THE CHEMICAL SPECIFICITY OF THE SCHULTZ TEST FOR STEROIDSLEWIS, P. R.; LOBBAN, MARY C.
doi: 10.1177/9.1.2pmid: 14464899
The colours given by a number of pure steroids under various strongly acid conditions have been studied both in the test tube and in model sections prepared in various ways. Aqueous sulphuric acid solutions containing ferric ions were found to give an intense blue-green colour with steroids closely related chemically to testosterone, and colours in the pink-mauve range with certain other steroids such as the oestrogens and pregnenolone. The blue-green colour has two sharp absorption bands in the red which make possible the conclusive identification of the testosterone group of compounds. A new modification of the Schultz reaction has therefore been devised: frozen sections of formalin-fixed material are mounted on slides, blotted dry, treated with a drop of 80% aqueous sulphuric acid containing 0.5% iron alum and the resulting colours observed. This procedure has several advantages over previous modifications: there is no charring and very little cytological distortion; the colours are stable for many hours, and their chemical specificity is more clearly defined. Cholesterol itself gives no colour, but preliminary oxidation (e.g. by preincubation in iron alum) leads to the formation of the typical blue-green colour. Various aspects of this new histochemical modification of the Schultz reaction are discussed.
AZO-COUPLING RATE OF ENTEROCHROMAFFIN WITH VARIOUS DIAZONIUM SALTSLILLIE, R. D.; GRECO-HENSON, JACQUELINE P.; CASON, JOSEPH C.
doi: 10.1177/9.1.11pmid: 14465283
The azo-coupling of enterochromaffin with some 30 diazomium salts has been explored, in regard to rate, concentration and pH. A technic of enterochromaffin cell counting in consecutive serial sections was employed and the counts related to those obtained in immediately adjacent serial sections reacted with the ferric ferri-cyanide reagent of Golodetz and Unna (6) as modified by Lillic and Burtner (8). This procedure enabled the equation of a considerable series of experiments with different diazotates on a succession of individual animals.Structure of the diazonium salt proved to have a considerable influence on the azo coupling rate. Of the nitroanilines, nitroanisidines and nitrotoluidines, those with the nitro group para to the diazonium radical coupled rapidly, those with ortho nitro groups slowly. Also p-anisidine was more rapid than the ortho isomer.In the specific cases of p-nitroaniline and of di-o-anisidinse fresh diazotates were found to be more rapid and effective couplers than commercial stabilized salts in similar concentrations. It is probable that other comparisons of the same sort would give similar results.While azo coupling increased in rapidity with higher diazo concentrations, increasing density of background reaction decreased the demonstrability of enterochromaffin, so that optimal concentrations were often 1-10 mM, but lower with "rapid" couplers or higher with slow.The second diazonium group of fast blue B is probably but little utilized in azo coupling with enterochromaffin under histochemical conditions, since di-o-anisidine treated with one molar equivalent of nitrous acid appeared almost if not quite as effective as when two molar equivalents were used.Fast blue B appears to fall into an intermediate group of diazotates, being neither as fast as the p-nitro derivatives nor as slow as fast blue RR, BB or VB, for example.
GROWTH, FLUORESCENCE AND METACHROMASY OF CELLS CULTURED IN THE PRESENCE OF ACRIDINE ORANGEWOLF, MERRILL K.; ARONSON, SAMUEL B.
doi: 10.1177/9.1.22pmid: 14007945
Four types of animal cells were cultured in perfusion chambers in medium containing acridine orange and serially observed by phase and fluorescence microscopy, so that cellular injury from toxic and photodynamic effects of acridine orange could be correlated with the variable fluorescence images of the cells. Spontaneous movements of pigment granules in healthy chorioidal and ciliary melanocytes provided a valuable criterion of cellular health. Dye concentrations of 10 –6 gm/ml permitted healthy outgrowth of brilliantly fluorescent cells for at least 20 days, but higher concentrations were lethal. Uninjured living cells had orthochromatic (green) fluorescence. Reversibly injured cells acquired metachromatic (red-fluorescent) cytoplasmic granules which might be identical with mitochondria or with the neutral red granules of Jackson. With irreversible photodynamic injury, ribonucleic acid of nucleoli and cytoplasm stained metachromatically; deoxyribonucleic acid of nuclei alnsost always remained orthochromatic. As degeneration ensued, cells lost first their metachromasy, finally all their fluorescence. The variable fluorescence images of living, injured, killed, degenerated and fixed cells stained with acridine orange may be explained according to the theory of variable stacking of bound dye molecules.
