Chemical structures and staining mechanisms of Weigert's resorcin-fuchsin and related elastic fiber stains

Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsin, Sheridan's crystal violet and their resorcinol-free analogues were investigated using reverse-phase and gel filtration chromatography, electrophoresis, and visible light spectroscopy. Their staining properties were also studied. It was concluded that 1) the staining components of Weigert's resorcin-fuchsin, orcinol-new fuchsin and their resorcinol-free analogues are all indamine oligomers, 2) resorcinol is required for the production of Sheridan's crystal violet, the staining components of which consist of crystal violet substituted by varying numbers of resorcinyl substituents, 3) the staining components of all preparations are cationic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachloroferrate anion (FeCl4-) and not as Fe or as a dye-chelate, and 5) since even the smallest Weigert's resorcin-fuchsin, orcinol-new fuchsin or Sheridan's crystal violet component has a conjugated bond number of 32, the observed staining of elastic fibers is only as expected.     

Dye Adsorption by Bacteria at Varying H-Ion Concentrations

Adsorption of hydrogen ions and dye cations by washed bacterial cells shows a reciprocal relationship. Apparently, H-ions and crystal violet ions are held by the cell at the same adsorption centers, and the influence of H⁺ on basic dye adsorption is one of direct competition or replacement The adsorption of H⁺ and acid fuchsin is similar in that an increase is noted as the pH of the suspension is lowered.     

Iodine131 Uptake in the Gram Staining Technic

The Hucker modification of the Gram staining technic, in which NaI¹³¹ was incorporated with the Gram's iodine solution, was performed as the basic procedure. The Gram positive test-bacteria were Staphylococcus aureus and Bacillus megaterium; the Gram negative were Escherichia coli and Pseudomonas aeruginosa. The uptake of I¹³¹ was measured after the addition of the Gram's iodine solution (NaI¹³¹) to the test-bacteria dried on a glass slide, after the decolorization process and after counterstaining. Radiation was measured by placing the slide under a GM-TGC-2 end-window counting tube after each procedure. The Gram positive test-bacteria retained approximately twice as much I¹³¹ after decolorization and counterstaining as did the Gram negative bacteria. In this, the basic technic, the uptake of I¹³¹ by the test bacteria appeared to be directly related to the crystal violet concentration in the primary staining solution. The uptake of I¹³¹ was not significantly altered by the time of application of the Hucker crystal violet staining solution (15-180 sec), or of the Gram's iodine (NaI¹³¹) solution (30-120 sec) or by the duration of the alcohol decolorization process (30-120 sec).

Variations (herein referred to as variations 2 and 3) of the basic procedure were carried out in which the primary staining solution contained crystal violet combined with NaI¹³¹ or Gram's iodine solution (NaI¹³¹). In variations 4 and 5 the effect of the order of application of the various staining reagents was investigated. In these variations (2-5) all test-bacteria were stained Gram negative. The initial uptake of I¹³¹ was decreased, though in variations 4 and 5 the percent retention of I¹³¹ was increased. In the staining of bacterial spores by different methods (variation 6), it was noted that the initial uptake and percent retention of I¹³¹ was greater than with the vegetative forms. When ovalbumin was stained by the Hucker technic and variations thereof, it was noted that the initial uptake of I¹³¹ was directly related to the protein (ovalbumin) concentration up to an ovalbumin concentration of 1%.     

Basic Fuchsin-Ferric Chloride: a Simplification of Weigert's Resorcin-Fuchsin Stain for Elastic Fibres

Weigert's resorcin-fuchsin stain for elastic fibres can be simplified by the omission of the resorcinol. The resulting “basic fuchsin-ferric chloride” gives results indistinguishable from those achieved with resorcin-fuchsin on tissues fixed in Bouin, Carnoy, Gendre, neutral formah, Susa or Zenker solutions. Gel filtration chroma tography on Sephadex LH20 showed that the staining components of basic fuchsin-ferric chloride had a high molecular weight, so selective staining of elastic fibres by this method may well be due to van der Waals attractions.     

