The use of aluminium lake of nuclear fast red in plant material succesively with alcian blue

The successive staining alcian blue/aluminium lake of nuclear fast red was proved a useful tool for studies on plant root tip. A simple and reliable procedure is given resulting in blue cell walls, almost colourless cytoplasm and red nuclei. Attempts were made to apply spectrophotometry and paper chromatography to overcome the confusions in manufacturers’ labelling of the dye and to check the lake formation.     

Histochemical demonstration of acidic glycosaminoglycans in the cell nuclei of the iris and other tissues.

Using histochemical methods, the presence of acidic glycosaminoglycans in the cell nuclei of 51 human irides and a series of monkey organs was demonstrated. In general, these substances are sensitive to testicular hyaluronidase and chondroitinase ABC and also to Streptomyces hyaluronidase, when using special staining methods. The specificity of testicular hyaluronidase was tested by inhibition with heparin. By simultaneously staining with alcian blue and Feulgen, acidic glycosaminoglycans can be distinguished from the nucleic acids. Sporadically, hyaluronidase-resistant substances with a specific acidic glycosaminoglycan stainability occur. We assume the existence of various acidic glycosaminoglycans in the cell nuclei. Aging changes were not traceable with constancy.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

Feulgen-positive staining of the cell nuclei in fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae

The Feulgen reaction and the staining of preparations with two DNA-specific fluorochromes, Hoechst 33258 and 4',6-diamidino-2-fenilindol (DAPI), were used to study the preservation of DNA in the fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae, Paramyrtacicarpus plurilocularis and Paramyrtaciphyllum agapovii collected in Yakutia (Siberia, Russia). It was shown that chromatin structures of the fossilized plants form stable red-purple complexes with the Schiff's fuchsin sulphuric acid reagent in situ . This coloration is specific for DNA, in particular, for the deoxyribose residues. It means that the cell nuclei of these 53–55 Myr old plants preserve a part of the deoxyribose backbone of DNA molecules. On the other hand, there was no, or only a very weak, staining of the cell nucleus with fluorochromes DAPI or Hoechst 33258, which specifically bind to the double-stranded DNA and do not bind to either the single-stranded DNA or RNA molecules. The stainability of fossil plant cell preparations with alcian blue shows that there are also polysaccharides containing carboxyl groups in the cell walls of fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 315–321.     

Alcian Blue Pyridine Variant-a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

Alcian Blue Pyridine Variant–a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

A comparative study of the effect of morphactin and Niagara on the leaf epidermis

Treatment of two-week-oldBrassica campestris andTrigonella foenum-graecum plants with morphactin andVicia faba, Antirrhinum orontium, andPapaver somniferum with Niagara, induced marked variations in the orientation and ontogeny of stomata and the epiderma cells. Morphactin—chlorflurenol at 12.5, 62.5, 125, 250, and 500 ppm, caused marked damage of the shoot apices and changes in the epidermal tissue, such as divisions of the guard cells, reduction in the size of the stomata, and epidermal cells. Niagara—ethyl-hydrogen-1-propylphosphonate at 100, 500, 1 000 5 000, and 10 000 ppm caused thickenings of the epidermal cell walls and differentiation of new meristemoids from the epidermal cells, contiguous stomata, and incomplete development of the guard cells.     

Contribution to the study on the reparative effect of exogenous DNA in the irradiated meristem ofVicia faba

The present report is focused on the study of participation of exogenous DNA in the process of postirradiation reparation of meristematic cells ofVicia faba primary roots. It is aimed at comparison of the positive reparative effect of isologous DNA with postirradiation action of heterologous DNA in its native, thermally denatured and DNAase-degraded forms, or DNA degraded by ultrasound, and with the effect of other biologically important macromolecules (RNA, histone, heparin, and dextran sulphate). For this purpose, the roots ofVicia faba irradiated by 150 r exposure were cultivated in solutions containing the above substances for an appropriate time interval. In squash slides both mitotic activity of the investigated cell population and frequency of postmetaphase chromosomal aberrations induced by radiation were evaluated. It was shown that a stimulation of cell division and reparation of chromosome damages were supported exclusively by isologous DNA. On the contrary, exogenously applied heterologous DNA increased postirradiation frequency of aberrations; maintenance of native structure of applied DNA was an essential condition for the above effect. Other macromolecules investigated on the course of postirradiation reparation ofVicia faba meristematic cells were without effect.     

