Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.     

鸡中枢淋巴器官肥大细胞的组织化学与形态学

对哺乳动物的,特别是啮齿动物和人类肥大细胞已有了比较深入的研究, 但关于家禽肥大细胞的研究很少.本研究旨在阐明鸡中枢淋巴器官中肥大细胞的组织化学与形态学特征.本研究证实Carnoy 氏液是鸡肥大细胞的优良的固定液,而中性缓冲福尔马林(NBF) 却阻断了大多数肥大细胞的着染力.甲苯胺蓝和阿尔新蓝是鸡肥大细胞的良好的染料,但阿尔新蓝能使更多的肥大细胞着染,虽然其也可使杯状细胞着染.作者的一种新的染色法, 长时间阿尔新蓝染色(LAB-S)可用于NBF固定的组织中肥大细胞的染色,因为其着染的细胞数与Carnoy 氏液固定甲苯胺蓝染色的细胞数无显著差异(P<0.001).在胸腺髓质中见有大量的肥大细胞,而胸腺皮质仅可见个别肥大细胞位于血管周围及小叶间结缔组织中.腔上囊的皮质与髓质中很少见有肥大细胞.肥大细胞有血管周围分布的倾向,但一个有趣的发现是血管内偶尔也有个别肥大细胞.电镜下可见肥大细胞的胞浆颗粒内充满无定形的颗粒状基质,但其电子密度有的较高,有的较低.少数胞浆颗粒内有旋涡状及网状亚微结构.但未见有人类肥大细胞胞浆颗粒内特征性的晶格状和卷轴状的亚微结构,也未见到在绵羊肥大细胞中描述过的特殊亚微结构.     

The toluidine blue-membrane filter method: absorption spectra of toluidine blue stained bacterial cells and the relationship between absorbance and dry mass of bacteria

Propan-2-ol did not leach dye from toluidine blue stained bacteria on membrane filters but ethanol did. The absorption spectra of toluidine blue stained cells of two Gram-positive and two Gram-negative organisms differed with the latter organisms exhibiting metachromasia. The results suggest that toluidine blue stains the cell envelope. Linear regression equations were derived for each of four organisms, Streptococcus cremoris, Lactobacillus bulgaricus, Pseudomonas fluorescens and Escherichia coli, relating absorbance at the peak of the absorption spectra and the mass of cells on the filters. With these equations it should be possible to determine mass of cells with an error between 3% and 7.5% depending on the organism. Since the regression equations are similar, the amount of toluidine blue retained per milligram of cells may be constant under standard conditions, irrespective of species.     

The Toluidine Blue-Membrane Filter Method: Absorption Spectra of Toluidine Blue Stained Bacterial Cells and the Relationship Between Absorbance and Dry Mass of Bacteria

Propan-2-ol did not leach dye from toluidine blue stained bacteria on membrane filters but ethanol did. The absorption spectra of toluidine blue stained cells of two Gram-positive and two Gram-negative organisms differed with the latter organisms exhibiting metachromasia. The results suggest that toluidine blue stains the cell envelope. Linear regression equations were derived for each of four organisms, Streptococcus cremoris, Lactobacillus bulgaricus, Pseudomonas fluorescens and Escherichia coli, relating absorbance at the peak of the absorption spectra and the mass of cells on the filters. With these equations it should be possible to determine mass of cells with an error between 3% and 7.5% depending on the organism. Since the regression equations are similar, the amount of toluidine blue retained per milligram of cells may be constant under standard conditions, irrespective of species.     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358 +/- 0.134 X 10(4) at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACT

Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Summary Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358±0.134×10⁴ at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.Supported by NIH grants EY00012, EY07035 and EY01583     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

Summary The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes showed higher electron density than control chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82 — for euchromatic arms — and 0.85 — for centromeric heterochromatin — were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Effect of temperature on the induced cotton effects of acid polysaccharide-basic dye complexes

The induced Cotton effect of some acid polysaccharide–thiazine dye complexes were studied at various temperatures. When the equivalent ratio of the anionic site of the polymer to the cationic dye was near unity, all the complexes examined showed remarkable Cotton effects corresponding to their absorption bands in the visible and ultraviolet regions, at neutral pH and room temperature. Although the structure of the hyaluronate complex resembles the carbohydrate backbone, the sign of the Cotton effects was opposite that of chondroitin sulfate complexes. When the temperature was raised, the hyaluronate–toluidine blue complex decreased the metachromatic shift in the absorption spectrum gradually; the amplitude of the Cotton effects of this complex decreased also. With a polysaccharide which has a carboxylate group as its only anionic site (such as carboxymethylcellulose or pectic acid), a similar effect of temperature was observed on its induced Cotton effects. With the chondroitin 4-sulfate–toluidine blue complex, some reverse of the metachromatic shift was observed at higher temperature. However, the amplitude of its Cotton effects decreased up to 70 °C, then the sign of the effects reversed and the amplitude subsequently increased. A similar inversion of the sign of the Cotton effects was found with the chondroitin 6-sulfate–toluidine blue complex, but at a lower temperature (about 40 °C). Charonin sulfate, a highly sulfated cellulose-like glucan from a marine mollusca, showed a marked metachromatic effect on methylene blue even at the elevated temperature. The induced Cotton effects of this complex were also affected by temperature and the inversion of the sign was observed. When the pH was lowered, the amplitude of the Cotton effects of the hyaluronate-toluidine blue complex decreased and diminished at pH 3, at room temperature. With chondroitin 4-sulfate, the induced Cotton effects were remarkable at such a low pH; however, the amplitude of the effects decreased with elevated temperature and no inversion was observed. The optical rotatory dispersion of the chondroitin 6-sulfate–toluidine blue complex was reversed by acidifying to pH 3, and the amplitude of the Cotton effects decreased by elevating the temperature.     

