Chemical structures and staining mechanisms of Weigert's resorcin-fuchsin and related elastic fiber stains

Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsin, Sheridan's crystal violet and their resorcinol-free analogues were investigated using reverse-phase and gel filtration chromatography, electrophoresis, and visible light spectroscopy. Their staining properties were also studied. It was concluded that 1) the staining components of Weigert's resorcin-fuchsin, orcinol-new fuchsin and their resorcinol-free analogues are all indamine oligomers, 2) resorcinol is required for the production of Sheridan's crystal violet, the staining components of which consist of crystal violet substituted by varying numbers of resorcinyl substituents, 3) the staining components of all preparations are cationic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachloroferrate anion (FeCl4-) and not as Fe or as a dye-chelate, and 5) since even the smallest Weigert's resorcin-fuchsin, orcinol-new fuchsin or Sheridan's crystal violet component has a conjugated bond number of 32, the observed staining of elastic fibers is only as expected.     

A methanol resorcin-fuchsin stain for elastic tissues and nuclei.

The staining properties of conventional ethanol resorcin-fuchsin and of methanol resorcin-fuchsin were compared. Formula; Dissolve 0.2 g of commercial resorcin-fuschin in 70 ml of methanol or ethanol, add 30 ml of water and 1 m1 of concentrated HC1; stain sections for 4 hours. Both solutions colored elastic and pseudoelastic fibers, cartilage and some mucins. Methanol resorcin-fuchsin also colored nuclei in methacarn- (methanol-chloroform-glacial acetic acid 6:3:1) and formalin-fixed tissues; this nuclear stain withstood counterstaining with picro-dye mictures. Zenker-fixed sections showed diffuse coloration with little or no contrast between nuclei and cytoplasm. Extraction with hot trichloracetic acid abolished binding of methylene blue, but binding of methanol resorcin-fuchsin by nuclei remained unaltered or was enhanced. Experiments with solvents containing various concentrations of methanol, ethanol or isopropanol indicated that the staining patterns of resorcin-fuchsin are determined by the nature and concentration of the alcohol. Methanol resorcin-fuchsin proved useful for simultaneous visualization of elastic tissues and nuclei.     

Effects of time and temperature during attachment of sections to microscope slides on immunohistochemical detection of antigens.

To study the effects of time and temperature on attachment of tissue sections to microscope slides, we examined the intensity of immunohistochemical staining of selected antigens in nine different neoplastic and normal tissues after attaching sections at different times and temperatures. Typically, both the temperature and time are minimized when tissue sections attached to slides; however, suboptimal times and temperatures during attachment may result in either loss of tissue due to poor attachment or the necessity for inconvenient staining regimens. Using standard immunohistochemical techniques, 5 microm tissue sections were attached at 58 degrees C for 1, 4 and 24 hr. In a separate study, 5 microm tissue sections were attached for 16 hr at 58, 68 and 80 degrees C. The intensity of staining decreased slightly when the tissue sections were heated at 80 degrees C for 16 hr, but there was little or no decrease when tissues were heated at 68 degrees C or lower for 16 hr, or at 58 degrees C for up to 24 hr.     

Chemical Structures and Staining Mechanisms of Weigert'S Resorcin-Fuchsin and Related Elastic Fiber Stains

Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsia, Sheridan's crystal violet and their resorcinol-free analogues were investigated using reverse-phase and gel filtration chromatography, electrophoresis, and visible light spectroscopy. Their staining properties were also studied. It was concluded that 1) the staining components of Weigert's resorcin-fuchsin, orcinol-new fuchsin and their resorcinol-free analogues are all indamine oligomers, 2) resorcinol is required for the production of Sheridan's crystal violet, the staining components of which consist of crystal violet substituted by varying numbers of resorcinyl substituents, 3) the staining components of all preparations are canonic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachlorofemte anion (FeCI₄^-) and not as Fe⁺⁺⁺ or as a dye-chelate, and 5) since even the smallest Weigert's resorcin-fuchsin, orcinol-new fuchsin or Sheridan's crystal violet component has a conjugated bond number of 32, die observed staining of elastic fibers is only as expected.     

