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.     

Chemical Structures and Staining Mechanisms of Weigert'S Resorcin-Fuchsin and Related Elastic Fiber Stains

Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsia, 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 canonic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachlorofemte anion (FeCI₄^-) 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, die observed staining of elastic fibers is only as expected.     

Fluorescence in Thelohania apodemi Doby, Jeannès and Rault, 1963, a microsporidian parasite of the brain of the fieldmouse, Apodemus sylvaticus]

Colonies of Thelohania apodemi in fresh preparations of the brain of the field mouse show an intense fluorescence with ultra violet radiation. This phenomenon is undoubtedly due to the presence of chitin in the spore envelope. It is to our knowledge the first time that such a phenomenon of fluorescence has been described in the Microsporida.     

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.     

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.     

Characterization of the photoactive GAF domain of the CikA homolog (SyCikA, Slr1969) of the cyanobacterium Synechocystis sp. PCC 6803

Phytochromes and bacteriophytochromes in plants and some species of bacteria, respectively, are photoreceptors that bind linear tetrapyrroles and can respond to red and far-red light signals in a reversible manner. A related but distinct photoreceptor candidate, CikA (denoted ScCikA), has been reported to reset the circadian clock in the cyanobacterium Synechococcus elongatus PCC 7942 after a dark pulse. However, recent studies have indicated that ScCikA does not function as a photoreceptor but as a redox sensor. Moreover, the Cys residue that covalently ligates the chromophore in phytochromes is not conserved in the ScCikA protein. On the other hand, the CikA homolog in Synechocystis sp. PCC 6803 (Slr1969, denoted SyCikA) retains this conserved Cys residue. In our present study, we have isolated the putative chromophore-binding GAF domain of SyCikA from Synechocystis and phycocyanobilin-producing Escherichia coli. Absorption spectra of both preparations showed two peaks in the UV and violet regions. Irradiation of these proteins with violet light yielded a broad peak in a yellow region at the expense of the peaks in the UV and violet regions. Interestingly, successive irradiation with yellow light did not revert these absorption spectra but a partial dark reversion to the original form was detected. These results suggest that SyCikA may function as a violet light sensor in Synechocystis.     

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.     

Demonstration of Lipofuscin and Nissl Bodies in Crystal Violet Stained Sections Using a Fluorescence Technique or Pyronin Y Stain

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures—Nissl bodies and lipofuscin—can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.     

Directed screening of bacteria--destructive agents of surface-active substances

A procedure for detection of bacteria destructing cationic surface active substances (CSAS) was developed. The procedure is based on changing colour of pyrocatechin violet, an organic dye from geen-blue in the presence of a CSAS to red-brown in its absence. After application of pyrocatechin violet to bacterial macrocolonies grown on a synthetic medium with a CSAS as the only source of C red-brown zones developed around the colonies which was probably indicative of the CSAS biodestruction. 55 strains of bacteria destructing undecylpyridinium bromide, a CSAS were isolated from water of sewage purification plants. The study of their spectrum with respect to CSASs of different chemical structure revealed 6 bacterial strains capable of destructing all the nine test preparations. It was shown that biodestruction of the CSASs was associated with the long carbon chain and depended on the presence of the substitute in the aromatic ring and branching in the side chain. Therefore, the procedure provides screening of microbes destructing CSASs and may be used for rapid determination of biodestruction of CSASs with different chemical structures.     

A new species of Leopoldia (Asparagaceae Scilloideae formerly Hyacinthaceae) from Iran

Leopoldia tabriziana Jafari (Hyacinthaceae) from northwest of Iran (East Azarbaijan province) is described here and illustrated by a line‐drawing. It is similar to L. comosa Parl . but differs in having violet (not cream) pedicels in the fresh fertile flowers, oblong‐campanulate (not oblong‐urceolate) fertile flowers, triangular‐obovate (not globose‐obovate) sterile flowers, dark violet (not beige) fertile flowers and dark violet (not pale violet) sterile flowers with much shorter pedicels. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The composition and structure of isolated chromosomes

1. The preparation of isolated chromosomes from liver, kidney, and pancreas has been described. 2. It has been shown that there is no gross cytoplasmic contamination in these preparations. 3. In a microscopic study of isolated chromosomes the same chromosomes have been found in different tissues of the same organism. Since individuality is one of the main characteristics of chromosomes, there can be little doubt that the preparations do, in fact, contain isolated chromosomes. 4. A quantitative study of staining with crystal violet shows that this basic dye competes with histone for the phosphoric acid groups of the DNA in chromosomes. The displacement of histone by protamine has been demonstrated. 5. Preparation of histone-free chromosomes has been described. Removal of histone does not affect the microscopic appearance of chromosomes. 6. The non-histone or residual protein has been prepared from histone-free chromosomes. The quantity of residual protein in a preparation of chromosomes is correlated with the amount of cytoplasm in the cells from which the chromosomes were prepared. 7. The microscopic appearance of chromosomes depends upon the association of DNA with residual protein. 8. Evidence has been given that in a chromosome there are two DNA-containing nucleoproteins; in one DNA is combined with histone, and in the other it is combined with residual protein.     

