Examining the microanatomy of various fetal body parts using the orcein-picroindigocarmine staining method

It is difficult to distinguish muscle, vessel, nerve, fascia, tendon and many other structures in fetal sections. The aim of the study reported here was to research the practicability of orcein-picroindigocarmine staining for distinguishing anatomic structures in histological sections containing complex structures. Histological serial sections of hand, foot, head and neck of four 18- to 20-week old fetuses were used. Bone matrix was stained bright blue, collagen fibers tones of green to blue, elastic fibers brown, cartilage matrix tones of pink, and chondroblasts blue. Muscles and erythrocytes were stained yellow to green, epidermis reddish brown, skin appendices including hair and nail stained light yellow to green.     

Plant Cuticle Staining with Bismarck Brown Y and Azure B or Toluidine Blue O before Paraffin Extraction

Transverse paraffin sections of mature greenwood stems of rose (Rosa x hybrida) and flowering dogwood (Cornus florida L.) were stained with Bismarck brown followed by azure B or tolnidine blue O. The Bismarck brown was replaced by thiazin dye metachromasia in all structures except the cuticle which remained brown or yellow. The interface between the cuticle and exterior cell walls of the epidermis was delineated clearly.     

Iodine as an Aid in Identification of Asci and Ascospores of Schizosaccharomyces octosporus

Transverse paraffin sections of mature greenwood stems of rose (Rosa x hybrida) and flowering dogwood (Cornus florida L.) were stained with Bismarck brown followed by azure B or tolnidine blue O. The Bismarck brown was replaced by thiazin dye metachromasia in all structures except the cuticle which remained brown or yellow. The interface between the cuticle and exterior cell walls of the epidermis was delineated clearly.     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

A Simple Polychrome Stain for Conventionally Fixed Epon-Embedded Tissues

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO₄ fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H₂O₂, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic' fibers. Intra cytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

Staining Tomato Fruit Cuticle and Exocarp Tissues

Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue 8GX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylinalcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at × 100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues magenta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.     

Dye plants of East Anatolia Region (Turkey)

Dye plants that are commonly used by the regional people of East Anatolia were studied. The following information was collected: local name of plants, the part of plants containing dye, obtained color, dying substances, and extraction of colors. As a result of area investigations carried out between 1994 and 2000, 50 taxa (used for dying wool yarns in the region) belonging to 38 genera and 26 families were determined from collected specimens By using the dying plants and mediator substances, 15 different colors and tones can be obtained. The main colors are yellow, green, olive green, black, red, blue, dark blue, brown, gray, beige, orange, khaki, mustard, purple, and smoke. The colors and their many different tones were observed on kilims and carpets that are woven in the East Anatolia region of Turkey     

Identification of lymphocyte subpopulations with a polymethine dye

Using the polymethine dye p-ethoxyphenyl-p-aminostyryl-1,3,3-trimethyl-3H-indolium chloride as an aqueous stain applied to specimens of peripheral blood or buffy coat fixed in FAA fixative, differential coloration of leukocytes was achieved using darkfield illumination. Neutrophils stained dark maroon and contained green granules, eosinophils contained bright blue granules, basophils revealed yellow and pink granules, and monocytes stained green with green and yellow vacuoles. In studies of purified lymphocyte subpopulations obtained in a cell sorter, T-helper cells stained red, T-suppressor cells were yellow orange, B-cells appeared yellow and often contained yellow annular structures in the cytoplasm, and natural killer (NK) cells stained green and contained large green granules. As a rapid screening technique for identification of T-helper and T-suppressor cells and their ratios in health and disease, the new polymethine stain may complement the more complex monoclonal antibody techniques for identification of these cells.     

Preference of the cabbage root fly, Delia radicum (L.), for coloured traps: influence of sex and physiological status of the flies, trap background and experimental design

Abstract. Catches of Delia rudicum (L.) (Diptera: Anthomyiidae) were compared in water traps that reflected predominantly wavelengths shorter (violet and blue traps) and longer (green and yellow traps) than 500 nm.Traps were positioned in choice and no-choice situations against backgrounds of bare soil and weeds in the field and against backgrounds of brown and green paper in the laboratory.The physiological status of the flies was modified in the laboratory by denying them access to food sources and oviposition sites.
Males discriminated significantly more clearly than females between yellow and blue traps.The discrimination between yellow and blue traps was significantly more pronounced when the traps were presented in the choice than in the no-choice situation in both sexes.Green background (weeds and green paper) was highly preferred for landing and thus competed with the traps to such an extent that few flies were caught when non-preferred violet and blue traps were sited on green backgrounds.Flies seldom landed on the brown background (soil and brown paper) which resulted in the relative increase of catches in the non-preferred violet and blue traps.The preference for yellow traps was innate even in young flies with immature egg-follicles.Females that were ready to lay eggs, even those deprived of an oviposition site till the age of 8 days, also preferred yellow traps.In the no-choice situation, flies deprived of food landed with the same frequency in yellow and blue traps.Food deprivation, however, did not affect preference for yellow traps over the blue traps presented in a choice situation.     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Differential dichrome staining of tissue culture monolayers: alternate dyes and possible mechanism.