Trichinella spiralis: effect of high temperature on infectivity in pork

Twenty gram samples of homogenized Boston shoulder from swine experimentally infected with Trichinella spiralis were sealed in plastic pouches, pressed to a uniform thickness of 2 mm, and subjected to water bath temperatures of 49, 52, 55, 60, and 63 +/- 0.5 C for intervals of 2 min to 6 hr, especially within the interval of 0 to 15 min. These times included a period of about 1 min at the start and a period of about 1 min at the end for temperature equilibration. Treated samples were rapidly chilled to 25 C and then digested in a 1% pepsin-HCl solution at 37 C for 18 hr to recover T. spiralis larvae. The recovered larvae were suspended in 2 ml saline; 1 ml of this suspension was introduced into the stomach of each of two rats. The linear equation, log (time) = 17.3 -0.302 (temperature), was calculated from the time required at each temperature for the inactivation of T. spiralis larvae. The correlation coefficient for that relationship was r = -0.994. Larvae heated in the meat to 55 C for 4 min retained their infectivity, but were rendered noninfective after 6 min at 55 C. At 60 C, larvae were not infective after only 2 min (zero dwell time); whereas at 52 C, 47 min were required to render the larvae noninfective. Larvae in meat heated to 49 C were infective after 5 hr but not after 6 hr. These data demonstrate that the destruction of infectivity of T. spiralis is time-temperature related.     

An Evaluation of Preparatory Procedures for Leaf-Tip Chromosomes Spreads of the Tea Plant (Camellia sinensis)

Well-spread metaphase plates for routine karyotype analysis can be obtained by treating the very young leaf-buds of tea shoots in a saturated aqueous solution of p-dichlorobenzene for 2-3 hr at 4-10 C, fixing in a 1:3:6 mixture of propionic acid, chloroform and ethanol for 6-12 hr, staining with 2% propiono orcein at about 80 C, and squashing in a drop of 1% propiono-carmine under a coverslip.     

Basic Fuchsin-Ferric Chloride: a Simplification of Weigert's Resorcin-Fuchsin Stain for Elastic Fibres

Weigert's resorcin-fuchsin stain for elastic fibres can be simplified by the omission of the resorcinol. The resulting “basic fuchsin-ferric chloride” gives results indistinguishable from those achieved with resorcin-fuchsin on tissues fixed in Bouin, Carnoy, Gendre, neutral formah, Susa or Zenker solutions. Gel filtration chroma tography on Sephadex LH20 showed that the staining components of basic fuchsin-ferric chloride had a high molecular weight, so selective staining of elastic fibres by this method may well be due to van der Waals attractions.     

The Spawning and Early Development of the Hawaiian Acorn Worm (Hemichordate), Ptychodera flava

The spawning and early embryogenesis of the hemichordate, Ptychodera flava, in Hawaii are described in detail and illustrated with photographs of living material. Natural spawning in the evenings of early December was induced by a shift of seawater temperature from about 22 degrees C to about 26 degrees C. The fertilized egg divides equally and slowly at first, reaching 8 cells at about 5 hr after insemination at room temperature (20-24 degrees C). Divisions then appear to become slightly unequal and by 9 hr the embryo has divided into about 100 cells. The blastocoel forms during cleavage as an irregular space that, when viewed from the side, tends to appear oblate and ultimately appear crescent-shaped as the vegetal plate thickens into the blastocoel. The archenteron forms at about 18 hr as a cleft beginning at the vegetal pole and extending into the vegetal plate. As development proceeds, the embryo expands and by 24 hr forms a typical deuterostome gastrula with an outer sphere of ectoderm and a inner tube of endoderm connected at the blastopore. An out-pocketing of the gut appears at the tip of the archenteron over the next 4 hr to form the protocoel which will become the proboscis coelom. Approaching 40 hr the gut becomes asymmetric and over the next few hr contacts the ectoderm to form a mouth. Hatching occurs during this time at about 45 hr of development. Morphogenesis continues to produce an early tornaria larva by about 60 hr.     