CORRELATION OF HISTOCHEMICAL, AUTORADIOGRAPHIC, AND MICRORADIOGRAPHIC DEMONSTRATIONS OF TISSUE CALCIFICATIONJOHNSTON, MURIEL E.; DURBIN, PATRICIA W.; ASLING, C. WILLET
doi: 10.1177/9.1.30pmid: N/A
1. The sensitivity and reliability of three representative histochemical tests for calcium (von Kóssa, ferrocyanide, and alizarin) have been compared by applying them to vertebral ossification centers in the rat fetus.2. The histochemical results were controlled by concurrent study of calcium45 autoradiographs and of soft x-ray microradiographs of the same or adjacent sections.3. Microradiographs and von Kóssa-positive reactions produced essentially identical representations of mineralized bone and cartilage.4. The von Kóssa reaction coincided with high grain density regions of calcium45 deposition in stripping-film autoradiographs but did not reflect the sparser densities.5. The ferrocyanide reaction showed gradients of staining density corresponding in many sites with the varying degrees of radiocalcium density. Faint ferrocyanide reactions extended beyond the limits of microradiographic mineral opacity.6. The resolution obtained with contact autoradiographs and alizarin dye-lake histological preparations was inadequate for study at high magnification.
CYTOLOGICAL LOCALIZATION OF GLYCOGEN IN CULTURED SKELETAL MUSCLEENGEL, W. KING
doi: 10.1177/9.1.38pmid: 13890229
The periodic acid Schiff stain was used to demonstrate the cellular location of glycogen formed and stored by chick embryo skeletal muscle cells newly grown in vitro without innervation. No glycogen was found in single myoblasts. In the myoblastic and less mature myocytic straps, there were irregular-sized granules of glycogen in the cytoplasm, especially in the perinuclear areas. Much glycogen was formed in the more mature cultured myocytic straps, and in a pattern like that of innervated adult skeletal muscle cells in vivo. There was glycogen in the perinuclear and subsarcolemmal cytoplasm and in alternating wide and narrow bands, tentatively interpreted as I and M bands respectively.
METAL CHELATE REACTION OF ENTEROCHROMAFFINLILLIE, R. D.
doi: 10.1177/9.1.44pmid: N/A
Enterochromaffin slowly binds ferrous ions at pH levels between 3.5 and 5.0 and gives them up again at low pH levels (1-2.5). Ferrous ions so bound may be demonstrated in situ by action of potassium ferricyanide, probably by acid dissociation of the chelate complex and precipitation of Turnbull's blue. This reaction is prevented by acetylation and tosylation, in agreement with the azo reaction. It is prevented by drastic oxidations but not by milder, sufficient to prevent azo coupling.This reaction was at first thought to indicate the presence of a substituent ortho to the phenolic hydroxyl of enterochromaffin, but in view of the occurrence of the reaction in model experiments with some para substituted phenols and phenylamines, after formaldehyde treatment, this conclusion must remain indefinite.Formaldehyde fixed serotonin model sections reacted relatively weakly, though giving strong reduction, azo and hematoxylin reactions.
MODIFICATIONS OF HISTOCHEMICAL TECHNIQUES FOR THE DEMONSTRATION OF CYTOCHROME OXIDASEBURSTONE, M. S.
doi: 10.1177/9.1.59pmid: 13875052
Four reagents were employed in conjunction with p-aminodiphenylamine as new histochemical substrates for cytochrome oxidase. The most useful[See figures in PDF file]substrates were 8-amino-1,2,3,4-tetrahydroquinone (I) and 8-hydroxy-1,4-naphthoquinone (III). (I) forms blue dyes and is recommended for general use, as well as for tissues of low activity (human gingiva). With the latter, the reaction may be augmented by the addition of cytochrome c.The dye produced from (III) has a strong affinity (substantivity) for tissue elements and resists dehydration with solvents.The new-substrates were useful in visualizing cytochrome oxidase activity in the digestive tract including oral mucosa, central nervous system, muscle and bone.
CYTOLOGICAL LOCALIZATION OF CHOLINESTERASE IN CULTURED SKELETAL MUSCLE CELLSENGEL, W. KING
doi: 10.1177/9.1.66pmid: N/A
The location of acetyl and butyryl thiocholinesterase activity was studied in cultured chick embryo skeletal muscle cells which were growing without being innervated. Muscle cells in all three stages of development, i.e., the single myoblast, the plurinucleated myoblastic strap, and the multinucleated myocytic strap, showed sites of thiocholinesterase activity as small brown granules located diffusely in the cytoplasm and accentuated in the perinuclear areas. In the well differentiated myocytic straps, thiocholinesterase was localized along the Z lines. No enzyme activity was found at the sarcolemma or in the nuclei. No thiocholinesterase-containing motor end-plate structures were found in any stage of development.