Schiff-Type Reagents in Cytochemistry. 2. Detection of Primary Amine Dye Impurities In Pyronin B and Pyronin Y(G)

Many batches of pyronin B (C.I. 45010), pyronin Y or G (C.I. 45005), and acridine red (C.I. 45000) produce positive Feulgen or PAS reactions when their 0.25% solutions are saturated with SO₂ and used on acid-hydrolyzed or periodate-oxidized tissue sections. These dyes behave as Schiff-type reagents and stain aldehyde-containing structures orange, brown, pink, red, or violet, depending on the particular batch used. The most frequent contaminants are violet and are nonfluorescent. Aldehyde groups are stained by these dyeSO₂ solutions as is shown by using unhydrolyzed controls in the Feulgen reaction and unoxidized controls in the PAS reaction, and by dye solutions lacking SO₂. Other procedures included reactions with aldehyde-blocking reagents, treatment with deoxyribonuclease and diastase, and extraction of nudeic acids with trichloroaeetic acid. The standard Schiff reagent was used in the Same procedures as a basis for comparing results. Since the Schiff-aldehyde reaction requires a dye with a primary amine group and since true pronins contain only secondary or tertiary amines, the positive histochemical results are evidently caused by dye contaminants possessing primary amine groups. The PAS reaction is more sensitive than the Feulgen reaction in detecting dye contaminants. Tissues used were chiefly formalin-fixed mouse intestine and ascites cells. Seventy-five commercial pyronins were studied from 21 different firms. Among 19 batches of pyronin B, 14 were found to contain primary amine dye contaminants. Among 39 batches of pyronin Y(G), 19 contained similar primary amine dye contaminants. Of the 8 batches of acridine red tested, 7 were found to contain primary amine dye contaminants. Nine commercial mixtures of methyl green-pyronin were studied and 4 were found to be likewise contaminated, but these reactive dye contaminants in them are apparently not associated with methyl green. A tabulated summary of the pyronin batches containing primary amine contaminants, and a list of sources and distributors of pyronin dyes are included.     

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.     

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.     

Stain Solubilities Part 1. Data in the Literature

The rather meager data found in the literature concerning the solubilities of the dyes used as biological stains is reviewed. Solubility data have been found concerning the following dyes: picric acid, martius yellow, crystal ponceau, methyl orange, tropaeolin O, orange II, Bismarck brown, Congo red, auramine, malachite green, fuchsin, methyl violet, gentian violet, crystal violet, methyl green, diphenylamine blue, aurin, corallin, phenolphthalein, flluorescein, eosin Y, iodo-eosin, methylene blue, alizarin, indigo carmine, and carmine. Much of this information is of questionable reliability. The writer is investigating the matter and his original data are to appear in subsequent papers.     

Stain Solubilities Part 1. Data in the Literature

The rather meager data found in the literature concerning the solubilities of the dyes used as biological stains is reviewed. Solubility data have been found concerning the following dyes: picric acid, martius yellow, crystal ponceau, methyl orange, tropaeolin O, orange II, Bismarck brown, Congo red, auramine, malachite green, fuchsin, methyl violet, gentian violet, crystal violet, methyl green, diphenylamine blue, aurin, corallin, phenolphthalein, flluorescein, eosin Y, iodo-eosin, methylene blue, alizarin, indigo carmine, and carmine. Much of this information is of questionable reliability. The writer is investigating the matter and his original data are to appear in subsequent papers.     

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.     

The Spectrophotometric Evaluation of Mixtures of Methylene Blue and Trimethyl Thionin

Spectrophotometric analysis affords the most convenient means for determining the proportion of methylene blue and trimethyl thionin (azure B) present in a mixture of these two dyes. The method proposed depends upon the determination of an “absorption ratio.” A suitable ratio for the purpose is that of the extinction coefficient at 640 mμ to that at 670 mμ. On account of the difference in absorption maxima of the two dyes, this ratio increases as the percentage of methylene blue decreases. The ratio value for eleven different mixtures is given and a graph is plotted from this data by means of which the proportions of the two dyes present in any mixture can be calculated from the absorption ratio determined as specified.     

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.     

Acid alcoholic brilliant cresyl blue stain for gastric and other mucins

Summary Some but not all samples of brilliant cresyl blue (6-methyl-7-dimethylamino-2-phenoxazin chloride) under C. I. No. 51010 in Conn's Biological Stains when dissolved at 1% level in 50–70% alcohol containing 1% concentrated (12 N) hydrochloric acid, stain (in 30 min) a wide variety of human and laboratory animal mucins blue black on an almost unstained background. The mucoprotein of the gastric surface epithelium and of the peptic gland neck cells of several species reacts strongly. A 16 hr 60° C methylation in 0.1 M methyl-sulfuric acid in methanol is required to block the staining of these gastric and some intestinal mucins, while 1–2 hr intervals suffice to prevent the staining of mast cells, cartilage and metachromatic sulfomucins generally. Saponification (1% KOH/70% alcohol, 20min) does not restore staining in either location group, indicating that sulfate mucins are probably reacting in both.Most other basic dyes fail to stain mucins from acid alcohol solutions: azure A, toluidine blue, resorcin blue, orcein, resorufin, azoresorufin brown, azolitmin, lacmoid, gallocyanin, Nile blue, methylene green, pararosanilin, crystal violet, Victoria blue R. Some staining occurred with one of three lots of Victoria blue B, with two lots of Victoria blue 4 R and with one lot each of Bernthsen's methylene violet, elastin violet PR and elastin purple PP.The stain may be preceded by the Feulgen reaction to give red nuclei, or followed by a brief collagen stain in an alcoholic acid fuchsin (0.05–0.1%), picric acid (1.5%) solution.Presented before the Symposium of the Histochemische Gesellschaft in Hamburg, 28. September 1968.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health.     

