Polychromatic Stains for Thin Sections of Beta Embedded in Epoxy Resin

Thin sections of leaves and anthers of Beta vulgaris L., fixed in glutaraldehyde-OsO₄ and embedded in epoxy resin, were stained with different stains at pH ranges from 5 to 9 at 50 C to select those that provided polychromatic staining of suitable intensity. The thionin derivatives, Azure B, Toluidine Blue O, and polychrome Methylene Blue provided adequate staining, as did the commercially prepared stain Paragon PS 1301. Azure B stain was superior for sugar beet 0.5μ monitor sections: cytoplasm appeared grey; nuclei, blue-gray; nucleoli, blue; chloroplasts, blue-green; primary walls, blue; and secondary walls, light blue. Choice of one of the stains mentioned probably would depend upon the plant material under study.     

Microspectrofluorometry of ultraviolet-inducible fluorescence in supravitally-stained ascites tumour cells

Synopsis Ultraviolet irradiation of tumour cells (Ehrlich tetraploid ascites tumour of mice, TO strain), supravitally stained with thiazine dyes (Azure II, Azure A, Methylene Blue, Toluidine Blue) or an oxazine dye (Brilliant Cresyl Blue), induces blue fluorescence in cytoplasmic bodies believed to be lipid droplets or lysosome-like bodies. Microspectrofluorometry of the inducible fluorescence in Ehrlich tumour cells gives bimodal excitation (340/394 nm) and emission (443/700 nm) curves.     

The composition of stains produced by the oxidation of Methylene Blue

Synopsis The composition of some stains produced by the oxidation of Methylene Blue has been studied by thin-layer chromatography. Various named methods for the production of Polychrome Methylene Blue, Azure A, Azure B, Azure C and Methylene Violet Bernthsen have been found to give complex mixtures of varying proporitions of up to eleven dyes. Ten of these, namely Methylene Blue, Azure B, Azure A,sym-Dimethylthionine, Azure C, Thionine, Methylene Violet Bernthsen, Methyl Thionoline, Thionoline and Thionol, have been identified by their visible absorption spectra. The remaining dye could not be identified. When used on a laboratory scale, these methods give stains of constant composition independent of the batch of Methylene Blue. Stain composition as revealed in the present study has been compared with that previously indicated by other, less effective, analytical techniques. Reasons are presented why the latter give equivocal results.     

A puzzling feature of Colloidal Iron positive and Alcian Blue negative material

Summary A puzzling feature of Colloidal Iron positive and Alcian Blue negative substance is encountered in yolk sac of young larvae of a fish —Tilapia mossambica. This yolk material is PAS positive (proved to be due to neutral mucopolysaccharide) and negative to Toluidine Blue, Azure A (pH 2 to 4.5), Aldehyde Fuchsin and AB pH 1. More work is necessary to establish the exact chemical nature of the CI positive material.     

Cytotoxicity, radiosensitization, and DNA interaction of platinum complexes of thiazin and xanthene dyes

Complexes of the platinum(II) tetrachlorodianion with positively charged nuclear dyes have been prepared in an effort to produce neutral molecules which could gain ready access to the nuclear DNA where the platinum(II) tetrachlorodianion could function as a radiosensitizing and a bifunctional alkylating agent. The thiazin dyes Thionin, Azure B, and Methylene Blue, the aminoxanthene dye Pyronin Y, and the thiazole dye Thioflavin have each been complexed to the platinum(II) tetrachlorodianion(PtCl4) in a ratio of 2:1(dye:PtCl4). Studies of the interaction of these complexes and of the dyes with the pBR322 plasmid superhelical DNA demonstrated that while each complex and dye readily associated with the DNA in a dose-dependent manner, only Pt(Thioflavin)2 and Thioflavin produced irreversible DNA changes (single-strand breaks). In exponentially growing EMT6 cells the cytotoxicity of these drugs was assessed in normally oxygenated and hypoxic cells at both pH 7.4 and 6.45. At concentrations ranging from 1 to 500 microM, Pt(Methylene Blue)2 was significantly more cytotoxic than the other thiazin dye complexes Pt(Thionin)2 and Pt(Azure B)2. The cytotoxicity of Pt(Thionin)2 and Pt(Methylene Blue)2 was increased in normally oxygenated and hypoxic cells at low pH. Both Pt(Pyronin Y)2 and Pt(Thioflavin)2 were more toxic than the thiazin complexes. Pt(Pyronin Y)2 was most cytotoxic to normally oxygenated cells at normal pH and hypoxic cells at low pH, while Pt(Thioflavin)2 was most cytotoxic to cells at low pH under both oxygenation conditions. In vitro studies of the radiosensitizing properties of these agents in EMT6 cells demonstrated that exposure to 100 microM for 1 h before and during irradiation (except for Pt[Thioflavin]2, which was assayed at 25 microM) resulted in enhancement rations of 2.5, 1.9, 1.5, and 1.5 for Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y)2, and Pt(Thioflavin)2, respectively, in hypoxic cells. In contrast, Pt(Methylene Blue)2 (and Methylene Blue) proved to be a radioprotector of normally oxygenated cells and did not sensitize hypoxic cells to the cytotoxic effects of radiation. In the FSaIIC fibrosarcoma in vivo administration of each drug at 100 mg/kg intraperitoneally (ip) 15 min prior to irradiation (except for Pt[Thioflavin]2, which was given at 1 mg/kg ip) showed that, with single radiation fractions of 10 and 20 Gy, dose-modifying factors of 2.1, 1.8, 1.5, and 1.2 were produced by Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y), and Pt(Methylene Blue)2, respectively, after correcting for growth delays induced by the drug alone. In comparison, misonidazole at 1 g/kg ip produced a dose-modifying factor of 1.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

