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PubMed1976

Prolonged methanol fixation of soluble mucosubstances in mucopolysaccharidoses.

A simple and efficient method for the demonstration of highly water soluble acid mucosubstances in cold microtone sections is described. It consists of prolonged treatment of cold microtome sections with methanol (for at least 1 h) and subsequent staining with 0.1% azure A in distilled water or in 30% methanol. The procedure is recommended particularly for the bioptical examination of mucopolysaccharidoses.

Azure Stains↗
PubMed1969

The organization of the nucleolus in meristematic plant cells. A cytochemical study.

The architecture of the nucleolus in Allium porum and Triticum vulgare meristematic cells has been investigated by means of digestions with various enzymes. After staining with azure B at pH4, plant nucleoli exhibit lighter regions which, under electron microscopy, correspond to the fibrillar zones characterizing these organelles. Evidence is presented indicating that these latter zones contain coarse convoluted filaments quite similar to the loops first demonstrated by La Cour (24) and which are assumed to originate from the nucleolar-organizing chromosomes. These coarse, 0.2micro wide filaments are remarkably resistant to the action of deoxyribonuclease, ribonuclease, pepsin, trypsin, or of various combinations of these enzymes and, moreover, they show insignificant incorporation of labeled thymidine even after long exposure to this DNA precursor. The clearing action of pepsin on different regions of the nucleolus lends support to the hypothesis that an amorphous material or matrix pervades the mass of this organelle. This effect is particularly striking within the particulate nucleolar zones themselves. Both ribonuclease and trypsin disorganize the RNP (ribonucleoprotein) nucleolar particles. The effect of the latter enzyme on the RNP particles is taken to indicate that they contain proteins particularly susceptible to trypsin which are essential for maintenance of their morphological integrity. Trypsin also interferes with azure B-staining of the nucleolar mass as a whole and, according to radioautographic data, extracts RNA throughout this organelle. Accordingly, the hypothesis is considered that RNA is complexed with proteins not only within the particulate nucleolar portions, as is already well known, but also in the fibrillar zones.

Autoradiography↗
PubMed1989

The influence of cationic thiazine dyes on eosin Y-uptake of red blood cells in Romanowsky-Giemsa type stains.

In the present study we have investigated the uptake of cationic thiazine dyes and of the anionic Eosin Y by red blood cells (RBCs). Blood smears were stained with Azure B-Eosin Y, Methylene Blue-Eosin Y, Thionin-Eosin Y and with the cationic and anionic dyes alone at varying concentrations. Dye content of erythrocytes was measured with a Vickers M 85a microdensitometer. Nuclear chromatin features of white blood cells were investigated with the IBAS 2000 image analyser. Azure B favoured Eosin Y uptake of RBCs remarkably, and vice versa. There was no clear indication of that type of molecular interaction which characterizes the generation of the colour purple on polyanions. Methylene Blue and Thionin left Eosin Y uptake unaffected, but contamination of the standard Azure B-Eosin Y stain with Methylene Blue obviously affected Azure B-Eosin Y uptake, probably by competition of Methylene Blue and Azure B binding in RBCs. Furthermore, Methylene Blue contamination of the standard stain increased the rate of error in image analysis of white blood cell nuclei due to variations of staining intensity. For cytophotometry and image analysis of blood smears the standard Azure B-Eosin Y stain is by far superior to the commercial stain which normally is contaminated with Methylene Blue and some of the lower azures.

Azure Stains↗
PubMed1985

Colorimetry for the stain technologist. IV. Analysis of the components of color difference.

Total color differences have been calculated for various pairs of stained microscopic substrates. The latter include azure B/eosin stained blood cells and Papanicolaou stained cells from the uterine cervix. Both the CIE L*u*v* and L*a*b* color spaces have been used. Total color differences have been analyzed in terms of lightness, hue and chroma components. Various discrepancies have been noted among these components, especially the chroma difference, for the two spaces. It is concluded that current color-difference formulae are less than perfect, although they can provide much useful information.

Azure Stains↗
PubMed1987

Standardization of the Romanowsky-Giemsa stain: the influence of staining time on the RG-staining pattern.

In this paper the influence of staining time on the staining pattern of Romanowsky-Giemsa (RG) type stains is investigated. Smears of rabbit bone marrow and of human venous blood were stained with azure B-eosin Y, azure A-eosin Y and with the cationic dyes alone under varying conditions of staining time and dye concentration. The stained smears were investigated by integrating microdensitometry. DNA-polyacrylamide (PAA) model films were stained with azure A-eosin Y, the extinction of the stained model films was determined by spectrophotometry. With increasing staining time the color of the cell nuclei changed from blue to an intense purple, the texture of the nuclear chromatin became more prominent. Prolonged staining resulted in over-staining of the cell nuclei with loss of a distinct chromatin texture. Besides such factors as dye concentration and pH of the staining solution standardization of staining time may be considered necessary for the reproducibility of the RG staining pattern.

Animals↗
PubMed1989

[G staining].

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Azure Stains↗
PubMed1985

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

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.

Azure Stains↗
PubMed1995

Azure B-eosin APAAP staining: a method for simultaneous hematological and immunological cell analysis.

Azure B-eosin APAAP staining allows simultaneous analysis of peripheral blood and bone marrow cells for hematological characteristics and immunological cell marker profiles. A defined sequence of staining procedures maintains characteristic components of the Romanowsky-Giemsa stain whereas cell antigens can be detected immunologically using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) detection system. Antigens are visualized by the staining product of the substrate-naphthol AS GR phosphate and variamine blue salt. The usefulness of the azure B-eosin APAAP method was demonstrated on blood and bone marrow smears of patients with various hematological disorders.

Alkaline Phosphatase↗
PubMed1984

Microspectrophotometric studies of Romanowsky stained blood cells. III. The action of methylene blue and azure B.

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.

Azure Stains↗
PubMed1979

Spectrophotometric and cytochemical studies on azures A, B and C.

The cytochemical use of azures A, B and C, propared with either N HCL and potassium metabisulphite or with sodium hydrosulphite in tissue sections were investigated. Both in situ absorption curves of nuclei stained with each of these dye-SO2 reagents as well as in vitro absorption data of acqueous solutions of the dyes are also presented. It has been pointed out that the mechanism of staining with azure A-SO2 and azure C Eosinate-SO2 is the same as that of the conventional Feulgen reaction with Schiff reagent but that of staining with azure B-SO2 is by the modified Feulgen reaction because this dye does not contain any primary amino group.

Animals↗