Methylene Violet (Bernthsen), by Zinc-Alkali-Chlorate Hydrolysis of Methylene Blue

Though Bernthsen's methylene violet (MV) is a common constituent of polychrome methylene blue, the hydrolytic oxydation of methylene blue to yield azure-free MV has been considered a difficult chemical reaction since the time of Bernthsen, who used Ag₂O in the hydrolysis. MV is qualitatively distinguished from azures by Bernthsen's criteria and the author's new tests: (1) light-excited isomeric change, (2) reactivity to acidity, (3) reaction with KCIO, and (4) reaction with Na₂SO₃ of azures in CHCI₃, while MV gives none. But MV shows (5) indicator properties at pH 4, while azures do not. For practical hydrolysis, treat methylene blue (10 parts by weight) and KCIO₃ (1 part) with 1-2 N NaOH to convert methylene blue to a mixture of MV and azures. Then dilute the solution, add a Zn salt and NaHCO₃ in excess of the amount needed to convert the NaOH to Na₂CO₃. Boil the solution gently for 1-2 hr. The end point of the reaction is found by pipetting a drop of reactant into 3% acetic acid in a test tube, adding CHC1₃ and extracting. The acetic layer should then be almost colorless while the CHC1₃ is colored intensely cherry red. After cooling, the precipitated dye is filtered and dried. This procedure gives good yields of a dye which meets the criteria given by Bernhsen. The peak of the absorption curve in solution, pH 4-11, is at 624 mμ (Bernthsen 625 mμ) and in acid solution, pH 0-4, 588 mμ (Biological Stains, 1953; 580 μ). The dye contains so little azures, that purification of the MV fraction obtained from the reaction mixture is unnecessary when it is used in the Wright-type Romanowsky stain. The remarkable staining effect of MV is its power to bring out red azurophil granules of monocytes and lymphocytes when used with eosinated thiazins in Wright's stain.     

Decontamination of aqueous solutions of biological stains.

Aqueous solutions of a number of biological stains were completely decontaminated to the limit of detection using Amberlite resins. Amberlite XAD-16 was the most generally applicable resin but Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-7 could be used to decontaminate some solutions. Solutions of acridine orange, alcian blue 8GX, alizarin red S, azure A, azure B, Congo red, cresyl violet acetate, crystal violet, eosin B, erythrosin B, ethidium bromide, Janus green B, methylene blue, neutral red, nigrosin, orcein, propidium iodide, rose Bengal, safranine O, toluidine blue O, and trypan blue could be completely decontaminated to the limit of detection and solutions of eosin Y and Giemsa stain were decontaminated to very low levels (less than 0.02 ppm) using Amberlite XAD-16. Reaction times varied from 10 min to 18 hr. Up to 500 ml of a 100 micrograms/ml solution could be decontaminated per gram of Amberlite XAD-16. Fourteen of the 23 stains tested were found to be mutagenic to Salmonella typhimurium. None of the completely decontaminated solutions were found to be mutagenic.     

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.     

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.     

Spectrophotometric and cytochemical studies on azures A, B and C.

The cytochemical use of azures A, B and C, propared with either N HCL and potassium metabisulphite or with sodium hydrosulphite in tissue sections were investigated. Both in situ absorption curves of nuclei stained with each of these dye-SO2 reagents as well as in vitro absorption data of acqueous solutions of the dyes are also presented. It has been pointed out that the mechanism of staining with azure A-SO2 and azure C Eosinate-SO2 is the same as that of the conventional Feulgen reaction with Schiff reagent but that of staining with azure B-SO2 is by the modified Feulgen reaction because this dye does not contain any primary amino group.     

Inactivation of Foot-and-Mouth Disease Virus by Interaction of Dye and Visible Light

The inactivation of foot-and-mouth disease virus was studied by means of the interaction of neutral red, Toluidine Blue, and methylene blue with visible light. The virus, Type A, strain 1, CANEFA of Argentine origin, was grown in tissue culture and tested in the crude and clarified state. Virus and dye were mixed and incubated together at 4 C for 45 min in the dark, or were mixed and immediately exposed to the visible light source without prior incubation together. Mixtures of crude virus and dye, under any of the experimental conditions used, did not inactivate more than 1 to 2 logs of viral infectivity when held in the dark or when exposed to light during a period of 45 min. Complete inactivation of virus was achieved when clarified virus and dye were mixed and immediately exposed to the visible light source for 15 min. Prior incubation of clarified virus and dye permitted inactivation by methylene blue only, whereas no incubation prior to exposure resulted in three of the dyes contributing to inactivation. A concentration of 6 μg of neutral red, Toluidine Blue, methylene blue, and crystal violet was used per milliliter of virus suspension. Crystal violet was not a good viral inactivator under the conditions of the experimentation. Inactive virus induced the formation of neutralizing antibodies in adult chickens and mice. The antibody titer stimulated by the antigen treated with methylene blue and visible light was probably significant.     

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.     

Qualitative Analysis by Thin-Layer Chromatography of Some Common Dyes Used in Biological Staining

Thin-layer chromatography will resolve impurities in commercial dyes, and will do so much faster than paper chromatography. Solvent systems consisting of (a) n-propanol: n-butanol: NH₄OH (conc.): H₂O—4:4:1:1; (b) n-propanol: NH₄OH (conc.): H₂O—8:1:1 on silica gel G plates; and (c) n-propanol: NH₄OH (conc.): H₂O-7:2:1 on Adsorbosil plates were found to be the most effective. Dyes studied were azure A, azure B, azure C, methylene blue, toluidine blue O, thionin, pyronin B, pyronin Y, methyl green, crystal violet amido black 10B and buffalo black (NBR).     

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.