Heterogeneity and Metachromasy of Some Commercial Anionic Dyes

The multicomponent character of all commercial anionic dyes tested (monoazo, disazo, indigoid and xanthene) was demonstrated by paper chromatography. On the basis of a reaction on filter paper, certain fractionated components of the dyes: aniline blue WS, benzoazurin, Bordeaux red, Congo red, cotton blue, chromotrope 2R, indigo-carmine, methyl-blau, soluble blue, and wasserblau showed a metachromatic response with the chromotropes, protamine and hexammine cobaltic chloride. The response of these same dye components with the chromotropes neomycin, polymyxin and viomycin was much weaker, and the alkaloids strychnine, codeine and cinchonidine could not elicit any metachromatic response. The hex-amminocobalt complex was the most effective of all the chromotropes studied, including protamine, both on filter paper and in aqueous solutions. Changes in color exhibited by the unchromatographed whole dyes such as alkali blue, alkali blue 6B, azoblau, Congo rubin, Hickson purple, isamine blue, orange G and trypan blue appear to be merely polychromatic effects because comparable changes are not shown by any of their chromatographically resolved components. In a solution system, the blue dyes, benzoazurin, cotton blue, indigo-carmine, methylblau, soluble blue, and wasserblau did not show definite visual changes in hue or in spectral shifts except with the hexamminocobalt complex, which induced a remarkable change in hue of all these dyes to a blue-violet or purple shade. A spectrophotometric study of methylblau has indicated that this change in hue is associated with a 25 mp shift of absorbance maximum to a lower wave length (hypsochromic effect). The filter-paper reaction between a dye component and a chromotrope is quite reliable and convenient for ascertaining a metachromatic response, since, unlike a reaction in solution systems, it is not affected by the unbound components of a reaction mixture. It is usable because water does not play any significant role in the metachromasy of anionic dyes. No correlation has been established between metachromasy and chemical constitution of anionic dyes.     

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.     

The cationic carbocyanine dyes Stains-all DBTC, and Ethyl-Stains-all, DBTC-3,3',9 triethyl.

A simple method is presented for distinguishing two closely related metachromatic carbocyanine dyes: Ethyl-Stains-all, a triethyl dye, and Stains-all, a diethyl methyl dye. This has become important since one lot of the triethyl dye was distributed erroneously under the diethyl methyl label. The dyes differ in solubility and in differential staining of macromolecules. Studies performed with both dyes are summarized.     

Ultrastructural chemistry of isolated molecules. Application to biological macromolecules. Study of metachromasia

The ultrastructural chemistry on isolated macromolecules enables to localize chemical function on spread molecules by means of reactions whose product is visible in electron microscopy (metal colloids, lectins, etc.). The reaction of linear polyanions with metachromatic dyes (bifunctional ligands) results in the formation of metachromatic complexes with a periodic structure : ligand molecules are intercalated between the polyanions arranged in parallel arrays; this structure is correlated with the metachromasy.     

An Evaluation of the Metachromasy of Anionic Dyes. II. Visual and Spectral Observations on Solutions

The reactions of 13 anionic dyes in solution with a basic protein (protamine), a cationic detergent, guanidine, histamine, procaine, quinine, and strychnine were examined visually and spectrophotometrically in order to distinguish metachromatic changes of the dyes. Disazo dyes (Congo red, benzopurpurin, but not trypan blue) were metachromatic; indigoid, triphenylmethane and xanthene dyes were not. The magnitude of metachromasy in this series of dyes was not great compared with cationic dyes, the shifts of absorbance maxima being only about 15 mμ against 90 mμ or more for some cationic metachromatic dyes. The most effective chromotropes were protamine and a cationic detergent. Agreement between visual observations on tissue sections, visual observations on solutions, and spectral observations on solutions was generally good.     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

Development of an automated SNP analysis method using a paramagnetic beads handling robot

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.     

Effects of differently hydrophobic solvents on the aggregation of cationic dyes as measured by quenching of fluorescence and/or metachromasia of the dyes

Aggregation of some cationic dyes leads to the appearance of their metachromatic spectra and/or quenching of their fluorescence. Ethanol and urea destroy metachromasia and enhance the fluorescence of such dyes by disaggregating them, suggesting hydrophobic bonds to be involved in their aggregation as in the formation of soap micelles or globular proteins. The ability of alcohols to disaggregate cationic dyes has been shown to be increased in the series methanol, ethanol, iospropanol and tertiary-butanol which is the order of increasing hydrophobic character of the alcohols themselves. Dimethyl urea is shown to be more effective than urea in destroying the metachromasia of toluidine blue, thus supporting the idea of hydrophobic bond to be involved in dye aggregation. Rhodamine 6 G undergoes quenching of its fluorescence in presence of a suitable polyanion but it is not metachromatic like acridine orange. Since only some specific cationic dyes undergo spectral changes with aggregation such changes seem to be the secondary effects of aggregation.Pool Officer, Scientists' Pool, Council of Scientific & Industrial Research (India), attached to the Bose Institute.     

