Nucleic acid staining with the methyl green-pyronin method. A comparison of the use of pure dyes and commercially available dyes

We compared the staining obtained using commercially available pyronin Y samples with that obtained using pure pyronin Y in a standardized methyl green-pyronin procedure. In addition, the importance of the dye content of the anhydrous dye was investigated by varying the dye content by the addition of pure pyronin Y to one of the commercially available pyronin Y samples. We found that, for routine histological work, commercially available pyronin Y samples may produce acceptable results provided the sample can be shown by spectrophotometry to contain at least 43% pyronin Y.     

An investigation of new commercial samples of Methyl Green and Pyronin Y

Summary New commercial samples of Methyl Green (Gurr Certistain), Pyronine G (Gurr Certistain) and Pyronin Y (Polysciences) have been investigated using spectrophotometry, thin layer chromatography and nuclear magnetic resonance, in addition to standardized simultaneous and sequential staining methods using purified Ethyl Green and pure Pyronin Y as reference dyes.The Methyl Green was found to be Ethyl Green contaminated with Crystal Violet. It did not have any advantages compared with Ethyl Green supplied by American dye companies. The Pyronine G sample was Pyronin Y with a high dye content that gave good staining results when used with purified Ethyl Green. Pyronin Y from Polysciences was found to be essentially pure Pyronin Y.     

Spectrophotometry of Dyes: 1. Methyl Green. 2. Pyronin

Comparisons of absorption peaks of seven samples of methyl green showed that two different types of the dye were represented. One type (2 samples) had the visible peak near 617 mμ; the other (4 samples) near 630 mμ, while one sample was intermediate in spectral characteristics. Using these findings as a means of differentiating between heptamethyl and hexamethylethyl pararosanil-in is suggested. The Y and B forms of pyronin were found to be readily distinguishable by comparing their absorption maxima (Y, 546 mμ, B, 557-8 mμ). A check on the application of Beer's law of dilution showed that it held (1-3 mg./liter) for pyronin and that the relative effect of dilution was a slow increase with pyronin but a rapid decrease with methyl green.     

Spectrophotometry of Dyes: 1. Methyl Green. 2. Pyronin

Comparisons of absorption peaks of seven samples of methyl green showed that two different types of the dye were represented. One type (2 samples) had the visible peak near 617 mμ; the other (4 samples) near 630 mμ, while one sample was intermediate in spectral characteristics. Using these findings as a means of differentiating between heptamethyl and hexamethylethyl pararosanil-in is suggested. The Y and B forms of pyronin were found to be readily distinguishable by comparing their absorption maxima (Y, 546 mμ, B, 557-8 mμ). A check on the application of Beer's law of dilution showed that it held (1-3 mg./liter) for pyronin and that the relative effect of dilution was a slow increase with pyronin but a rapid decrease with methyl green.     

Schiff-Type Reagents in Cytochemistry. 2. Detection of Primary Amine Dye Impurities In Pyronin B and Pyronin Y(G)

Many batches of pyronin B (C.I. 45010), pyronin Y or G (C.I. 45005), and acridine red (C.I. 45000) produce positive Feulgen or PAS reactions when their 0.25% solutions are saturated with SO₂ and used on acid-hydrolyzed or periodate-oxidized tissue sections. These dyes behave as Schiff-type reagents and stain aldehyde-containing structures orange, brown, pink, red, or violet, depending on the particular batch used. The most frequent contaminants are violet and are nonfluorescent. Aldehyde groups are stained by these dyeSO₂ solutions as is shown by using unhydrolyzed controls in the Feulgen reaction and unoxidized controls in the PAS reaction, and by dye solutions lacking SO₂. Other procedures included reactions with aldehyde-blocking reagents, treatment with deoxyribonuclease and diastase, and extraction of nudeic acids with trichloroaeetic acid. The standard Schiff reagent was used in the Same procedures as a basis for comparing results. Since the Schiff-aldehyde reaction requires a dye with a primary amine group and since true pronins contain only secondary or tertiary amines, the positive histochemical results are evidently caused by dye contaminants possessing primary amine groups. The PAS reaction is more sensitive than the Feulgen reaction in detecting dye contaminants. Tissues used were chiefly formalin-fixed mouse intestine and ascites cells. Seventy-five commercial pyronins were studied from 21 different firms. Among 19 batches of pyronin B, 14 were found to contain primary amine dye contaminants. Among 39 batches of pyronin Y(G), 19 contained similar primary amine dye contaminants. Of the 8 batches of acridine red tested, 7 were found to contain primary amine dye contaminants. Nine commercial mixtures of methyl green-pyronin were studied and 4 were found to be likewise contaminated, but these reactive dye contaminants in them are apparently not associated with methyl green. A tabulated summary of the pyronin batches containing primary amine contaminants, and a list of sources and distributors of pyronin dyes are included.     

The Identification and Purification of Pyronin and Rhodamine Dyes

The purity of 27 commercial pyronin and rhodamine samples was studied by thin-layer chromatography and visible spectroscopy. Seven different red dyes were detected and separated. The chemical identities of 6 of these were established by nuclear magnetic resonance spectroscopy. The identities of samples sold as rhodamine B and rhodamine 6G were as labelled 8 out of 9 times, the pyronin (G)Y samples were as labelled 5 out of 8 times and the 10 samples sold as acridine red, pyronin B, rhodamine 36 and rhodamine S were always incorrectly labelled. The dextrin and salt contents of the dyes were determined by solvent extraction of the dye with dry methanol or ethanol. Amounts of dextrin and salt varied from none to nearly 90%. Practical methods for identification, separation of coloured components and removal of dextrin and salt are given.     

