential Oral Contraceptive Agent

One hundred and seventy-eight patients were placed on a new sequential oral contraceptive preparation (Ortho-Novum SQ) and observed through a total of 1785 months in an effort to determine the effectiveness of the agent and its acceptability to patients. Patients with varicose veins and/or previous thrombophlebitis were not excluded from the study. The most frequent side effect was “breakthrough bleeding” which occurred in 19 patients. One patient became pregnant while apparently taking the tablets as directed, and another was already pregnant when started on the drug. However, in spite of at least four weeks'' continued ingestion after conception, both gave birth to normal female infants. Chloasma, noted in four patients while on combination oral contraceptives, cleared on this medication. There was no aggravation of thrombophlebitis or varicose veins. The acceptance of this sequential preparation by patients was excellent.