THE EFFECT OF SHORT PERIODS OF HIGH TEMPERATURE DURING DAY AND NIGHT PERIODS ON PEA YIELDS

Karr , E. J. (Ohio State U., Columbus), A. J. Linck , and C. A. Swanson . The effect of short periods of high temperature during day and night periods on pea yields. Amer. Jour. Bot. 46(2) : 91-93. Illus. 1959.—The effect of high temperatures during periods of relatively short duration (3-4 days) at various stages following anthesis at the first bloom node was studied in relation to yield of peas at this node. Except for the periods of differential temperature treatments, the plants were maintained in a standard environment room (24°C., light, 12 hr.; 15°C., darkness, 12 hr.). Three different temperature regimes during the treatment periods were studied: high day temperature—standard night temperature (32°—15°C.) ; standard day temperature—high night temperature (24°—30°C.) ; and high day and night temperatures combined (32°—30°C.). The data reveal the existence of a relatively well-defined thermal-sensitive period, with maximal sensitivity to high day temperatures occurring at about 9-11 days from full bloom, and maximal sensitivity to high night temperatures occurring about 6-9 days from full bloom. High night temperatures proved more critical, resulting in a maximal reduction of 25% in yield, as opposed to about 8% for high day temperatures. The effect of high day and night temperatures combined tended to be roughly additive.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

Methods for Accelerating the Fluorescent-Antibody Test for Rabies Diagnosis

The time required to perform the fluorescent-antibody test for rabies was reduced by eliminating acetone fixation of the brain impressions and by incubating the conjugate-impression reaction at room temperature for only 10 min. Elimination of the preliminary acetone fixation had no effect on the diagnosis of impression smears from 246 mammalian brains by immunofluorescence. Staining at 37 C for 30 min and staining at room temperature for 10 min were found to be equally effective in the examination of impression smears from 161 brain samples. The procedure, as modified, shortens the time required for the diagnosis of rabies by immunofluorescence from about 5.5 hr to approximately 45 min.     

Effect of Fixatives on Silver Impregnation of Plant Cells

The kind of fixative and duration of fixation modify the affinity of plant cell structures, as shown by a 10-15 hr impregnation at 70 C in 2% aqueous AgNO₂, and a 1-2 hr reduction at room temperature by a 1:1 mixture of 10% formalin and 1% hydroquinone. Cytoplasmic staining was enhanced by fixing in salts of heavy metals, in buffered 6.5% glutaraldehyde, and in 0.5% picric acid. Nuclear staining was prominent after mixtures of glutaraldehyde and hydroquinone, after formalin and pyrogallol, and after acetone, propylene glycol or ether. Nucleolar staining was favored by fixing in 10% formalin, in 5% formalin containing 0.5% hydroquinone, in 50% ethanol containing 0.5% pyrogallol, or in ethylene glycol. Chromosome staining was favored by fixation in 50% acetic or propionic acid, in 2% trichloroacetic acid, and in methanol or ethanol. The best morphological preservations were seen after 50% acetic acid, 6.5% glutaraldehyde, or the 5% formalin-0.5% hydroquinone mixture.     