Mast cell "disappearance" in chronic murine graft-vs-host disease (GVHD)-ultrastructural demonstration of "phantom mast cells"

Mast cells were studied during the induction of chronic graft-vs-host disease (GVHD) induced in mice across minor histocompatibility barriers. B10.D2 spleen cells (or control BALB/c cells) were injected into irradiated (600 rad) BALB/c recipients. Serial skin biopsies were taken over 26 days, during which time changes occurred resembling scleroderma, namely, dermal fibrosis, a mononuclear cell infiltrate, and loss of fat and appendages. Mast cells, when stained with toluidine blue, "disappeared" from GVHD, but not from control skin. Ultrastructural analysis showed that mast cells in GVHD skin were indeed present but underwent degranulation. Some mast cells showed only pale expanded sacs, indicating granule depletion. Because these cells could not be seen by toluidine blue staining but were plainly present, we have called them "phantom mast cells." Cellular activation occurred in many GVHD mast cells as shown by increased cytoplasmic activity, with numerous Golgi complexes, ribosomes, granular endoplasmic reticulum, and small vesicles. No identifiable mast cells were seen after day 19. No significant changes were seen in the mast cells of syngeneic control mice. We believe that immunologic processes in chronic GVHD cause a slow release of mast cell granule contents, which is different from anaphylactic degranulation. The depleted mast cells (invisible by toluidine blue staining) are also activated, perhaps in an attempt to replete their stores of granule contents. We discuss the relation of mast cell changes to fibrosis.     

Membrane potential-dependent binding of polysialic acid to lipid monolayers and bilayers

Polysialic acids are linear polysaccharides composed of sialic acid monomers. These polyanionic chains are usually membrane-bound, and are expressed on the surfaces of neural, tumor and neuroinvasive bacterial cells. We used toluidine blue spectroscopy, the Langmuir monolayer technique and fluorescence spectroscopy to study the effects of membrane surface potential and transmembrane potential on the binding of polysialic acids to lipid bilayers and monolayers. Polysialic acid free in solution was added to the bathing solution to assess the metachromatic shift in the absorption spectra of toluidine blue, the temperature dependence of the fluorescence anisotropy of DPH in liposomes, the limiting molecular area in lipid monolayers, and the fluorescence spectroscopy of oxonol V in liposomes. Our results show that both a positive surface potential and a positive transmembrane potential inside the vesicles can facilitate the binding of polysialic acid chains to model lipid membranes. These observations suggest that these membrane potentials can also affect the polysialic acid-mediated interaction between cells.     

不同固定液和染色方法显示山羊子宫内肥大细胞效果的比较

目的探讨用不同固定液和染色方法对显示处于间情期山羊子宫肥大细胞的影响。方法用四种不同的固定方法,应用改良甲苯胺蓝（MTB）染色法和阿尔辛蓝-番红花红（AB-S）染色法显示处于间情期山羊子宫肥大细胞。结果山羊子宫组织采用Carnoy氏液固定,MTB和AB-S染色对所有的肥大细胞均可获得良好的染色反应,但10％中性福尔马林,4％多聚甲醛,Bouin氏液固定的组织仅有少量肥大细胞着染。结论MTB和AB-S染色法均是山羊子宫肥大细胞良好的染色方法。     

Rapid Single-Solution Polychrome Staining of Semithin Epoxy Sections using Polyethylene Glycol 200 (PEG 200) as a Stain Solvent

Rapid, onestep polychromatic staining of 0.75-1.5 μm epoxy sections of glutaraldehyde-osmium fixed tissues can be obtained with mixtures of basic fucbsin and toluidme blue O in alkaline polyethylene glycol ZOO (PEG ZOO). Sections are attached to slides by heating at 100 C for 45 seconds and stained at that temperature for 2-3 minutes with a solution consisting of PEG 200 (50 ml), 0.2 N KOH (0.75 ml), basic fuchsin (1.7 gm), and toluidine blue O (0.3 gm). Red-blue balance and selective staining of different structures can be controlled by varying the amount of toluidine blue added. After rinsing with 10% acetone and rapid drying, sections are covered with immersion oil or mounting medium and a cover-slip. Total time from cutting of a section to finished preparation is less than 6 minutes. This staining solution is stable, does not produce precipitates on the sections, and does not wrinkle or lift the sections from the slides.     

