ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : V. WHY DOES AZURE B PENETRATE MORE READILY THAN METHYLENE BLUE OR CRYSTAL VIOLET?

Glass electrode measurements of the pH value of the sap of cells of Nitella show that azure B in the form of free base penetrates the vacuoles and raises the pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, but the dye salt dissolved in the sap does not alter the pH value of the sap. It is concluded that the dye penetrates the vacuoles chiefly in the form of free base and not as salt. The dye from methylene blue solution containing azure B free base as impurity penetrates and accumulates in the vacuole. This dye must be azure B in the form of free base, since it raises the pH value of the sap to about the same extent as the free base of azure B dissolved in the sap in vitro. The dye absorbed by the chloroform from methylene blue solution behaves like the dye penetrating the vacuole. These results confirm those of spectrophotometric analysis previously published. Crystal violet exists only in one form between pH 5 and pH 9.2, and does not alter the pH value of the sap at the concentrations used. It does not penetrate readily unless cells are injured. A theory of "multiple partition coefficients" is described which explains the mechanism of the behavior of living cells to these dyes. When the protoplasm is squeezed into the sap, the pH value of the mixture is higher than that of the pure sap. The behavior of such a mixture to the dye is very much like that of the sap except that with azure B and methylene blue the rise in the pH value of such a mixture is not so pronounced as with sap when the dye penetrates into the vacuoles. Spectrophotometric measurements show that the dye which penetrates from methylene blue solution has a primary absorption maximum at 653 to 655 mµ (i.e., is a mixture of azure B and methylene blue, with preponderance of azure B) whether we take the sap alone or the sap plus protoplasm. These results confirm those previously obtained with spectrophotometric measurements.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : V. RESEMBLANCES BETWEEN THE LIVING CELL AND AN ARTIFICIAL SYSTEM IN ABSORBING METHYLENE BLUE AND TRIMETHYL THIONINE.

The rate of diffusion through the non-aqueous layer of the protoplasm depends largely on the partition coefficients mentioned above. Since these cannot be determined we have employed an artificial system in which chloroform is used in place of the non-aqueous layer of the protoplasm. The partition coefficients may be roughly determined by shaking up the aqueous solutions with chloroform and analyzing with the spectrophotometer (which is necessary with methylene blue because we are dealing with mixtures). This will show what dyes may be expected to pass through the protoplasm into the vacuole in case it behaves like the artificial system. From these results we may conclude that the artificial system and the living cell act almost alike toward methylene blue and azure B, which supports the notion of non-aqueous layers in the protoplasm. There is a close resemblance between Valonia and the artificial system in their behavior toward these dyes at pH 9.5. In the case of Nitella, on the other hand, with methylene blue solution at pH 9.2 the sap in the artificial system takes up relatively more azure B (absorption maximum at 650 mµ) than the vacuole of the living cell (655 mµ). But both take up azure B much more rapidly than methylene blue. A comparison cannot be made between the behavior of the artificial system and that of the living cell at pH 5.5 since in the latter case there arises a question of injury to cells before enough dye is collected in the sap for analysis.     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.     

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue greater than azure B greater than azure A greater than toluidine blue greater than thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap alpha-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascorbate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.     

The Azure Dyes their Purification and Physicochemical properties. II. Purification of Azure B

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or plychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polyineric adsorbent (XAD-2) the acetate-formate anions are exchanged for chloride. Regeneration of both columns is possible: KMnO₄, Na₂S₂O₄ and water are run through column A, 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

The azure dyes: their purification and physicochemical properties. II. Purification of azure B.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

Metal contaminants in commercial thiazine dyes.

The iron, potassium, sodium and zinc content of commercial samples of the thiazine dyes azure A (C.I. 52005), azure B (C.I. 52010), azure C (C.I. 52002), methylene blue (C.I. 52015), new methylene blue (C.I. 52030), polychrome methylene blue, thionine (C.I. 52000) and toluidine blue (C.I. 52040) have been determined by atomic absorption spectrophotometry. The metal concentration varied widely in the 38 samples examined--iron, potassium, sodium and zinc together comprised between 0.02% and 25.35% of individual samples.     

Metal Contaminants in Commercial Thiazine Dyes

The iron, potassium, sodium and zinc contents of commercial samples of the thiazine dyes azure A (C.I. 52005), azure B (C.I. 52010), azure C (C.I. 52002), methylene blue (C.I. 52015), new methylene blue (GI. 52030), polychrome methylene blue, thionine (C.I. 52000) and toluidme blue (C.I. 52040) have been determined by atomic absorption spectrophotometry.

The metal concentrations varied widely in the 38 samples examined—iron, potassium, sodium and zinc together comprised between 0.02% and 25.35% of individual samples.     

