Alum Hematoxylin Research Articles

Differential collagen stain by an acid fuchsin, iron, flavianic acid mixture. A note on ferrous sulfate hematoxylin.

AbstractSubstitution of flavianic acid (50 mM) or of a stoichiometric mixture of naphthol yellow S and hydrochloric acid, in place of picric acid in Van Gieson type mixtures gives deeper yellow colors to cytoplasm, muscle and erythrocytes. Higher concentrations of acid fuchsin can be used with consequent greater density of collagen fiber staining and improved contrast.The familiar weakening of hematoxylin nuclear stains by exposure to Van Gieson mixtures can be largely avoided by inclusion of 0.1 M ferric chloride in the Van Gieson mixture. Alum hematoxylin can then be used in place of the unstable iron hematoxylin solutions, and the iron hematoxylin effect is attained by the iron postmordanting in the Van Gieson bath. A ten minute prestain in an alum hematoxylin containing 0.5% hematoxylin is adequate but density can be enhanced by longer staining or by staining at higher temperature; 5–10 min at 60°C is suggested. The iron containing flavianic or picric acid Van Gieson staining baths should be restricted to three minutes; longer exposures gradually weaken nuclear staining.Substitution of 0.1 M copper sulfate for the iron in the Van Gieson bath also yields dark gray to black nuclei. Aluminum chloride (0.1 M) has an effect similar to the control hydrochloric acid, while the chromium ion seems quite inferior, even to the control HCl mixture.A ferrous sulfate hematoxylin ripened overnight with a small amount of ferric chloride gives excellent progressive nuclear staining, adequate in 2–5 minutes, and not excessive in 30 minutes. The solution gradually deteriorates in 6–8 weeks.

A system linking the third ventricle with the pars tuberalis of the rhesus monkey.

IN the course of studies of the hypothalamus of the rhesus monkey, we have investigated a restricted area of specialized ependymal cells. This area lies anterolaterally in the tuber cinereum (see Fig. 1), and is distinguished by the long processes of the ependymal cells which extend to the region of the pars tuberalis; neighbouring ependymal cells are cuboid, without processes. The elongate ependymal cells stain deeply with the Gomori chrome–alum–haematoxylin technique and may be followed with ease using the optical microscope. They are not evident after silver impregnation techniques (Bodian's silver protargol method or Bielschowsky's technique), nor are they detectable after performic acid–alcian blue staining; they can, however, be demonstrated by cresol fuchsin and aldehyde fuchsin and the periodic acid–Schiff (PAS) method.

A FIBER APPARATUS IN THE NUCLEUS OF THE YEAST CELL

The structure and mode of division of the nucleus of budding yeast cells have been studied by phase-contrast microscopy during life and by ordinary microscopy after Helly fixation. The components of the nucleus were differentially stained by the Feulgen procedure, with Giemsa solution after hydrolysis, and with iron alum haematoxylin. New information was obtained in cells fixed in Helly's by directly staining them with 0.005% acid fuchsin in 1% acetic acid in water. Electron micrographs have been made of sections of cells that were first fixed with 3% glutaraldehyde, then divested of their walls with snail juice, and postfixed with osmium tetroxide. Light and electron microscopy have given concordant information about the organization of the yeast nucleus. A peripheral segment of the nucleus is occupied by relatively dense matter (the "peripheral cluster" of Mundkur) which is Feulgen negative. The greater part of the nucleus is filled with fine-grained Feulgen-positive matter of low density in which chromosomes could not be identified. Chromosomes become visible in this region under the light microscope at meiosis. In the chromatin lies a short fiber with strong affinity for acid fuchsin. The nucleus divides by elongation and constriction, and during this process the fiber becomes long and thin. Electron microscopy has resolved it into a bundle of dark-edged 150 to 180 A filaments which extends between "centriolar plaques" that are attached to the nuclear envelope.

Retarded Embryo Sac Development in Irradiated Clones of Malus silvestris Hort. cv. ‘Golden Delicious’

SEVEN ‘Golden Delicious’ apple clones propagated from buds irradiated at 5,000 r. in a cobalt-60 source were selected for high, intermediate, and low pollen abortion. Pollen abortion was 4.9 per cent in a typical check clone, and varied in the irradiated clones from 10.4 per cent to 84.8 per cent. Blossoms collected at anthesis were sectioned serially at 10µ thickness and stained with Heiden-hain's iron alum haematoxylin for embryo sac examinations.