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.     

A Selective Stain for Elastic Tissue (Orcinol-New Fuchsin)

A selective stain for elastic tissue (designated orcinol-new fuchsin) is described. Two grams of new fuchsin (C.I. No. 678) and 4 gm of orcinol (highest purity) are added to 200 ml of distilled water and the solution boiled for 5 min. Then 25 ml ferric chloride solution (U.S.P. IX) are added and the solution is boiled 5 min longer. The precipitate is collected and dissolved in 100 ml 95% ethanol. This is the staining solution. Sections are deparaffinized and brought to absolute ethanol, stained for 15 min at 37 °C with orcinol-new fuchsin, differentiated for 15 min in 70% ethanol, dehydrated, cleared and covered as usual.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

Elastic Tissue Staining

It has been found that the addition of dextrin to samples of crystal violet and basic fuchsin employed in the prepararation of the elastic tissue stain after the technic of Weigert makes more sure a satisfactory final product. A modification of the original Weigert technic employing crystal violet or a mixture of crystal violet and basic fuchsin is offered as providing a better color contrast both visually as well as photographically. Crystal violet alone affords a bright greenish-yellow elastin while the addition of basic fuchsin results in a darker stain shading into dark blue as the proportion of basic fuchsin is increased.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

Studies in Gram Staining

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuchsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal viokt-stained organisms with alcoholic safranin (0.25%) for 15 scc will distinguish Gram-positive bacteria (viokt) from Gram-negative bacteria (pink).

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily dccolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet

ABSTRACT. We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca²⁺ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca²⁺ uptake by digitonin-permeabilired epimastigotes. Crystal violet also induced Ca²⁺ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca²⁺ uptake and the (Ca²⁺-Mg²⁺)ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca²⁺ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.     

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.     

The demonstration of two acid groups in juxtaglomerular granules with basic triphenyl methane dyes,aldehyde-fuchsin and schiff's reagent

Summary Oxidation and bromination of mouse kidney JG cell-granules result in the production of cysteic acid from cystine; cysteic acid is capable of taking up rapidly and selectively certain basic triphenyl methane dyes including aldehyde fuchsin at lower pH levels.After treatment with periodic acid, bromine and hydrochloric acid, the JG granules or the nuclear chromatin also take up the basic triphenyl methane dyes (including aldehyde fuchsin) which contain amino groups, probable as a result of the production of aldehyde groups. Basic triphenyl methane lacking amino groups does not react with aldehydes.Some substance present in JG granules could be stained by aldehyde fuchsin after prior oxidation; HCl methyl violet 2B was taken up both with or without prior oxidation. Only strong methylation completely abolished these affinities which were restored after demethylation. These reactions are attributed to cystine.The staining of JG granules with dilute aldehyde fuchsin and dilute methyl violet 2B is not affected by oxidation, bromination, aldehyde blocking and hydrolysis; these reactions are abolished by mild methylation, but restored by subsequent saponification. These staining properties are due to the presence of carboxylic acid in JG granules.The positive PAS reaction of JG granules is due to the presence of 1.2-glycol in the same granules.     

A methanol resorcin-fuchsin stain for elastic tissues and nuclei.

The staining properties of conventional ethanol resorcin-fuchsin and of methanol resorcin-fuchsin were compared. Formula; Dissolve 0.2 g of commercial resorcin-fuschin in 70 ml of methanol or ethanol, add 30 ml of water and 1 m1 of concentrated HC1; stain sections for 4 hours. Both solutions colored elastic and pseudoelastic fibers, cartilage and some mucins. Methanol resorcin-fuchsin also colored nuclei in methacarn- (methanol-chloroform-glacial acetic acid 6:3:1) and formalin-fixed tissues; this nuclear stain withstood counterstaining with picro-dye mictures. Zenker-fixed sections showed diffuse coloration with little or no contrast between nuclei and cytoplasm. Extraction with hot trichloracetic acid abolished binding of methylene blue, but binding of methanol resorcin-fuchsin by nuclei remained unaltered or was enhanced. Experiments with solvents containing various concentrations of methanol, ethanol or isopropanol indicated that the staining patterns of resorcin-fuchsin are determined by the nature and concentration of the alcohol. Methanol resorcin-fuchsin proved useful for simultaneous visualization of elastic tissues and nuclei.     