Hydrogenase activity of the methylotroph, Pseudomonas methylica]

The cells of Pseudomonas methylica, strain 2, cultivated in a medium containing methanol, displayed the activity of hydrogenase in the exchange reaction (D2--H2O) and in the absorption of H2 in the presence of methylviologen, azocarmine, methylene blue, and ferricyanide. The rate of H2 utilization was highest in the presence of methylviologen. Cell extracts absorb H2 in the presence of methylviologen, NAD, and NADP, but much faster in the presence of flavin mononucleotide. The bulk of the hydrogenase remains, during centrifugation of the initial cell extract (3,000 g), in the soluble fraction (144,000 g). The absorption of oxygen by the cell suspensions and the incorporation of 14C of formiate into the cells are stimulated by H2. The cells, however, cannot grow in the autotrophic conditions at the account of molecular hydrogen and CO2.     

A study on the killing by light of photosensitized cells of halobacterium salinarium

Summary The present study aimed at a demonstration of a protecting effect of carotenoids against photokilling of Halobacterium salinarium in the presence of exogenous photosensitizer. In comparative experiments with a colorless mutant and the carotenoid-containing wild type the use of toluidine blue as photosensitizer gave erratic results. It seems probable that the erratic results were due to the varying ability of cells from one culture to another to reduce toluidine blue to a leuco form which is inactive as photosensitizer. The use of phenosafranine as photosensitizer gave consistent results; this dye has a lower oxidation-reduction potential than toluidine blue and is less easily reduced to a leuco form by the cells. When aerated cultures were exposed to light in the presence of phenosafranine the colorless cells were killed; the carotenoid-containing cells were not so affected. It can therefore be concluded that carotenoids protect the cells against photokilling under these conditions.The killing of the colorless cells was closely parallelled by an extensive lysis of the cells; the structure of the carotenoid-containing cells was barely affected by the exposure. There are reasons to believe that the carotenoids are located in or on the cell envelope. It thus seems likely that the cell envelope is a site of the photochemically induced damage.     

Histological, Chromatographic and Spectrophotometric Studies of Toluidine Blue

Chromatographic analysis of commercial batches of toluidine blue shows these to be dye mixtures. Histologically, some samples were found to be poor metachromatic dyes. These unsatisfactory stains contained blue dyes with little or no metachromatic properties as well as a metachromatic fraction. On the other hand, contaminating dyes in histologically satisfactory samples had poor staining qualities and hence did not interfere with the color produced by the metachromatic fraction.

Chromatographic fractionation of different commercial batches of toluidine blue yielded identical, homogeneous metachromatic dyes. These purified dyes had a peak absorption at 615 mμ in contrast to that of purified azure A whose peak absorption was at 622.5 mμ.     

Association of polyphosphate with protein in freeze-substituted sclerotia ofSclerotinia minor

Summary In freeze-substituted sclerotia stained with aqueous toluidine blue O, metachromatic material was found throughout the cytoplasm in discrete granules. It was also distributed evenly throughout spherical and elongate protein bodies. This material stained at low pH and was extracted by cold acid, indicating that it was polyphosphate. Retention of metachromatic material was much greater than previously reported in chemically fixed, conventionally processed sclerotia. X-ray microanalysis of dry-cut, unstained sections of freeze-substituted sclerotia confirmed that phosphorus was distributed evenly throughout the protein bodies and was not localised in discrete granules but phosphorus levels in the cytoplasm were very low. It is concluded that polyphosphate is lost during conventional preparation procedures but retained in dry-cut, unstained sections of freeze-substituted material. However, when freeze-substituted sections were stained with toluidine blue O, water soluble polyphosphate was extracted and subsequently precipitated in the cytoplasm as polyphosphate granules. Therefore it is considered that polyphosphate granules are an artefact, and that protein bodies are the major site for storage of phosphorus in this fungus.Abbreviations STEM scanning transmission electron microscope - ER endoplasmic reticulum     

Salivary glands of heteromyid rodents, with a summary of the literature on rodent submandibular gland morphology

The principal parenchymal elements of the submandibular glands of the heteromyid rodents Dipodomys merriami, Perognathus longimembris, Perognathus fallax, Perognathus penicillatus and Perognathus baileyi consist of acini, granular tubules and striated ducts. Acinar cells of the four species of Perognathus are aniline blue, PAS (magenta) and Alcian blue (pH 2.5) positive and metachromatic with toluidine blue and safranin. The granules of the tubule cells are orthochromatic and react with aniline blue, orange G, the PAS reagent (deep pink) and the tryptophan indicator, xanthydrol. Acinar and tubule cells of D. merriami exhibit similar reactions except for the Alcian blue stain. Acinar cells of D. merriami do not react with Alcian blue. Submandibular glands of D. merriami exhibit a sexual dimorphism of the granular tubules. There is little observable difference between the sexes in the species of Perognathus but the ratio of granular tubules to acinar elements, the degree of hypertrophy of the tubules, and the amount of mucosubstance and protein (granules) contained in their cells are different in the four species studied. Since these desert rodents have similar habitats and habits, the differences observed between the two heteromyid subfamilies studied, as well as among the four members of a single subfamily, suggest that these are inherent species variations rather than variations of adaptation to environment.     

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.