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.     

Spectrophotometric Characteristics and Assay of Biological Stains IV. the Phenyl Methane Dyes

This paper continues the investigation of the assay and spectral properties of biological stains. The phenyl methane dyes, auramine O, basic and acid fuchsins, methyl violet, crystal violet, methyl green, and anilin blue W. S., may all be assayed by the spectrophotometric method. Of these, methyl green has been found unstable both in solid form and in solution, hence color densities of this dye must be measured promptly after preparation.     

The Mechanism of the Gram Reaction I. The Specificity of the Primary Dye

Dyes of all major types were tested for their suitability as the primary dye in the Gram stain. When a counterstain was not used, some dyes of all types were found to differentiate Gram-positive from Gram-negative organisms. When a counterstain was used, these dyes were found to vary greatly in their suitability. Those dyes found to be good substitutes for crystal violet were: Brilliant green, malachite green, basic fuchsin, ethyl violet, Hoffmann's violet, methyl violet B, and Victoria blue R. All are basic triphenylmethane dyes. Acid dyes were generally not suitable. Differences in the reaction of Gram-positive and Gram-negative cells to Gram staining without the use of iodine were observed and discussed but a practical differentiation could not be achieved in this manner. Certain broad aspects of the chemical mechanism of dyes in the gram stain are discussed.     

Methods for the Standardization of Biological Stains: Part IV

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group 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: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

A Modification of the Technic of Handling Chick Embryos

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group 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: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Removal of basic dyes from aqueous medium using a novel polymer: Jalshakti

Studies were carried out to remove basic dyes such as safranine T, methylene blue, crystal violet, light green, brilliant milling violet and patent blue VS from their aqueous solutions using biodegradable polymeric absorbent material, viz., Jalshakti (JS). Results showed that 93% safranine T, 98% methylene blue and 84% crystal violet were adsorbed on JS relative to their initial concentration (10 mg L(-1)). The optimum pH was found to be 6.0+/-0.5 and smaller size of particle of JS resulted better adsorptive removal of the dyes. IR spectroscopic and potassium ion release studies revealed that basic dyes were selectively removed through adsorption-ion-exchange mechanism involving carboxylic groups and K+ ions of JS.     

Gene-Controlled Resistance to Acriflavine and Other Basic Dyes in Escherichia coli.

Nakamura, Hakobu (Konan University, Kobe, Japan). Gene-controlled resistance to acriflavine and other basic dyes in Escherichia coli. J. Bacteriol. 90:8-14. 1965.-The genetic determinant controlling the sensitivity of Escherichia coli K-12 W1895 to the basic dyes acriflavine, methylene blue, toluidine blue, crystal violet, methyl green, and pyronine B appears, from results of mating experiments, to be located between the marker governing the utilization of lactose and the origin of genetic transfer. The determinant controlling this resistance to basic dyes does not control resistance to acid dyes. After the introduction of the resistance gene into merozygotes, acriflavine resistance is not established immediately but develops slowly.     

Contribution to the Staining of Neuroglia

The chemistry of Weigert's glia staining method is critically discussed. An investigation of the Heidelberger Victoria blue staining method has shown that Victoria blue may be replaced by other phenylmethane dyes as methyl violet, ethyl violet, and crystal violet. It was found that the exposure of the stained section to sunlight is an oxidation process. Artificial ultra violet rays or chemical oxidation agents give the same effect. Frozen sections fixed in formalin or alcohol may be stained in a concentrated aqueous solution of any of the above mentioned phenylmethane dyes, dried, and exposed to ultra violet rays for 30 minutes, then treated with 1/10 N. iodine solution, differentiated in xylol anilin and cleared in xylol. The glia cell body as well as the fibrils are clearly differentiated from the nervous elements and connective tissue.     