INDUCTION OF POLYPLIODY BY KINETIN IN TRITURUS VIRIDESCENS.

IN 1962 Buckley, Witkus and Berger1 reported that kinetin induced mitotic divisions throughout the digestive tract of the salamander, Triturus viridescens. It was also reported that these were diploid divisions. The effect followed exposure for 9 days to kinetin and was most evident in the duodenum. Since this was one of the first clear cases of mitotic stimulation in animal tissues by kinetin, a more detailed examination was made of the duodenum. As in the previous work1, the animals were immersed in a 10 mg per cent solution of kinetin for 9 days. During the period of treatment the animals were not fed and the solutions were changed every third day. After 9 days exposure the animals were returned to spring water and allowed a 9-day period of recovery. Immediately after treatment and after daily periods of recovery the tissue was fixed, sectioned and stained using the standard iron alum haematoxylin technique. Acetoorcein smears were also made of the same regions.

Comparative Studies of Macroscopic and Microscopic Features of Spermatophores of Some Plethodontid Salamanders

The macroscopic and microscopic structure of spermatophores of 13 species representing five plethodontid salamander genera are described. Spermatophores of representative species of Desmognathus, Eurycea, Gyrinophilus, and Pseudotriton consist of a nonadhesive gelatinous stalk surmounted by a milky-white, dome-shaped cap with a smooth outer surface. The cap is composed of a mass of amorphous material in which the spermatozoa are imbedded. Plaques of homogeneous material stained orange-red with alizarin red and blue with alum hematoxylin are distributed irregularly throughout the cap. After application of von Kossa's method for calcium, the plaques are colorless. Spermatophores of representative species of Plethodon are generally mushroom shaped. The sperm cap is fairly rigid in comparison with those of the other genera. Microscopically, the cap appears to be surrounded by a capsule of concentric membranes. The capsule is stained orange-red by alizarin red and blue by alum hematoxylin. It is colorless after von Kossa's method. Stalks of freshly deposited spermatophores of all genera studied bear numerous cross-striations on the outer surface. Microscopically the stalks appear to be composed of highly tortuous, thickened threads of material. The spermatophores of Plethodon differ most markedly from those of the other genera studied.

Cytology of Rhodotorula glutinis.

Thyagarajan, T. R. (Cornell University, Ithaca, N. Y.) and H. B. Naylor. Cytology of Rhodotorula glutinis. J. Bacteriol. 83:127-136. 1962.-The structure and manner of division of nuclei in actively dividing cells of Rhodotorula glutinis were studied with the phase contrast microscope. The nucleus consists of a dense central body, surrounded by a shell of optically uniform material of low density. The entire structure is enclosed within a nuclear membrane. Various fixation and staining techniques were employed to confirm the observations made from living cells. Since the dense central body is Feulgen-negative and is readily stained by iron alum hematoxylin, it is identified as the nucleolus. The material surrounding the nucleolus has no marked affinity for hematoxylin but is Feulgen-positive and stains intensely with Giemsa and basic fuchsin. The nucleus appears to divide by a process of elongation and constriction during which roughly half of the nucleolus, along with the surrounding chromatin, passes into the bud. The nuclear membrane was found to persist during all stages of division. Vacuoles were seldom observed in actively dividing cells. The nucleus of R. glutinis is similar in structure to the nuclei of higher organisms, but its behavior during division is quite different.

The Life History of Scaberia Agardhii Greville

Scaberia agardhii is an endemic species in southern Australia. Growth is initiated by means of a three-sided apical cell which segments and forms the meristoderm, the cortex, and the medulla. Slender, scale-like, and verruciform segments occur. The plant is monoecious and the conceptacles are bisexual. On germination two primary rhizoids are formed. Scaberia is included within the Cystoseiraceae. ent parts of southern Australia. No complete account of the genus is available. Gruber's observation on the presence of a three-sided apical cell was based on a fragmentary specimen (1896). This study, based on adequate fresh, liquid-preserved, and dried material, was undertaken to obtain detailed morphological, anatomical, and embryo- logical information of the genus, and so to clarify its systematic position. Material and Methods In most cases the material was preserved in Karpechenko solution (Papen- fuss modification) or in 4 per cent, formal in sea water. After washing five or more times in sea water the material was dehydrated in the usual manner in seawater-distilled water-alcohol mixtures. Johansen's method (195 1) of em- bedding was carried out after dehydration. Microtome sections of various thicknesses between 4 and 12 fx were cut and stained in 0-5 per cent, iron alum haematoxylin. Material collected for embryological studies was brought into the laboratory either immersed in sea water or in plastic bags. Within an hour from the time of collection or immediately conceptacles (fertile segments) were washed thoroughly in sea water and placed in Petri-dishes, or on slides in filtered sea water. The conceptacles were removed after 2 hours and the water was changed gently. Either three times filtered sea water or Schreiber or Erd- schreiber solutions were used as culture media. The zygotes, attached on the dishes or on slides, were rinsed with the culture solution to remove debris.