Topo-optical reactions on the membrane of the human red cell ghost (author's transl)]

Previous studies have proved that the thiazin dyes toluidine blue, azure A, azure B, 1.9-dimethyl methylene blue and the quinolin dyes N,N'-diethylpseudoisocyanine chloride, N,N'-6,6'-dichlorpseudoisocyanine chloride are suitable for topo-optical reaction on the membrane of the red blood cells. In the present study the applicability of the thiazin and quinolin dyes on the membrane of the human red cell ghost was examined. Optical analysis revealed that the thiazin dyes are bound in radial position to the membrane, while the quinolin dyes are bound parallel to the membrane's plane.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

Adsorption of metal complex dyes from aqueous solutions by pine sawdust

An attempt to alleviate the problem caused by the presence of metal complex dyes, mostly used in textile industries, in the textile effluents was undertaken. The effects of adsorbent particle size, pH, adsorbent dose, contact time and initial dye concentrations on the adsorption of metal complex dyes by pine sawdust was investigated. Acidic pH was favorable for the adsorption of metal complex dyes. A contact time of 120 min was required to reach the equilibrium. The experimental isotherm data were analyzed using the Langmuir, Freundlich and Temkin equations. The equilibrium data fit well the Langmuir isotherm. The monolayer adsorption capacities are 280.3 and 398.8 mg dye per g of pine sawdust for Metal Complex Blue and Metal Complex Yellow, respectively. The results indicate that pine sawdust could be employed as low-cost alternative to commercial activated carbon in aqueous solution for the removal of metal complex dyes.     

Standardization of the Romanowsky staining procedure: an overview

Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.     

Purification of Thionin, Azure A, Azure B and Methylene Blue

Column and paper chromatography of four thiazin dyes revealed both inorganic and organic impurities. In thionin, azure A, azure B and methylene blue, sodium and other metal cations were found as inorganic impurities. The analysis for organic impurities revealed that the dyes were mixtures; specifically each dye contained one or more of the other dyes as impurities. Inorganic impurities were detected by ashing the dyes in the presence of H₂SO₄ and chromatographing the sulfate salts on paper. They were removed by filtration through ion exchange resins. Organic impurities were detected by paper chromatography and removed by column chromatography on Woelm's neutral alumina.     

Mechanisms of Giemsa banding

Summary We investigated the capability of individual thiazins in Giemsa mixtures (methylene blue and azures A, B, and C) and of two related dyes (toluidine blue and thionin) to produce G-banding. We further tested the effects of variations of buffer composition and concentration, dye concentration, and staining time.G-banding was produced by all of the dyes at low concentrations, although differences were noted. Overall, methylene blue and azure B produced the best banding, azures A, C, and toluidine blue produced moderately good banding, and thionin produced poor banding. This order did not appear to be altered essentially by different treatments. The optimal conditions for G-banding for all dyes and treatments included the use of (1) 0.025–0.05M phosphate buffer, (2) dye concentrations of 0.002%–0.005%, and (3) staining times of 6–15 min.     

The Azure Dyes: Their Purification and Physicochemical Properties. I. Purification of Azure A

A method is described for the separation of azure A from commezcial samples of polychrome methylene blue. Up to 300 mg of the pure dye may be isolated in this way. The method is based on chromatography using columns 90 cm high, 7 cm in diameter, loaded with 3 g of polychrome methylene blue. The absorbent is silica gel, the eluent a mixture of acetic and formic acid.

Poor solubility of the dye acetate in water necessitates dissociation of the acetate by alkalinization and subsequent conversion of the dye to the chloride with diluted Ha. Demethylation that occasionally occurs during this procedure is negligible. Pure azure A does not spontaneously demethylate under ordinary conditions.     

Thiazin Dyes in Supravital Staining of nerve Fibers

Supravital staining by thiazins of segments of small intestine and mesentery of young dogs was studied with reference to specificity for nervous tissue. Attempts to secure a purer form of methylene blue by alumina adsorption and alcohol elution of the commercial, medicinal dye yielded a product which appeared to be structurally different from the original dye. The treated dye had absorption maxima from 620 to 655 mμ in contrast with 665 for the untreated. Small nerve bundles were stained by the treated dye after 2 to 4 hours of immersion, but staining was always incomplete. Staining by untreated methylene blue was compared with that by the leucobase, thionol, methylene green, toluidine blue, new methylene blue and the azures. It was concluded that the specificity for nerve fibers resides mainly in the =N(CH₃)₂Cl radical, although some specificity appears to be effected by the methyl groups on the trivalent nitrogen, since azure A (dimethyl) and azure C (mono-methyl) stained weakly, but thionin did not. Methylene green showed some specificity but stained weakly. The leucobase was less active than the reoxidized dye obtained from it.     

Additional Schiff-Type Reagents for Use in Cytochemistry

Twenty-four new Schiff-type reagents were discovered in a survey of 140 different dyes. These dyes include acid fuchsin, acridine yellow, acriflavine hydrochloride, azure C., Bismarck brown R, Bismarck brown Y, celestine blue B, chrysoidine 3R, chrysoidine Y extra, cresyl violet, crystal violet, gentian violet, methylene blue, neutral violet, phenosafranin, phosphine GN, proflavine, toluidine blue O, and toluylene blue. Positive results obtained with crystal violet and a few samples of methylene blue are considered due to impurities. Various chemical extractions, aldehyde blocking reagents, and enzymatic treatments were used to verify the aldehyde specificity of the above dye-SO₂, reagents as well as azure A, brilliant cresyl blue, neutral red, safranin O, and thionin which have been mentioned by other workers. These reagents were tested in the Feulgen reaction for DNA and the PAS reaction for polysaccharides. Absorption curves were obtained from individual nuclei stained for DNA. The absorption peaks ranged from 450 mμ, to 630 mμ. depending on the dye studied. The Feulgen reaction could be followed by the PAS reaction or vice versa in mouse intestine using reactive dyes of complementary colors. The evidence indicates that a potential Schiff-type reagent must have at least one free NH₂ group on the dye molecule.