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

A Cresyl Fast Violet Stain for Bacteria and Fungi in Tissue

The cresyl fast violet staining method was modified to eliminate differentiation. Paraffin sections from tissues fixed in Zenker-formol were stained in a 1% aqueous solution of cresyl fast violet (Chroma), adjusted to pH 3.7 with acetic acid, washed in running tap water, dehydrated and covered. Because basophilia increases with time of fixation or storage in formalin or Kaiserling's fluid, dilution of the dye solution to 0.5-0.1% is recommended for such material. Bacteria, nuclei, Nissl substance, and lipofuscin were colored dark blue; fungi, blue to purple; and cytoplasm and muscle fibers, light blue. Collagen and reticulum fibers were only faintly stained. Thus, microorganisms were easily visible against the lightly colored background. In formalin-fixed material, bile pigment was colored olive green. Because this method does not require differentiation, it gave uniform results even in the hands of different users. Little or no fading was observed in sections stored for more than 2 yr.     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle

Using frozen sections from human muscle biopsies, we assessed the value of Nile blue and Nile red, two fluorescent probes, as stains for lipid droplets in normal and pathological skeletal muscle fibers. In normal muscle, lipid storage disorders, and mitochondrial myopathies, Nile blue stained the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red-stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibers and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies.     

A Triple Stain for Deoxyribonucleic Acid, Polysaccharides and Proteins

Tissues from mammalian, amphibian, insect and plant sources were fixed in formalin or in Carnoy's, Helly's, or Smith's fluid and processed in the usual manner to mounted paraffin sections. These were stained by the sequence: Feulgen reaction (using azure-A-Schiff reagent); periodic acid Schiff (with basic-fuchsin-Schiff reagent); and a 0.02% solution of naphthol yellow S in 1 % acetic acid. Nuclei stained blue to green; polysaccharides, red; and proteins, yellow. Thus, deoxyribonucleic acid, vicinal hydroxy and hydroxy-amino groups, and free basic groups of proteins were demonstrated selectively. Of the tissues tested, stomach, intestine, kidney, thyroid gland and plant root tips were most strikingly stained.     

Staining Paraffin Embedded Sections of Scald of Barley before Paraffin Removal

Staining of paraffin embedded sections with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differentiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalis. Crystal violet, toluidine blue O and aniline blue also successfully stained fungal structures of R. secalis in barley leaf tissues. Staining of embedded sections before paraffin removal allows simple processing of a series of sections, saves time and reduces solvent consumption.     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

On the histochemical differences of aldehyd-fuchsin positive material in the fibres of the hypothalamo-hypophyseal tract of Rana temporaria

Summary Using a modified Alcian blue/Alcian yellow method, differences in stainability in the fibres of the preoptic-hypophyseal tract of Rana temporaria were observed. Near the preoptic nucleus most of the fibres stained yellow, in the tract the fibres stained lightgreen or showed green inclusions on a yellow background, whereas at the fibre-endings a colour switch to blue-green was found. This colour switch was the most pronounced in the neural lobe, where the neurosecretion stained blue-green, whereas in the fibre-endings in the outer zone of the median eminence a colour switch to green was noticed.The results can best be explained with the assumption that a modification or transformation of the neurosecretion occurs during its transport from the preoptic nucleus to the fibre-endings.Literature related to this hypothesis is discussed.     

Chlorantine fast green BLL as a stain for callose in oak phloem

Sections of oak bark were stained with chlorantine fast green BLL, used as a 0.25% aqueous solution. All tissues were unstained, except for local deposits of material associated with phloem cell walls, which stained deep green. This green-staining material also stained specifically with resorcinol blue and with the aniline blue fluorescence technique, the usual histochemical tests for callose. The chlorantine fast green-staining material was removed from sections by treatment with a beta-1,3-glucan hydrolase. It is concluded that chlorantine fast green BLL stains callose in plant sections and is a useful additional stain for the histochemical detection of this polymer.