Chromosomal elimination induced by 6-aza-uracil.

DURING preliminary work in this Laboratory dealing with the effects of some antimetabolites on cell division in Allium cepa, it was noted that 6-aza-uracil brings about chromosomal elimination. This elimination occurred at metaphase and anaphase. Root tips from bulbs after reaching a length of 2–3 cm. were treated for 6 hr. with a 1 per cent aqueous solution. The solution was prepared by first grinding the powder and dissolving it in warm tap water. In order to clarify the location of the spindle attachment region of the chromosomes the root tips were treated with a 1 per cent aqueous solution of colchicine for a half-hour prior to fixation. On each preparation the number of normal metaphases, anaphases and telophases and the number of these stages showing elimination were counted. The eliminated chromosomes were studied in detail to determine whether or not the same chromosomes were always eliminated. Aceto-orcein smears and sections stained in iron alum haematoxylin were used.

The peracetic-orcein-Halmi stain: a stain for connective tissues.

A selective stain useful for the study of connective tissues is described. The stain demonstrates elastic and oxytalan fibers as well as fibrils in mucous connective tissues previously undescribed. Reticular fibers are not stained. The stain may be used on sections that have been fresh frozen or fixed in formalin or ethanol. Sections are deparaffinized, washed in absolute ethanol, oxidized in peracetic acid 30 min, washed in running water, stained in Taenzer-Unna orcein 15 min, 37°C, differentiated in 70% ethanol, washed in running water, stained in Lillie-Mayer alum hematoxylin 4 min, blued in running water, and counterstained 20 sec in a modified Halmi mixture of 100 ml distilled water, 0.2 gm light green SF, 1.0 gm orange G, 0.5 gm phosphotungstic acid and 1.0 ml glacial acetic acid. Sections are rinsed briefly in 0.2% acetic acid in 95% ethanol, dehydrated and mounted.

Cytochemistry of pineal lipids in rat and man.

Lipid cytochemistry studied in 130 rat pineals demonstrates two common types of parenchymal cells which may be involved in an endocrine secretory activity. The first cell type is characterized by its content of abundant lipid droplets, composed primarily of ethanol-soluble carbonyl lipids coated with phospholipid. The second cell type is distinguished by its content of a phospholipid cytoplasmic matrix containing few droplets on vacuoles. This cell type appears to be identical with the cell staining with orange G or phloxine in a modified chrome alum hematoxylin technique described previously. Parenchymal cells containing lipid occur in seven human pineals of diverse ages; and in the older pineals interlobular cells containing lipid occur as well. The lipid in human parenchymal cells differs from that in the type 1 parenchymal cells of the rat by, (1) forming smaller droplets, (2) containing a greater amount of neutral lipid and less carbonyl lipid, and (3) being associated with pigment fomation in adults and older people.

Phosphorylation as a histochemical procedure.

(1) The use of phosphoryl chloride (phosphorus oxychloride) in pyridine as a reagent for phosphorylation of tissue components is described. (2) Phosphorylation of many carbohydrate-containing substances is obtained by this method, including mucins, connective tissue (elastic tissue, reticulum, collagen, bone matrix), some glycolipids, glycogen and other tissue components. (3) Certain chemical entities that can be phosphorylated probably do not contain carbohydrate, but are lipids (either unsaturated or incompletely esterified), or proteins with free sulfhydryl, hydroxyl, or amino groups. (4) Phosphorylation reduces metachromasia of some tissue components (e.g. cartilage matrix), but enhances that of many others. The basophilia and metachromasia of phosphorylated components persist when staining is done in strongly acid solutions. (5) The significance of direct leukofuchsin positivity of many phosphorylated substances, and of relatively red staining of such substances with alum hematoxylin, and aldehyde-fuchsin, is not certain.

Regional differences of the pancreatic islet.