Staining Reactions of some Anionic Disazo Dyes and Histochemical Properties of the Red Impurity in Trypan Blue

Some staining properties of 10 anionic disazo dyes are clarified by comparison with previous chromatographic analysis. Trypan blue contains both blue and red components and the purified blue fraction displays no color shifts in tissue sections. Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B and Niagara sky blue 6B generally resemble trypan blue. Congo red is a metachromatic dye and the only known example among anionic dyes of established purity whose color shows shifts in tissue sections and also in solutions with certain basic compounds. Other red dyes (Congo corinth, trypan red and vital red) are not metachromatic. The red dye impurity of trypan blue selectively stains nuclei which are pycnotic, degenerating or undergoing no further division. This reaction is apparently related to basic protein content. Other reactions of the red fraction of trypan blue (mammalian erythrocytes, blood plasma) are not fully explained on this basis.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Mechanisms of chromosome banding

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.     

Simultaneous histochemical demonstration of capillaries and muscle fibre types

Immunohistochemical staining of fibronectin is proposed as a good method for demonstrating capillaries in skeletal muscle tissue. The reaction superimposed on the histochemical reaction for myofibrillar ATPase enables simultaneous demonstration of fibre typing and capillary supply on the same tissue section. The method is quick and makes possible automated image analysis.     

Direct recording of metachromatic spectra in a model system of polyacrylamide films

Synopsis The metachromatic staining of polyacrylamide films containing different glycosaminoglycans is described. This model system made direct recording of metachromatic curves possible, under circumstances comparable to those of stained sections under the microscope, with virtually no interference of the corresponding orthochromatic peaks.The staining was carried out under standardized conditions (of buffer concentration, pH and temperature) and was shown to follow the Lambert-Beer law. The metachromatic peaks obtained with this system are listed for seven basic dyes, each complexed with seven different glycosaminoglycans.     

The staining of elastic fibres with Direct Blue 152. A general hypothesis for the staining of elastic fibres

Summary Elastic fibres may be stained by a number of dyes, e.g. Direct Blue 1 (C.I. 24410), Direct Blue 10 (C.I. 24340), Direct Blue 15 (C.I. 24400), Direct Blue 152 (C.I. 24366) and Direct Violet 37 (C.I. 24370). A convenient method using Direct Blue 152 has been developed which is specific for elastic fibres. The method is simple and allows the demonstration of other connective tissue fibres. Staining of elastic fibres is unimpaired by numerous blocking procedures or by changes in dyebath pH. These properties are shared by several standard elastic fibre stains.As the Direct dyes and several of the standard elastic fibre stains possess numerous aromatic rings a wide range of dyes containing varying numbers of aromatic rings were examined for ability to stain elastic fibres. No association was observed between the ability to stain elastic fibres and dye class, formal charge or the presence of hydrogen bonding groups. Staining was, however, definitely associated with the presence in the dye molecule of 5 or more aromatic rings. This suggested that van der Waals forces of attraction may be responsible for elastic fibre staining both by Direct dyes and the standard elastic fibre stains. Staining of elastic fibres as a side-effect in many procedures is similarly explicable.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.     

Specificity and Sensitivity of Insulin Staining by Aldehyde Fuchsin, Pseudoisocyanin and Toluidine Blue

Aldehyde fuchsin, pseudoisocyanin and toluidine blue, histochemical dyes reported to be specific for insulin-containing granules of the pancreatic beta cell, were applied to insulin fixed in polyacrylamide gel by disc electrophoresis. Two major and four minor bands were resolved as demonstrated by staining with amidoschwarz; only the two major bands, were stained by aldehyde fuchsin. The addition of serum did not affect this reaction. Serum or insulin components gave no metachromatic reactions to the other stains. Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.     

Metachromasy: an experimental and theoretical reevaluation

Non-chromotropic substances such as fibrin and gelatin and most tissue and cellular structures stain orthochromatically with internal dye concentrations of such metachromatic dyes as methylene blue and toluidine blue which, if in solution, would be metachromatic. Therefore, at ordinary levels of staining these substances depress the natural tendency of these dyes to change color. However, at elevated levels of dye-binding metachromasy eventually occurs. This phenomenon is explained on the basis of the distribution of dye-binding sites. In these substrates, by contrast with chromotropic substances, many binding sites are too far removed for dye interaction, consequently the interaction frequency can become high enough to produce a color change only as saturation of the available sites is approached. It is also shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color. A concept of metachromasy is proposed in which the interaction between water molecules and suitably approximated dye ions plays an essential role. The experimental studies are described against a background of the history and evolution of ideas on metachromasy. The literature is reviewed and reassessed particularly from the physicochemical viewpoint.