White-rot fungus <Emphasis Type="Italic">Ganoderma</Emphasis> sp.En3 had a strong ability to decolorize and tolerate the anthraquinone,indigo and triphenylmethane dye with high concentrations

The ability of the white-rot fungus Ganoderma sp.En3 to decolorize different kinds of dyes widely applied in the textile and dyeing industry, including the anthraquinone dye Remazol Brilliant Blue R (RBBR), indigo dye indigo carmine and triphenylmethane dye methyl green, was evaluated in this study. Ganoderma sp.En3 had a strong capability of decolorizing high concentrations of RBBR, indigo carmine and methyl green. Obvious reduction of Chemical Oxygen Demand was observed after decolorization of different dyes. Ganoderma sp.En3 had a strong ability to tolerate RBBR, indigo carmine and methyl green with high concentrations. High concentrations of RBBR, indigo carmine and methyl green could also be efficiently decolorized by the crude enzyme of Ganoderma sp.En3. Different redox mediators such as syringaldehyde, acetosyringone and acetovanillone could enhance the decolorization capability for higher concentration of indigo carmine and methyl green. Different metal ions had little effect on the ability of the crude enzyme to decolorize indigo carmine and methyl green. Our study suggested that Ganoderma sp.En3 had a strong capability for decolorizing and tolerating high concentrations of different types of dyes such as RBBR, indigo carmine and methyl green.     

用凝胶电泳发现肝素与染料之间的相互作用

目的:观察肝素与23种染料之间的相互作用.方法:利用淀粉琼脂糖凝胶电泳及聚丙烯酰胺凝胶电泳,观察染料加肝素后电泳行为的变化.结果:淀粉琼脂糖凝胶电泳中染料加肝素后多数结果是泳速变慢,有一些留在原点,也有前移、后退者.二氯荧光素、荧光红、荧光素、甲酚红、苯胺蓝、溴酚蓝、茜素红S、胭脂红、亮绿、氨基黑10B、丽春红G、曙红、苯胺黑、氯酚红加肝素后泳速变慢.亚甲蓝、灿烂甲酚蓝、甲基紫加肝素后电泳留在原点.刚果红、洋红加肝素后电泳变化不明显.四氯荧光素、氯化硝基四氮唑蓝加肝素后电泳荧光看不清.聚丙烯酰胺凝胶电泳中溴酚蓝加入肝素后泳速明显变慢,随着肝素量增加溴酚兰的泳速加快.与淀粉琼脂糖凝胶电泳结果一致,但它的电泳结果更明显.结论:本次所研究的23种染料,几乎都能与肝素发生相互作用,给它们的进一步深入研究打下基础.     

Zum Farbsehvermögen von Hausziegen (Capra hircus L.)

Tests on male goats were designed to determine their capacity for colour vision. The colours yellow, orange, blue, violet and green were tested against gray nuances of like brightness. Goats were found to be able to distinguish between colours and gray nuances. The rate of errors increased in the order: orange, green, red, yellow, violet, blue.     

Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme

The ability of the white-rot fungus Lentinula (Lentinus) edodes to decolorize several synthetic dyes was investigated using solid state cultures with corn cob as substrate. Cultures, containing amido black, congo red, trypan blue, methyl green, remazol brilliant blue R, methyl violet, ethyl violet and Poly R478 at 200 ppm, were completely decolorized after 18 days of incubation. Partial decolorization was observed in the cultures containing 200 ppm of brilliant cresyl blue and methylene blue. High manganese peroxidase activity (2600 U/g substrate), but very low lignin peroxidase (<10 U/g substrate) and laccase (<16 U/g substrate) activities were detected in the cultures. In vitro, the dye decolorization was markedly decreased by the absence of manganic ions and H2O2. These data suggest that manganese peroxidase appear to be the main responsible for the capability of L. edodes to decolorize synthetic dyes.     

A Differential Stain Favorable to the Diagnosis of Neisserian Infection

A differential Gram stain has been evolved which incorporates the combined features of the original Gram and Pappenheim methods. National Aniline crystal violet and new methyl green and pyronin are the dyes preferred. The iodine mixture of Kopeloff and Beerman is a satisfactory mordant and Merck's pure technical acetone is an excellent differentiating agent. A system is established by means of the dyes and reagents which form a physicochemical equilibrium, provided pure dyes are employed, and the technic is carried out with precision. Gram-positive bacteria are coated by means of buffered crystal violet solution and the iodine-sodium hydroxide solution precipitates the crystal violet from other substances. The dye-iodine precipitate is readily dissolved by pure acetone. Iodine green, a pure derivative of crystal violet has the effect of noninterference in the technic and has selective action upon nuclear substance. Pyronin has affinity for Neisserian organisms primarily and acts as an inert substance upon most other proteins, (except cytoplasm of eosinophils, lymphocytes, plasma cells, and endothelial cells). The following technic is recommended:

Stain air-dry films 3 to 5 minutes in a 1% solution of crystal violet in 10 parts of Clark and Lubs' phosphate buffer of pH 6.6 to 7.0 and 90 parts water. Decant and flush with 2% iodine in N/10 NaOH. Decant and decolorize in acetone 10 seconds or less. Air dry and counterstain 1 1/2 to 2 minutes with methyl-green-pyronin (2 parts 2% aqueous methyl green National with one part 0.3% aqueous pyronin yellowish). Wash and air dry. Oil of Bergamot is preferable to xylene as a clearing agent. Best results are obtained if each slide is handled separately as for staining blood films.     

Basic Two-Dye Stains for Epoxy-Embedded 0.3-1 μ Sections

Dyes used in the 3 methods recommended are: I, thionin and acridine orange (T-AO); II, Janus green and Darrow red (JG-DR); III, methyl green and methyl violet (MG-MV). The first 2 methods were two-solution stains, applied in sequence; the third, required only one solution since methyl violet is present in commercial methyl green. Staining solution and timing was as follows: Method I. 0.1% thionin in a 45% ethanolic solution of 0.01 N NaOH, 5 min at 70 C; rinsing in water and followed by 1 min in a 1% aqueous solution of acridine orange made up in 0.02 N NaOH, also at 70 C, then washed, and dried on slides. Method II. 0.5% Janus green in aqueous 0.05 N NaOH, 5 min at 70 C; rinsing in water then into 0.5% Darrow red in 0.05 N NaOH (aq.), 2 min at 70 C., washing, and drying on slides. Method III. 1% methyl green (commercial, unpurified) in 1% aqueous borax for 15-20 min at 20-25 C, washing and attaching to slides. All staining was performed by floating the sections on the staining solutions, all drying at 70 C, and mounting in a resinous medium. T-AO gave blue to violet cytoplasmic structures, darker nuclei which contrasted strongly with yellow connective tissue and the secretion of goblet cells. JG-DR resembled a hematoxylineosin stain, but by shortening the staining time in DR to 0.5-1 min, collagenous and elastic tissue retained more of the green dye. MG-MV gave dark green nuclei in light green cytoplasm, with collagenous and elastic tissues in blue to violet. As with most methods for staining ultrathin sections, thicknesses of less than 1 μ required longer staining times.     

A Technic for Preparing Frozen Sections

A combination of the Gram-Pappenheim stains for the examination of gonorrheal pus, cellular exudate and paraffin sections of formalin-fixed tissues has been described elsewhere (Scudder and Lisa, 1931). The crystal violet solution was made stable for the first time by employing phosphate buffers on the acid side of neutrality, and a stable counterstain was prepared for the first time from National Aniline dyes, ethylated methyl green and pyronin yellowish. Original findings were demonstrated by means of color plate I and II (Scudder, 1931) to show gonococci, pneumococci and cells in smears, and formalin-fixed tissue brought down to water in the usual way. A new color plate is published herewith to show the microscopic appearance of cells, Gram-positive and Gram-negative bacteria, higher bacteria, fungi and spermatozoa in the study of genitourinary and gynecological cases. The method has a value in the field of medical jurisprudence. Crystals were well demonstrated, especially those resulting from sulfa drug therapy. The National Aniline methyl green batches numbered NG 10, 11, 13 to 19, and their batches of pyronin numbered NP 5 to 10 were found consistently stable. Earlier dyes were found either too purple or too blue for the technic and the most satisfactory dyes were found to require a ripening time of several days and could be prepared in amounts of from 1 to 4 liters and stored indefinitely without preservatives.     

Comparison of historic Grübler dyes with modern counterparts.