Simard (1945) found an almost complete disappearance of the A cell in a subcutaneous pancreatic transplant of one dog which had been operated upon following the method of Ivy and Farrell (1926). Our interest in the A cell in relation to Glucagon led us to investigate the possibilities offered by Simard]s work. In the present study we have confirmed Simard]s observations that the transplanted segment was devoid of A cells and have concluded that regional differences in islet cell composition were responsible for the absence of A cells in the transplants. MATERIAL AND METHODS Eight dogs weighing between 20 and 50 pounds and two mature cats were used. Animals were killed with nembutal and their pancreas completely removed. All tissues unless otherwise specified, were fixed in Zenker-Formol, embedded in paraffin, and cut at 2.5 m Some sections were stained with Masson]s trichrome and Gomori]s Chrome Alum hematoxylin following a procedure previously described (Bencosme, 1952)

牛馬胎児生殖腺の組織学的所見

The gonads of 9 male and 9 female fetuses of the cattle were used in this study. The fetuses are 5.7 to 45.0 cm in "crown-rump length" in males and 16.3 to 38.0 con in females. The gonads were fixed in 10% formalin and ZENKER-formol. Routine alcohol dehydration and paraffin embedding were practiced. Sections were cut at 7μ. stained with hematoxylin-eosin or HEIDENHAIN's iron alum hematoxylin, and after MASSON's trichrome method, VAN GIESON'S method and BIELSCHOWSK Y-MARESCH reticulm method. The following observations were made.Testis: There is a cuboidal layer of germ epithelium in the testis of a 5.7-cm-long fetus It becomes flattened gradually with ages. With the vascularization in the tunica albuginea, there is a remarkable increase of connective tissue fibers in the vascularized areas. The seminiferous tubules are distinctly limited by a membrana propria, exterior to which is a thin layer of small connective tissue cells forming a capsular investment. The tubules consist of two kinds of cells, i. e., indifferent cells and germ cells. The former is numerous and the latter a few. Both cells in the tubules have a tendency to increase in number, the numerical ratio between them being scarcely altered in all testes used. Interstitial cells are found sparsely at first and then form cluster-like masses with gradual vascularization.Ovary: In 16.3-cm-to 18.5-cm-long fetuses, the sex cords having a broad hand-like appearancee contain many small indlfferent cells and a few germ cells. Primary follicles increase gradually in the inner part of the cortex. In the bodies of 25-cm-and 27-cm-long fetuses, the sex cords manifested a degenerated appearance. Meanwhile, in a fetus grown 30.0-cm-long, the remarkable change is noticed with the increase in replenished sex cords. Therefore, it is presumed that the sex cords have newly been formed descending from the germ layer, or reconstructed from the parenchymal tissues in these growing stages. No interstitial cells have been found in any ovaries used.The origin of the interstitial cells in the testisand of the medullary cords in the ovary is also. discussed briefly.

The effect of methylation on basophilia.

A mixture of 0.05 N HCl and methanol is capable of completely inhibiting thionin and azure A metachromasia of intestinal mucin, cartilage and mast cell granules after treatment for 2 hours at 58°C. or 48 hours at room temperature. Slight reduction is noted after 2 weeks at 3°C. Cytoplasmic basophilia is completely destroyed after 4 hours of methylation at 58°C. or 48 hours at room temperature. Pancreatic acinar cell cytoplasm appears more resistant, requiring 8 hours at 58°C. and longer than 9 days at room temperature. Methylation at 3°C. is ineffective in inhibiting this basophilia even after 2 weeks. Nuclear and nucleolar basophilia with aniline dyes (azure A and thionin) requires 24 hours of methylation at 58°C. or 14 days at room temperature before there is complete destruction. There is no effect produced by methylation at 3°C. for 2 weeks. Alum hematoxylin staining of these structures is unaffected by the methylation procedure, indicating the different nature of action of this dye complex from the aniline dyes used. Reversal of the methylation procedure or demethylation can be accomplished with 0.5% KMnO4 for ½ hour at room temperature provided the methylation has not been too prolonged. Other oxidants such as periodic, peracetic and chromic acids as well as aqueous HCl and alkali (borax solution) fail to reverse the blockade. The substitution of 0.1 N HCl for the usual 0.05 N HCl in the methylation procedure brings about effective blockade in approximately ½ the time required by the 0.05 H NCl methanol. Despite the removal of cytoplasmic and nuclear basophilia by this method sections treated concomitantly and stained with the Feulgen nucleal procedure demonstrate strong Feulgen reactions indicating selectivity for the acid portion of the nucleic acid involved. Methylation after fixation of tissue in a chromate containing fixative (Zenker acetic) requires more prolonged treatment to inhibit cytoplasmic basophilia than is required after Formalin or 95% alcohol fixation. Otherwise the reactions occur similarly after either of the three fixatives. The positive periodic acid-Schiff reaction of colonic mucin, mixed tumor stroma and cartilage is completely inhibited by prior methylation for 96 hours at 58°C. and the reaction of gastric and small intestine mucins is only moderately reduced at this period. At room temperature no alteration is observed after methylation for 21 days. The positive reaction of human mast cell granules is abolished after 72 hours of treatment at 58°C. and is only moderately affected after treatment for 21 days at room temperature.