Shell colour variation in Bullia digitalis, a sand-dwelling, intertidal whelk (Gastropoda: Prosobranchia)

Bullia digitalis is an intertidal whelk that lives on sandy beaches in South Africa. It is highly variable in shell colour, with individuals varying from white to dark brown. This paper describes shell colour variation of B. digitalis at seven sites, along a 230 km coastline east of the Cape Peninsula. Seven colour forms were found: striped, violet, banded violet, banded brown, orange, pale yellow and white. These forms are probably genetically determined morphs. The striped form is the most common at all sites, constituting 53–62% of each sample. The violet is the second most common morph. Its frequencies are remarkably stable at 15–17%. The striped form blends well into the sandy environment and may therefore be of considerable cryptic value in concealing B. digitalis from predators. The violet form is highly conspicuous. Its stable frequency throughout the study area may represent a genetic balance that is not relevant to any visual advantages of the violet colour.     

Neuron Staining by Basic Dyes Versus Basic Metal-Dye Complexes; Differences Shown by Histochemical Blocking Reactions

Various blocking procedures were applied to sections of paraffin-embedded, formalin-fixed cat spinal cord. Treated sections and untreated controls were stained with cresyl violet acetate or gallocyanine-chrome alum. Although both dyes have been said to stain by simple salt formation it was found that staining was affected differently for each dye by the blocking procedures, and also that staining of neuron nuclei differed in the controls. In these, the cresyl violet acetate stained only the nucleoli within the nucleoplasm whereas gallocyanine-chrome alum stained much more material of unknown composition and function. It is proposed that if cresyl violet acetate and other basic dyes stain by salt linkage, and can be specific for nucleic acid and other highly acid materials, then gallocyanine and other basic metal dye complexes can not be specific for nucleic acid and do not stain by a simple salt linkage.     

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.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

Spectrophotometric determination of folic acid in pharmaceutical preparations by coupling reactions with iminodibenzyl or 3-aminophenol or sodium molybdate-pyrocatechol

Novel coupling reagents are used for the simple and sensitive spectrophotometric determination of folic acid either in pure form or in its pharmaceutical preparations. The methods are based on the probable diazotization of the p-aminobenzoylglutamic acid obtained after reductive clevage of folic acid, followed by either coupling with iminodibenzyl to give a violet product with lambda(max) of 580nm or coupling with 3-aminophenol to produce an orange yellow-colored product with lambda(max) of 460nm. Sodium molybdate and pyrocatechol are used in the third method and the pale red-colored product formed has a lambda(max) of 490nm. The methods are highly reproducible and have been applied to the determination of folic acid in tablets and the results compare favorably with the official method. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

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.     

Inhibition of Protein Synthesis and Amino Acid Transport by Crystal Violet in Trypanosoma cruzi

ABSTRACT. [³⁵S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [³⁵S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [³H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.     

Phenotypic plasticity in Passiflora suberosa L.(Passifloraceae): induction and reversion of two morphs by variation in light intensity.

Leaf morphology may vary considerably even within a branch of Passiflora suberosa plants. Leaves are of a typical green type in shaded areas, but in open fields turn into violet, and apparently have greater thickness and trichome density. The proximate causes and the adaptive meaning, if any, for the existence of the violet morph are still unknown. By cultivating P. suberosa clones under two light regimes (total and partial exposure to sunlight), we consecutively induced (first year) and then reversed (second year) the appearance of the violet morph. We evaluated the corresponding changes in morpho-anatomic and chemical leaf characteristics. Plants that were grown under partial sunlight had a greater size and did not alter their green color, but those grown under total sunlight changed into violet, were smaller in size and their leaves were tougher, thicker, and had a greater number of trichomes. The violet morph had increased anthocyanins and phenolic derivatives. It also showed cellular hypertrophy, a greater number of cell layers in the mesophyll, and a lignified pericycle. Since these morphs are interchangeable by changing light conditions, we inferred that they are not determined by genotypic diversity, but are mainly a result of a physiological response to light stress, and thus part of P. suberosa phenotypic plasticity.