Seventeen Grübler dyes produced in Germany between 1880 and 1939 were examined in this study. These dyes were: fuchsin-bacillus, diamond fuchsin, fuchsin S acid, rubin S, safranin O water soluble, safranin yellowish water soluble, methyl eosin, Sudan III, scarlet R, auramine, orange G, aniline blue, pyronin, carmine, lithium carmine, hematein and aurantia. Spectrophotometry and staining characteristics were used to determine the maximum absorbance and efficacy of each dye in common staining techniques. The spectral curves and staining characteristics of these dyes compared well with modern dyes used as controls. Fuchsin bacillus and diamond fuchsin are synonyms for basic fuchsin. Fuchsin S acid and rubin S are synonyms for acid fuchsin. The scarlet R sample was the same as the Sudan III. The two safranins were the same. The basic fuchsin samples were unsuitable for preparation of Schiff's reagent. Both basic fuchsin and pyronin samples were less concentrated than modern counterparts. It is noteworthy that the dyes worked well after up to 100 years in storage, and this observation indicates that dyes can have a long shelf life when stored in cool, dry, air-tight conditions.     

The Combined Gram-Pappenheim Stain as Modified for Films and Paraffin Sections

A combination of the Gram-Pappenheim stains for the examination of gonorrheal pus, cellular exudate and paraffin sections of formalin-fixed tissues has been described elsewhere (Scudder and Lisa, 1931). The crystal violet solution was made stable for the first time by employing phosphate buffers on the acid side of neutrality, and a stable counterstain was prepared for the first time from National Aniline dyes, ethylated methyl green and pyronin yellowish. Original findings were demonstrated by means of color plate I and II (Scudder, 1931) to show gonococci, pneumococci and cells in smears, and formalin-fixed tissue brought down to water in the usual way. A new color plate is published herewith to show the microscopic appearance of cells, Gram-positive and Gram-negative bacteria, higher bacteria, fungi and spermatozoa in the study of genitourinary and gynecological cases. The method has a value in the field of medical jurisprudence. Crystals were well demonstrated, especially those resulting from sulfa drug therapy. The National Aniline methyl green batches numbered NG 10, 11, 13 to 19, and their batches of pyronin numbered NP 5 to 10 were found consistently stable. Earlier dyes were found either too purple or too blue for the technic and the most satisfactory dyes were found to require a ripening time of several days and could be prepared in amounts of from 1 to 4 liters and stored indefinitely without preservatives.     

Methyl green and its analogues bind selectively to AT-rich regions of native DNA.

Methyl green has long been used as a DNA stain in histochemistry. The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting. At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T. At higher concentrations the dyes bound to almost all available DNA sites. Models, with and without intercalation are discussed to account for the specific binding.     

Differential staining of the cell cycle of plant cells using safranin and indigo-picrocarmine.

A trichrome staining technique using safranin-indigo-picrocarmine (SIPC) can be used to distinguish the various stages of the cell cycle in onion root tip. When the tissue was fixed first in formalin followed by picric acid and stained in SIPC, a clear differentiation of interphase nuclei into four color classes, viz., green, orange, red and brown can be recorded. Replacing crystal violet for safranin produces a similar pattern of differentiation of interphase nuclei into green, light blue, blue and deep blue. Autoradiographic study using 3H-thymidine as a DNA precursor demonstrates the reliability of the SIPC staining technique. All the orange and red nuclei are found to be labelled and therefore are in S phase of the cell cycle. Almost all the green nuclei are unlabelled and may be assigned to G1. The larger brown nuclei which are mostly unlabelled can be considered in G2 phase.     

Method of counterstaining preparations for immunofluorescent studies of the pituitary]

To elucidate nonfluorescent structural elements of the hypophyseal parenchyma for immunofluorescent investigations, properties of some dyes most commonly applied for hypophysis staining have been studied. Such dyes as paraldehide-fuchsin, light green, orange G, chromotrop 2R, hematoxylin, eosin, fuchsin, azocarmin possess their own intensive luminescence and block immunofluorescence completely. Some other dyes (trypan blue, bromthymol blue, aniline blue, malachite green, methyl green) though not blocking immunofluorescence, they do not reveal hypophyseal cellular elements distinctly enough. Good results have been obtained with 0.3% water solution of toluidine blue, 0.5% solution of methylene light blue, methylene blue, as well as with Gram--Weigert's staining and with gallocyanin after Einarson. For special staining of corticotropocytes, the authors recommend 0.1% solution of bromphenol blue in barate buffer, pH 8.2.