A New Digenetic Trematode (Cephalouterina Dicamptodoni n. g, n. sp., Pleurogenetinae) from the Pacific Giant Salamander

During the summers of 1950 and 1951, the first author examined about 50 specimens of the Pacific giant salamander, Dicamptodon ensatus (Eschscholtz), collected near Portland, Oregon. About half of these were found to harbor a species of intestinal fluke with peculiar anatomical features. Although resembling trematodes assigned to the subfamilies PLEUROGENETINAE and CEPHALOGONIMINAE in some respects, this species could not be placed in any of their genera. The authors therefore propose for it the new genus Cephalouterina with Cephalouterina dicamptodoni n. sp. as type species. The specimens were fixed in either Bouin's fluid, formalin-alcohol-acetic or corrosive-acetic acid solution. They were stained with borax carmine or Ehrlich's hematoxylin, cleared in xylene or terpineol, and mounted in Clarite. Serial sections were stained with Galigher's alum hematoxylin and eosin. All measurements are in millimeters with averages in parentheses. We wish to express our thanks to Mr. Kenneth Neiland for his help in obtaining the salamanders utilized in this study and to Dr. R. M. Cable for critical reading of this paper.

Modifications in Bowie's Staining of pepsinogen granules

Modifications of Bowie's technic for staining the pepsinogen granules of the body chief cells in the gastric glands, and a method of preparing the stomach of small rodents (e.g. the rat) for histo-topographical studies are described. The modifications involve counter-staining with aqueous alum hematoxylin, the use of the neutral synthetic mounting medium Permount, and changes in the timing of fixation, staining and differentiation.

Cestodes of California Gulls

There is but little information available regarding the helminthology of the California coast; therefore the following account of some cestodes found in gulls of this region may be of interest. Sixty-six birds were examined, fifty-six of which contained one or more parasites, an infection of 84.8o%. The worms have been examined in both fresh and preserved material, in whole mounts and sections. The fixatives employed were alcoholic-sublimate-acetic and alcoholic Bouin, with a few specimens fixed in 10%o formalin. The stains used were aceto-carmine and Ehrlich's hematoxylin for whole mounts, and for sections the latter and iron alum hematoxylin. All measurements in this paper are expressed in mm. and were made on preserved material except as stated in the text.

An Azocarmine stain for Differential cell Analysis of the rat Anterior Hypophysis

A method of staining is described which is especially designed to facilitate differentiation of the cell types of the rat anterior hypophysis. Fixation in Zenker-formol solution is recommended. Pre-staining of the nuclei by a short immersion in alum hematoxylin is followed by mordanting in anilin alcohol and a 45 minute period of staining in azocarmine solution at 60d`C. The counterstains, acid green and orange G, are dissolved in clove oil to avoid destaining of the azocarmine.

Basic Fuchsin as a Nuclear Stain for Fungi

Negative or weak Feulgen reactions have been obtained in many plant organisms. The three procedures for staining with basic fuchsin presented in this paper were developed in consequence of failure to obtain adequate Feulgen reactions in cytologic studies on certain fungi pathogenic for man, and because of the difficulty encountered in obtaining iron alum hematoxylin preparations which do not have a murky aspect. Clear and beautiful preparations can be obtained by the following technics, which appear to hold tremendous promise for studies of nuclear phenomena in the fungi. Ohlmacher (1895) was the first and, we believe, the only author to use formaldehyde as a mordant for basic fuchsin staining. He applied his methods to bacteria and tissues. The studies of DeLamater and Ulrich extend, but only partially confirm, the initial observations of Ohlmacher.