Standardization of Staining in Giycosaminoglycan Histochemistry: Alcian Blue, its Analogues, and Diamine Methods

Glycosaminoglycans are identified in tissue sections by various histochemical techniques including staining with alcian blue and its analogues, such as cuprolinic blue and cupromeronic blue, or with high and low iron diamine methods. The variation in staining results is particularly confusing in the case of alcian blue, where not only are several different brands of alcian blue available but also several different staining protocols are used. If the results obtained by these techniques are compared, they often do not match. We have developed a dot blot technique for quality control of glycosaminoglycan histochemistry to standardize the staining protocols. This staining technique enables his-tochemists to test particular batches of alcian blue or its analogues for selective glycosaminoglycan staining, thus improving control of histochemical results. The results obtained using the dot blot assay indicate that it is necessary to test each batch of dye individually to obtain valid results in glycosaminoglycan histochemistry.     

A macerating ammonium oxalate fixative and schedule for some root meristems

0.25 % ammonium oxalate in a mixture of ethyl alcohol, chloroform, formaldehyde, acetic acid and hydrochloric acid works as a very fast and efficient macerating fixative for the root meristems ofAllium cepa, Allium sativum, Ornithogalum thyrsoides, Pisum sativum andVicia faba. Slicing the tip region of excised meristems hastens spindle poison pretreatment, fixation and staining. Owing to the dissolution of the middle lamella and eliminating a physical barrier to permeability an extremely rapid fixation and staining schedule involving several stains has thus been developed.     

Staining Tomato Fruit Cuticle and Exocarp Tissues

Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue 8GX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylinalcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at × 100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues magenta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.     

Histogenesis and localization of non-specific esterase in root tip

A procedure was developed for satisfactory freeze-sectioning of root tips. The use of Ca formol-fixed material kept and frozen in Holt's syrup is recommended. The existence and different localization of 2 fractions of non—specific esterase was verified in root tips ofVicia faba. The same results were revealed in fixed and unfixed material. The dynamics ofin situ reaction was followed with respect to optimal incubation time. The results with substrates of different chain length support the existence of 2 fraction of the studied enzyme, none of which, concerning substrate specificity, is a lipase. It follows from the present studies inVicia faba and other species (Cucurbita pepo, Lupinus albus, Pisum sativum Zea mays), that non-specific esterase localization is not directly given by histogenesis.     

Chromosome aberrations caused by the effect of ultrasound in the meristematic cells ofvicia faba

In our present work the formation of chromosome aberrations has been studied in dependence on the tima interval between sonication and fixation of the primary root tips of Vicia faba. Maximum occurrence of aberrations was recorded immediately after sonication. The results of our experiments pointed to the fact that the frequency of the induced changes was independent on the sonic waves intensity within the range of 0-2—3-0 W/cm² and on ultrasond treatment duration within the range of 1—20 min. Studies of the distribution of chromosome abnormalities caused by ultrasound between the large and small chromosomes of theVicia faba meristematic cells in various time intervals showed that the frequency of the aberrations in both chromosome groups was proportional to its total metaphase lengths. Analysis of the type of aberrations observed in various time intervals after sonication indicated the simultaneous formation of chromosome and chromatide abnormalities.     

The accentuators,pH and the principles of the effect in histological staining

A study was made of the influence of accentuators (phenol, aniline) and pH on the stainability of vegetable tissue (roots ofAllium cepa L.) with light green and methylene blue with reference to the theory of the electrostatic basis of staining. — The results obtained on the whole confirmPischinger’s theory of the electrostatic basis of staining for light green only, but with methylene blue the situation was found to be much more complicated. Only the alkalization of a watery solution of methylene blue increases its staining ability, whereas the accentuators markedly disturb staining. Prischinge’s theory is thus not of general validity, but applies only to one factor of this complicated process. Electrostatic influences acting on staining ability are not so important as other factors in this complicated process in some dyes.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

