Thin Layer Chromatography of Certain Preformed Metal Complex Dyes Used in Biological Staining

Thin-layer chromatographic systems are described for the analysis of various preformed metal complex dyes (aluminon-chromium(III), carminic acid-aluminum, carminic acid-chromium(III), carminic acid-iron(III), oelestine blue-chromium(III), gallamine blue-chromium(III), gallocyanin-chromium(III), hematein-aluminum, hematein-chromium(III), purpurin-aluminum) and their parent dyes. Certain of there dyes have also been analysed by agar-gel electrophoresis or gel-filtration chromatography. The merits of the three analytical methods are discussed.     

Detection of Griseofulvin and Dechlorogriseofulvin by Thin-Layer Chromatography and Gas-Liquid Chromatography

A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

The History of Staining Logwood Dyes

Except for the cochineal derivatives, logwood extract was the first of the important modern stains to be employed in histology. Certain other natural dyes, such as madder and indigo, had been used earlier, but they are of little significance in discussing the history of staining, because none of them nor even alizarin, the derivative of madder, are of any appreciable significance in these days of synthetic dyes. Hematoxylin, on the other hand, still continues a very important stain, and it has played an interesting part in the history of staining.     

The History of Staining Logwood Dyes

Except for the cochineal derivatives, logwood extract was the first of the important modern stains to be employed in histology. Certain other natural dyes, such as madder and indigo, had been used earlier, but they are of little significance in discussing the history of staining, because none of them nor even alizarin, the derivative of madder, are of any appreciable significance in these days of synthetic dyes. Hematoxylin, on the other hand, still continues a very important stain, and it has played an interesting part in the history of staining.     

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.     

Simplified Approach to Identification of Aerobic Actinomycetes by Thin-Layer Chromatography

A system has been developed for the identification of aerobic actinomycetes in the clinical laboratory based on analysis of whole cells for diaminopimelic acid and carbohydrates and on the ability of the organism to decompose casein, tyrosine, and xanthine media. The whole-cell analyses were performed by a simple thin-layer chromatographic procedure that is described. Eighteen reference cultures were correctly identified and, subsequently, 35 isolates from clinical material were grouped by using this system. The method is well suited for use in routine clinical laboratories.     

Studies on Textile Dyes for Biological Staining II. A Trichrome Stain for General Use

This trichrome staining procedure differentially stains elastic fibers, collagen fibers and mucin. Gomori's aldehyde-fuchsin is used for elastic fibers; fast yellow TN is the component used for collagen and cytoplasm; pontacyl blue black SX is the nuclear stain. Procedure: Paraffin sections to water; aldehyde-fuchsin, 30 min; 70% ethanol; distilled water; 0.75% pontacyl blue black SX in 1.5% K.₂Cr₂O₇, 15 min; tap water; 70% ethanol to wash off all free dye; 2% fast yellow TN in 95% ethanol, 5 min; dehydrate, clear and cover.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Selective Staining of Endocrine Cells by Basic Dyes After Acid Hydrolysis

Staining of tissue sections by basic dyes after immersion in hot hydrochloric acid (0.2 N for 3-10 hr at 60 C) provides a means for selective detection of many endocrine cells. The acid hydrolysis suppresses diffuse basophilia, mainly due to RNA, DNA and acid polysaccharides, and increases the basophilia of secretory granules in endocrine cells, due, at least in part, to the proteins they store. After such treatment, toluidine blue or azur A (0.01-0.005% in 0.02 M McIlvaine buffer, pH 5) or pseudoisocyanin (0.02% in distilled water) heavily stain A and D cells of pancreatic islets, enterochromaffin and nonenterochromaffin endocrine cells of the gastrointestinal mucosa, thyroid parafollicular or C cells, pituitary basophil cells and adrenalin-secreting cells of the adrenal medulla.     

Chromatography and Biological Stains: III. Comparison of the Fat Staining Efficiency of Fractions of Commercial Sudan III Separated by Column Chromatography

Fractions of commercial Sudan III which were separated by column chromatography were compared for fat staining efficiency. Paraffin sections of chromated mouse liver tissue and sections of both fresh and formalin-fixed rat liver tissue or Musca domestica larvae, cut with the freezing microtome, were used. Evidence is presented that a sample of very highly purified Sudan III has no ability to render a fat stain in fresh, formalin-fixed or chromated tissue. However, certain other fractions from the commercial sample, some completely devoid of Sudan III, had good staining characteristics. It is concluded that some substance or substances, other than Sudan III, is responsible for the staining action of the commercial dye.     