NILE BLUE, ANILINE BLUE AND NEUTRAL RED AS INDICATORS OF LIPOLYSIS

SUMMARY: Dyes which have been used to detect lipolysis (Nile Blue, Aniline Blue and Neutral Red) and others (Methylene Blue, Toluidine Blue and Thionin) were critically examined for their inhibitory effects in both liquid and solid media. It was confirmed that Nile Blue would inhibit the Gram-positive bacteria tested, even at 2 × 10⁻⁵. Gram-negative bacteria were not inhibited at 1 × 10⁻⁴ and the colour change in fat media was good. Aniline Blue and Neutral Red did not inhibit Gram-positive organisms at 1 × 10⁻⁴ and the colour change was moderately good. The other three dyes were not toxic at 2·5 × 10⁻⁵, but there was no colour change. When butter fat saturated with the precipitated bases of Nile Blue, Basic Fuchsin, Crystal Violet or Malachite Green was incorporated in peptone-Yeastrel agar, the resulting media were toxic to Gram-positive bacteria. Fat saturated with the bases of Aniline Blue or Neutral Red permitted growth, but the colour change with lipolytic species was not striking, being one of intensity only. Satisfactory growth and colour changes were obtained using butter fat stained with the inert dye Waxolene Green in medium containing Neutral Red in the aqueous phase, and with butter fat stained with the oxazine base of Nile Blue in agar containing Aniline Blue.     

Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.     

On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of Azures B-Eosin Y stain as compared with other thiazine dye-Eosin Y combinations

Summary After incorporation into a polyacrylamide matrix, the biopolymers DNA, RNA, heparin, hyaluronic acid, collagen and the synthetic polymers poly(U) and poly(A, U) were stained with the pure thiazine dyes, Methylene Blue, the Azures and Thionin alone and combined with Eosin Y. Satisfactory spectrophotometric agreement was obtained between the staining reactions of the biopolymers in the artificial matrix and those in their natural surroundings. This was especially true with respect to the specificity of the Azure B-Eosin Y dye-pair, which is based on the generation, on suitable substrates, of a purple colour, the Romanowsky-Giemsa effect (RGE), with an absorbance maximum near 550 nm. In the model experiments, DNA, heparin, hyaluronic acid and collagen were found to be RGE-positive and poly(U), poly(A, U) and RNA RGE-negative.A theory of RGE is proposed which complies with the new and earlier observations: after saturation of available anionic binding sites and aggregate formation by Azure B, electron donor acceptor complexes are formed between Eosin Y and Azure B via hydrogen-bridge formation of the aminosubstituent proton of Azure B and between Eosin Y and the biopolymer surface. Charge-transfer complex formation may also account for the qualitative identity of Azure B-Eosin Y and Azure A-Eosin Y spectra of substrates, which are coloured purple. Quantitatively, Azure A-Eosin Y is less efficient in giving RGE. The generation of RGE is time-dependent. Equilibrium staining is attained after about 120 h. The implications of the results for the biological application of Romanowsky-Giemsa staining are discussed briefly.     

Histochemical evidence for lipoidal material in secretory granules of rat salivary glands

Synposis The granules of parotid acinar cells and submandibular granular tubule cells of rats contain one or more periodic acid-Schiff positive substances that are extracted during fixation with lipid solvents or acidic solutions or if frozen sections are stained in aqueous solutions. The granules in these cells can be stained by Schmorl's reaction, Luxol Fast Blue and a permanganate-Aldehyde Fuchsin sequence. The results obtained with these stains after a variety of fixation procedures strongly suggest that the secretory granules of these two cell types contain several components and that in parotid acinar and submandibular granular tubule cells, at least one of these components is a lipoidal substance.     

The Giemsa-11 technique for species-specific chromosome differentiation

Summary Oxidizing Methylene Blue and adding the reaction products to Eosin Y and Azure B makes possible a highly reliable Giemsa-11 technique for discrimination of chromosomes in hybrid cells according to their parental origin. This staining can be combined in a sequential procedure with a fluorescent banding technique allowing the exact identification of the chromosomes.     

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

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 microns. 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.     