Mikrospektralphotometrische Untersuchungen an lebenden Zellen der Schuppenblattoberepidermen vonAllium cepa nach Toluidinblau 0- und Nilblauchlorid-Färbung

Zusammenfassung Aus spektralphotometrischen und Elektrophorese-Untersuchungen geht hervor, daß bei Toluidinblau 0 und Nilblauchlorid im physiologischen pH-Bereich vorwiegend einwertige Kationen vorliegen. Nach eigenen Messungen betragen die Dissoziationskonstanten zwischen einwertigem Kation und Farbbasenmolekül für Toluidinblau 0 1,74 · 10^–12 (pKa=11,76) und für Nilblauchlorid 2,81 · 10^–10 (pKa=9,55).Die Abhängigkeit der Absorptionsmaxima wäßriger Farbstofflösungen konstanten pH-Wertes von der Farbstoffkonzentration deutet darauf hin, daß bei Toluidinblau 0 und Nilblauchlorid eine Assoziation der einwertigen Kationen erfolgt.Die Färbung der typisch leeren Zellsäfte und der Zellwände mit Toluidinblau 0 bzw. Nilblauchlorid ist ein reiner Konzentrationseffekt. Die Farbstoffkonzentration wird bei den leeren Zellsäften durch den Ionenfallenmechanismus, bei den Zellwänden durch Elektroadsorption stark erhöht (maximal 1/500 bis > 1/500), so daß eine Assoziation der einwertigen Kationen stattfindet.Bei den typisch vollen Zellsäften beruht die Speicherung auf einer salzartigen Komplexbildung zwischen den Farbkationen von Toluidinblau 0 bzw. Nilblauchlorid und den zelleigenen Flavonolen.In den Zellsäften mit geringerem Flavonolgehalt liegen nebeneinander monomère, dimere Kationen und Moleküle des Toluidinblau 0- bzw. Nilblauchlorid-Flavonol-Komplexes vor.

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Microspectrophotometric investigations of cells from the living upper epidermis of the bulb scale ofAllium cepa after staining with toluidine blue 0 and nile blue chloride

Summary Spectrophotometric and electrophoretic investigations show that in a physiological range of pH toluidine blue 0 and nile blue chloride predominantly exist as univalent cations. According to measurements performed by the present author the dissociation constants regarding the equilibrium between the univalent cation and the dye base molecule are 1.74 · 10^–12 (pKa=11.76) for toluidine blue 0 and 2.81 · 10^–10 (pKa=9.55) for nile blue chloride. At a constant pH the absorption maxima of aqueous solutions depend on the dye concentrations. Therefore one may assume that toluidine blue 0 and nile blue chloride form associations of univalent cations.The staining of typically empty cell saps and cell walls by toluidine blue 0 and nile blue chloride respectively, is a pure effect of concentration. In empty cell saps the dye concentration is remarkably increased by the ion trap mechanism; in cell walls an intense accumulation is caused by electro-adsorption (the maximum concentration being 1/500 to > 1/500) resulting in an association of univalent cations.In typically full cell saps accumulation is caused by the formation of salt-like complexes of the dye cation of toluidine blue 0 and nile blue chloride with flavonoles native to the cell. In cell saps of a lower flavonole concentration molecules as well as monomeric and dimeric cations of toluidine blue 0 and nile blue chloride exist along with their flavonole complexes.

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Herrn Prof. Dr.Horst Drawert in Verehrung und Dankbarkeit zu seinem 60. Geburtstag gewidmet.

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Herrn A. Soumpos danke ich für die Umzeichnung der Abbildungen.     

Glucose-6-phosphate dehydrogenase isoenzymes in root growth zones ofVicia faba

The specific activity of glucose-6-phosphate dehydrogenase (G-6-PD) in growth zones ofVicia faba roots is increasing with cell maturation and differentiation. Changes in the total activity of G-6-PD are not associated with a change in the number of G-6-PD isoenzymes. Five G-6-PD isoenzymes were found in all root growth zones. Some differences were found in the activity of individual isoenzymes.     

A permanent cell viability assay using alcian blue.

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with "centrifugal cytology" to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.