Characterization of Petroleum-Contaminated Soils by Thin-Layer Chromatography with Flame Ionization Detection

Petroleum hydrocarbons from 20 soils from refineries or other industrial sites were extracted with a mixture of chloroform and methanol (1:1, v/v), and the extracts were analyzed by thin layer chromatography with flame ionization detection (TLC/FID). The TLC/FID procedure has been used widely in biological and medical research but generally has been underutilized in environmental chemistry. The analysis method involved spotting a small volume of sample extract (typically 1 to 3?µl) on ten silica-coated quartz rods, and chromatographically separating constituents in the spots using solvent systems of increasing polarities (hexane, toluene, and dichloromethane + methanol). We achieved complete separation of saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes from the hydrocarbon-contaminated soils with this method. Analysis of the separated constituents by TLC/FID also allowed quantification of aromatic and aliphatic hydrocarbons without interference from soil biogenic lipids. A simplified version of the method permitted excellent separation of aliphatics +aromatics (forming a single peak) from resins and asphaltenes. The procedure is rapid (complete analysis of ten samples in about 1?h after extraction). Thus, the method seems well suited for synoptic surveys or screening and characterizing numerous samples prior to using more detailed and costly analyses.     

Separation and Purification of Monogalactosyl and Digalactosyl Diglycerides by Thin-Layer Chromatography

Six kinds of glycolipid of monogalactosyl and digalactosyl diglyceride were separated and purified from the wheat and the natural latex of chilling-tolerant and chilling-sensitive variety by two kind solvent systems and twice one-dimensional thin-layer chromatography. Besides, the compositions of fatty acid of hydrophobic side chain of six kinds of glycolipid were compared. The indices of unsaturation of fatty acid of hydrophobic side chains in the glycolipids of wheat flour, in the glycolipids of natural latex of chilling-tolerant, variety and in the digalactosyl diglycerides, are much bigger than those, respectively, in the glycolipids of natural latexes, in the glycolipids of chilling-sensitive variety and in the monogalactosyl diglycerides.     

Detection of Arginine Dihydrolase in Nonfermentative Gram-Negative Bacteria by Use of Thin-Layer Chromatography

Thin-layer chromatography was used to detect the presence of the arginine dihydrolase system in nonfermentative gram-negative bacteria. The test was positive for the fluorescent pseudomonads as well as Pseudomonas maltophilia, whereas other Pseudomonas sp., Mima, and Herellea were negative. This procedure can be completed in a few hours and may be useful in the clinical laboratory.     

藏药翼首草薄层鉴别方法研究

本文对藏药翼首草薄层鉴别方法进行系统考察。试验以熊果酸为对照品,采用单因素试验方法,对影响翼首草薄层色谱的因素进行了考察,筛选最佳薄层鉴别方法。结果采用硅胶G板,薄层板置展开缸中饱和10min,以10%硫酸乙醇溶液为显色剂,105℃加热至斑点清晰,置日光和紫外光灯(365 nm)下检视,所得的薄层色谱分离效果较好。研究结果表明本法分离效果好、重复性好,可用于藏药翼首草的质量控制。     

A Histochemical Examination of the Staining of Kainate-Induced Neuronal Degeneration by Anionic Dyes

Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy-or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)₂ method; all staining was blocked by benzil but was relatively refractory to deamination by HNO₂. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainite-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.     

Tissue Staining by Disazo Dyes, Teratogenicity and the Haemoglobin-Nitrite Sensitivity Reaction

Five disazo dyes related to trypan blue but differing in molecular structure by substitution, replacement or addition of radicals have been tested for their action in increasing the sensitivity of haemoglobin to oxidation by nitrites. None has been found to be as active as trypan blue itself. The activity of these dyes is not related to their ability to cause generalised tissue staining in vivo. Four dyes of known redox potential, neutral red, phenol-indophenol, phenol blue and janus green, have also been tested for their action on the haemoglobin-nitrite sensitivity reaction. Their activity is not related to their redox potential.