Mature Cereal Grain Endosperm: Rapid Glass Knife Sectioning for Examination of Proteins

A simple, rapid procedure was developed by which the quality of endosperm protein in cereal grains could be evaluated by microscopic observation of the subcellular protein structure. Kernels with vitreous endosperm were sectioned without pretreatment at 3-4 μ with a glass knife. Floury endosperm tissues were fixed in 10% glutaraldehyde, pH 7.4, for 16 hr at 5 C, and boiled to gelatinize the starch. After drying, this tissue was sectioned as for vitreous endosperm. Sections were mounted on gelatin-coated slides and destarched with a-amylase. To identify sites of prolamine, alcohol-soluble protein was extracted for 1 hr at about 70 C with 80% ethanol. The subcellular proteins were stained with either iodine vapor or various organic dyes. Proteins were differentiated by a combination of staining and mounting in selected high refractive index liquids.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

The Effects of Hot Water Treatments on Survival of Heterodera schachtii

Cysts of Heterodera schachtii were treated in a water bath at constant temperatures ranging from 45 - 62.5 C for 1 sec to 28 hr. Treated and untreated cysts were incubated 8 weeks in sugarbeet root diffusate at 24 C to measure emergence of surviving larvae. Within the temperature range of 49 - 54 C, the minimum lethal temperature was proportional to the log time of treatment. No larvae emerged from cysts exposed 10 sec at 60 C. Although treatment of cysts for 8 hr at 45 C significantly reduced emergence, increasing the treatment period to 28 hr did not completely suppress emergence.     

Camwood (Baphia nitida) Extract as a Histological Stain for Interglobular Dentine, Striated Muscle, and Counterstaining

The shavings of the dried heartwood of the tree Baphia nitida are ground to a fine powder, and 6 gm of the powder are extracted in 100 ml absolute ethanol at 27-30 for 6-24 hr. The extract is filtered with Whatman No. 1 paper and stored in a screw-capped bottle. For staining the interglobular dentine of nondecalcified sections of formlin-fixed teeth, sawed cross sections 20-30 μ thick were dehydrated in ethanol and stained in the undiluted extract for 6-12 hr at room temperature. The interglobular dentine was stained a bright golden brown on a pale brown background. For staining striated muscle, the extract was diluted 1:1 with distilled water and filtered. After mordanting formalin-fixed paraffin sections with 0.25% KMnO₄ for 5 min, and bleaching with 5% oxalic acid for 10 min, they were washed in water and stained for 2-24 hr at room temperature. The striations were stained light to deep golden brown. For use as a counterstain, a 1:6 dilution of the original extract was required. When applied after haematoxylin for 15-30 min, it stained tissue components in varying shades of golden brown with distribution comparable to that produced by 1% eosin.     

Metabolism, Macromolecular Synthesis, and Nuclear Behavior of Cryptococcus albidus at 37 C

The temperature-sensitive events which prevent Cryptococcus albidus from growing at 37 C were investigated. Cultures incubated at 37 C immediately after inoculation did not increase in optical density nor in cell numbers, and by 24 h 90% of cells in such cultures were deformed and dead. When cultures in log phase were shifted from 23 to 37 C the optical density increased but the cell numbers did not. Morphological observations revealed that the increase in turbidity at 37 C represented enlargement and distortion of cells without appreciable replication. Uptake and incorporation of (14)C-leucine were similar at 23 and 37 C. There was no difference in (14)CO(2) evolution from cells at either temperature. Uptake and incorporation of adenine-8-(14)C into RNA was slightly lower in cells incubated at 37 C. There was, however, a 60% reduction in incorporation of adenine-8-(14)C into DNA after 3 hr at 37 C. Nuclear staining revealed that nuclear migration did not occur in cells incubated at 37 C. Thus the data indicate that both adenine incorporation into DNA and nuclear migration prior to nuclear division by C. albidus are temperature sensitive.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

A Useful Variant of the Movat Pentachrome I Stain

A simplified and improved staining procedure based on the Movat pentachrome I stain has been devised by substituting the van Gieson procedure for the saffron du Gatineau and woodstain scarlet portions of Movat's method, and by the use of commercially prepared resorcin-fuchsin for the staining of elastica.     

A method for staining pollen tubes in pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsin, 6 ml; 1% aqueous aniline blue, 4 ml; 1% orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45 +/- 2 C. They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45 +/- 2 C, hydrolyzed in the clearing and softening fluid at 58 +/- 1 C for 30 min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.