Fixative-Stain Sequences for Selective Demonstration of Juxtaglomerular Cells

The effects of 31 fixatives, containing alcohol, acids, formalin and metallic salts, and representing many of the standard fixatives, were observed for selectivity and intensity of staining of juxtaglomerular granules in mouse kidney. Four staining methods: 1:400,000 aqueous methyl violet 2B; Bowie's ethyl violet-Biebrich scarlet; 1:200,000 aldehyde fuchsin; and periodic acid-Schiff were used. Fixatives containing HgCl₂, trichloroacetic acid or formalin were found to be the most satisfactory for subsequent staining of the granules.     

细菌脱色酶TpmD对三苯基甲烷类染料脱色的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行有效脱色的脱色酶,命名为TpmD。该酶的亚基分子量为29.4kDa,等电点为5.6。该酶催化上述4种三苯基甲烷类染料脱色反应的适合温度为40~60℃,适合pH范围为5.5~9.0。动力学参数测定结果显示TpmD对结晶紫、碱性品红、灿烂绿及孔雀绿的Km值分别为24.3、40.65、4.2、68.5μmol-1.L-1,Vmax值分别为19.6、74.1、82.8、115.6μmol.L-1.s-1。结晶紫为该酶的最适反应底物。TpmD催化的脱色反应依懒于NADH/NADPH及分子氧的存在,显示该酶属于NADH/NADPH依赖型的氧化酶类。这是国内外首次关于细菌中三苯基甲烷类染料脱色酶酶学性质的描述。     

Standardization of the Feulgen-Schiff technique

Summary Four fuchsin analogues (Pararosaniline, Rosaniline, Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches.Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 m.Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

New rapid technique for counting microorganisms directly on membrane filters

The proposed technique is a modification of classical procedures for counting micoorganisms directly on membrane filters. The technique consists of clearing the filter with immersion oil, paraffin oil or cedar oil prior to staining with crystal violet, carbol fuchsin or malachite green. Millipore filters (0.1 micron pore size, VC type) were found to be superior to other filters with regard to the contrast between microorganisms and filter surface.     

Basic Fuchsin as A Nuclear Stain

Five distinct nuclear stains and staining procedures which utilize basic fuchsin as the dye have been studied, compared and tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi.

Aqueous basic fuchsin has been shown to be an excellent, though impermanent, stain with which to study the nuclei of this and other fungi. The conditions under which formaldehyde acts as a mordant for basic fuchsin and produces a permanent nuclear stain have been established.

Comparison of crystal violet and basic fuchsin suggests that the mordanting action of the aldehyde operates through the para-amino groups of the dye. Certain other basic dyes were not mordanted by formaldehyde.

Gentle acid hydrolysis of the tissues has been found to be essential both to the specificity of the dye as a nuclear stain and to the mordanting effect of the aldehyde.

The possible relationship of these observations to the Feulgen reaction is discussed. A protocol for the method developed is presented.     

Dye Complexes Produced in Gram Staining

Complexes of crystal violet and iodine containing (a) one molecule and four atoms, and (b) one molecule and two atoms respectively, were prepared and their solubility in alcohol (95%) determined. Both complexes were only slightly soluble in alcohol, the former being less soluble than the latter. The solubility decreased with decreasing concentration of alcohol, and also on storage. Complexes of malachite green, basic fuchsin and Victoria blue B with iodine were also prepared, and complexes of each dye with picric acid. Two complexes of different composition could be obtained from crystal violet and picric acid, and from Victoria blue and iodine. Complexes with basic fuchsin were much more soluble in alcohol (95%) than complexes with the other dyes tested. Dye-mordant complexes have some of the properties of organic charge-transfer complexes.