Mechanism of Intranuclear Crystal Formation of Herpes Simplex Virus as Revealed by the Negative Staining of Thin Sections

Structural alterations induced in HeLa cells by herpes simplex virus and the mechanism whereby the virus is formed in the nucleus in crystal arrays were studied by electron microscopy with both the usual and negatively stained sections. Aggregates of granular and filamentous material were observed in the cytoplasm of infected cells with both sections. On the other hand, no remarkable alterations in appearance of the cytoplasmic ground substance were observed with the usual sections of infected cells. However, the cytoplasmic ground substance of infected cells when negatively stained consisted of granular material which was different in appearance from the spongy material constituting the cytoplasmic matrix of uninfected cells. In the nucleus of infected cells, complexes consisting of round bodies, amorphous material, aggregates of uniform granules in rows, and viral crystals were often observed near the nuclear membrane in both types of sections. Examinations of the granular aggregates with negatively stained sections suggested that each granule represents a subunit and that the several adjoining subunits (approximately eight) constitute the requirement for formation of a single viral capsid with a core. Thus, rapid and simultaneous formation of the core and capsid within the aggregate would replace the rows of the granules with the viral crystal. The advantages of negative staining of thin sections for visualization of fine structural alterations are discussed.     

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.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.     

A mechanistic study of the histochemical reactions between aldehydes and Basic Fuchsin in acid alcohol used as a simplified substitute for Schiff's reagent

Synopsis For the identification of polysaccharides after periodic acid oxidation or of DNA after acid hydrolysis, a solution of 0.5% w/v Basic Fuchsin in acid alcohol (water-ethanol-concentrated hydrochloric acid 80:20:1 by volume) may be used instead of Schiff's reagent. Sections are stained in the Fuchsin solution for 20 min, after which the unreacted dye is washed off with ethanol. Except for its yellower colour the Fuchsin staining is almost indistinguishable from Schiff's reagent staining.Histochemical blocking studies indicated that the Fuchsin stain, like Schiff's reagent, reacts with aldehyde groups or subsequent oxidation products. The results of studies of model systems (cellulose film oxidized by periodic acid and also of aqueous formaldehyde solution) in which infra-red spectroscopy and, where appropriate, chromatography were used are consistent with the initial coloured products being azomethines which may react further to produce coloured secondary amine derivatives.     

An improved method for the demonstration of fibrin in biopsies of rabbit skin homografts

Summary Tissues embedded in resin are convenient for routine use when the presence or absence of fibrin in them is to be confirmed using the electron microscope. To visualize fibrin using the light microscope, sections (1.0–2.0 m) from such specimens should be stained with Methylene Blue-Azure II-Basic Fuchsin (MBBF). Staining with MBBF is more controllable than with other methods and it requires only two short staining steps. Compared with Giemsa, MBBF provides a polychromatic, as opposed to a monochromatic end-result, sharply contrasting fibrin (blue) against collagen (pink-violet).     

The cytochemical localization of lysozyme in Paneth cell granules

Synopsis The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue is so strong that any other alcianophilia (e.g. of acid mucosubstances in goblet cells) can be removed selectively by washing withN-cetylpyridinium chloride and counterstaining with Basic Fuchsin and Nuclear Fast Red.     

Quantitative microspectrophotometry of RNA in plant tissue

Synopsis Chemical estimation of nucleic acid, essentially RNA, in fixed tissue from Jerusalem artichoke tubers, coupled with an examination of the types of RNA in the fixed tissue by gel electrophoresis, demonstrates that ribosomal and soluble RNA are preserved in this tissue after various fixation procedures including methanol, ethanol-acetic acid and aqueous formaldehyde. Tissue fixed in ethanol-acetic acid or formaldehyde is resistant to loss of nucleic acid by aqueous extraction but tissue fixed in all three standard fixatives loses nucleic acid in citrate buffer under conditions used for Azure B staining. The presence of Azure B in the buffer does not wholly prevent this loss. Tissue fixed in formaldehyde or mixed fixatives containing formaldehyde is resistant to loss of nucleic acid during treatment with EDTA to obtain cell suspensions.Preliminary experiments with Azure B, Gallocyanin chrome-alum and Methylene Blue showed that the Gallocyanin technique is the most practicable for demonstrating RNA cytochemically. Its specificity was confirmed by ribonuclease extraction of the tissue. Optimum staining conditions, requiring treatment of the tissue in Gallocyanin chrome-alum solution overnight at 40°C, are established for the artichoke tissue and for paraffin sections of pea shoot apices.     

A new method for myofibrillar Ca++-ATPase reaction based on the use of metachromatic dyes: its advantages in muscle fibre typing

A new method for Ca++-ATPase reaction in human muscle fibres is presented as an alternative to previous ATPase stains. The method is based on the use of metachromatic dyes, namely Azure A and Toluidine Blue, and has the advantages of speed, ease of performance and production of an elegant and clearcut fibre typing. The method distinguishes fibre types because of their metachromatic or orthochromatic staining, due to their different content of phosphate after incubation in the reaction medium. The comparison of serial sections stained by cationic dyes and by ammonium sulphide revealed close correspondence of fibre typing. Fibre type differentiation was also obtained with Acridine Orange; however this method was less reproducible.