Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Romanowsky dyes and Romanowsky-Giemsa effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky-Giemsa staining.

A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations. Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18,100 cm-1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB approximately 1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions. The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly oriented dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Understanding Romanowsky staining. 2. The staining mechanism of suspension-fixed cells, including influences of specimen morphology on the Romanowsky-Giemsa effect

Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.     

Gallocyanin chromalum as a nuclear stain in cytology. I. A cytophotometric comparison of the Husain-Watts Gallocyanin chromalum staining protocol with the Feulgen procedure

Summary In the present study, the staining characteristics of the Gallocyanin chromalum technique devised by Husain and Watts are compared with the Feulgen reaction. Liver imprints, blood smears, and cervical smears were fixed in ethanol and stained with either the Husain and Watts Gallocyanin chromalum reagent or the Feulgen-Schiff reagent. The slides were then post-treated with 70% ethanol-HCl pH 1.0, or with phosphotungstic acid for 0.5–30 min. The integrated optical density of cell nuclei was measured with a VIDAS image analyzer. In the material stained with the Husain and Watts procedure, some Gallocyanin chromalum was removed from the nuclei in the early phase (5 min) of all the post-treatment steps, followed by a plateau phase where the integrated optical density remained constant for 30 min. In this phase, the nuclear absorbance was highly reproducible and of the same size regardless of the post-treatment. Both the Husain and Watts procedure and the Feulgen-reaction gave quantitative staining of DNA. The Gallocyanin chromalum stain after Husain and Watts is a quick staining procedure for quantitative evaluation of DNA in cytological material. Proper rinsing of the slides is necessary for a good reproducibility of results.     

A new bacterial staining method involving Gram stain with theoretical considerations of the staining mechanism.

In order to investigate the mechanism of Gram staining of bacteria, tests with anionic dyes followed by treatment with cationic octyltrimethylammonium (OTMA) were carried out. The study revealed that tetrabromophenolphthalein ethylester (TBPE) gave the most reliable staining of Gram-negative bacteria with negative staining of Gram-positive bacteria. Tests on many species of bacteria showed that TBPE positive bacteria were Gram-negative and vice versa, without exception.     

The new and reproducible Papanicolaou stain. Morphologic and spectrophotometric observations on the influence of stain composition on staining results

The morphologic and spectroscopic characteristics of a new and reproducible modification of the Papanicolaou stain are briefly described. The main features of this modification are (1) replacement of the natural dye hematoxylin by the synthetic and chemically well defined dye thionin, (2) introduction of an alcoholic counterstain consisting of eosin gamma and fast green FCF only and (3) employment of alcoholic solutions only. The absorption characteristics of hematoxylin and thionin bound to chromatin are influenced by the cytoplasmic counterstaining, especially by the two green dyes, the absorption peaks of which are close to those of the nuclear stains. The implications of these results for visual and automated cytologic diagnosis are briefly discussed.     

Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. I. Sialomucins and sulphomucins (singly or in simple combinations)

Synopsis This study is concerned with the staining of epithelial acid glycoproteins by Alcian Blue at various pH levels. A detailed analysis of the effect of pH on Alcian Blue staining of epithelial tissues at selected sites was made. Alcian Blue was combined with the periodic acid-Schiff technique, the Alcian Blue being used at pH levels between 2.6 and 0.5.Animal salivary glands, human foetal tracheal gland and epithelial goblet cells of the adult bronchial mucosa were chosen for study because the nature of their acid glycoprotein is known and is relatively simple.In sites containing sialomucin alone, no Alcian Blue staining took place below pH 1.5. A difference was demonstrated between sialidase-sensitive sialomucin which stained only between pH 2.6 and 1.7, and sialidase-resistant sialomucin which stained between pH 2.6 and 1.5. Two types of sulphomucin were identified: the usual one stained with Alcian Blue at all the pH levels studied, and the other, in the canine gland, stained only at the most acid pH levels, that is, between pH 1.5 and 0.5.     

Cobalt thiocyanate as a stain for basic proteins and other organic bases on thin sections.

Thin sections in mouse mast cells and thymic cells are stained with cobalt thiocyanate a compound known to form insoluble complexes with organic bases. Chromatin, nucleolus, ribosomes and mast cell granules are contrasted. Different blockade reactions and enzymatic digestions indicate the staining corresponds to the basic protein amino-groups. The silver methenamine reaction stains the same cellular structures. However, the specificity control reactions show the staining mainly corresponds to protein sulphydryle groups and in a lesser extent to aldehyde and polyanions.     

Biological structure of the Pacific Ocean as compared with two other oceans

Homologous habitats (biotopes, geosystems) are similar to eachother in their position among other habitats (biotopes, geosystems)of the whole ocean and in physical processes controlling theirformation. In pelagic species with discontinuous ranges, theirindividual populations live in homologous habitats in 90% ofcases. For some species the margin of the Pacific tropical zoneis more similar to the equatorial zone of the other two oceans.Due to its size, the Pacific Ocean is characterized by (1) amaximal development of latitudinal zonation in the distributionof species and a suppression of circum-continental zonation;(2) of all the oceans, it has the most developed oceanic zonerelative to distant neritic ones and, within it, a well developedequatorial part; and (3) the Pacific east-equatorial distantneritic area is the most developed one. ^*An address to the XIV Pacific Science Congress, Khabarovsk,August 1979.