The acridine dyes: Their purification,physicochemical, and cytochemical properties

Summary The present investigation was designed to allow a critical comparison of the cytochemical behaviour of commercially available acriflavine dye samples and pure acriflavine and proflavine dyes, regarding their application in automated cell analysis. Thin layer chromatography, NMR-spectroscopy and mass-spectrometry were applied for the identification of the dye composition.This study includes (1) a column chromatographic technique for the purification of larger dye quantities, (2) the investigation of the photodecomposition of different dye samples, and (3) the evaluation of the influence of various acriflavine/proflavine dye concentrations (1.6·10^–3–4·10^–6 mol/l) on to the emission spectrum of stained unhydrolyzed and hydrolyzed chicken erythrocytes.The commercially available acriflavine dye samples showed a much higher reduction in fluorescence intensity than the pure dyes, whereby proflavine faded less than acriflavine. Photodecomposition is markably influenced by dye impurities. Fluorescence emission spectra were registered at various acriflavine and proflavine dye concentrations for unhydrolyzed and hydrolyzed chicken erythrocytes in order to investigate the dye-dye interaction and the behaviour of the cellular DNA-dye complex. Proflavine showed a similar spectral behaviour as acriflavine. The dye concentration-dependent spectral behaviour of the DNA-dye complex of these fluorochromes seems to be a very critical factor. A comparison of quantitative fluorescence measurements can only be performed by staining cells with the same dye quality, because automated cytology requires reproducible information of cells in machinesensible terms.This investigation was supported by a grant from the Bundesministerium für Forschung und Technologie (01 VH 065)     

Comparisons of Pyronin Dyes Obtained from Various Commercial Sources. 1. History

The history of pyronin dyes is discussed, beginning with the synthesis of pyronin Y(G) in 1889. The chemical structures of the dyes are given in addition to references to literature describing methods of synthesis. The early histological use of pyronins is described as well as the distribution and use of pyronins in the U. S. A. and Europe. Further discussion is devoted to the use of pyronins for histochemical demonstration of ribonucleic acid, especially in relation to sources, dye variability, contamination, and substitution by rhodamines. Additional studies with improved pyronins are advocated to evaluate histochemical staining mechanisms and to investigate possible quantitative uses.     

Dextrin and salt as impurities of histological dyes

Summary The dextrin and salt contents of 18 common histological dyes (usually obtained from more than one supplier) were estimated by solvent extraction of dye from the commercial sample with organic solvents such as ethanol, iso-propanol and methanol. The procedure is also suitable for preparing large quantities of dextrin and salt-free dyes. The dye contents of the commercial dye samples varied widely:many commercial dyes were pure but nearly half the samples contained less than 75% dye. The solvent insoluble residues from 10 commercial pyronin and rhodamine dyes were studied in more detail. Tests for dextrin and for NH ₄ ⁺ , Cl^– and SO ₄ ^2– ions were applied and the dextrin:salt ratio determined by combustion. Only 3 of the residues contained appreciable amounts of salt. Certain samples contained organic materials other than dextrin.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

Comparative purity of commercially available sodium dodecyl sulfates: A simple criterion

The purity of various commercially available sodium dodecyl sulfates was checked by gas chromatographic analysis of the fatty alcohols obtained from the acid hydrolysis of the alkyl sulfates. The content of tetradecyl sulfate in these samples was found to vary from 0 to 31% as impurity, while the content of the decyl sulfate homolog was ～2% in all the samples. Accurate critical micelle concentration measurements are a sensitive means of detecting impurities, especially those of higher chain-lengths. These measurements have indicated the presence of large amounts of tetradecyl sulfate impurity in one of the “pure” samples.In the course of work on the determination of the binding of large amounts of various detergents to proteins (1), we have discovered that some of the commercially available “pure” sodium dodecyl sulfates fall far short of any conceivable standards of purity. Although one might expect rather small amounts of inorganic salts and organic compounds such as unsulfated alcohols, the major impurities found are the homologous sodium decyl sulfate and sodium tetradecyl sulfate.In this communication, we will report only the degree of impurity arising from decyl and tetradecyl sulfates in some of the commercially available samples of “ 99 1/2% pure” sodium dodecyl sulfates. Such large contents of tetradecyl sulfates have been found that even methods simpler than the more sensitive ones (i.e., gas chromatography of the fatty alcohols after acid hydrolysis) reveal their presence.     

Pyronin Y as a fluorescent stain for paraffin sections

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green–pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogenbreak granules.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.     

Spectrophotometric characteristics and assay of pure pyronin Y

The spectrophotometric characteristics of analytically pure pyronin Y have been investigated. Addition of metal ions (Fe3+, Zn2+, and Mg2+) and of dextrin were shown not to influence the absorption characteristics. The composition of the solvent strongly influenced the value of the extinction coefficient. Aqueous ethanolic solutions with a content of about 50% ethanol gave higher epsilon-values than those found for more concentrated ethanol solutions. The difference can be explained by the existence of a solvent-solute complex in the less concentrated ethanol solutions. A new spectrophotometric assay is proposed using the epsilon-value 11.7 X 10(4) lmol-1 cm-1 found in aqueous ethanol (52%) as standard.     

Pyronin Y For Staining Stripped Autoradiographs Prepared from Plastic Embedded Sections of Rat Tissues Containing Plutonium

Autoradiography prepared by the stripping film technique from 1 μm sections of semithin plastic embedded liver and bone tissues were poststained for examination with a 1% pyronin Y solution. The use of this dye avoids heating the tissue section and the overlying photographic emulsion. It also allows the staining of the tissue section without excessive uptake of the stain by the gelatin of the stripping film.     

The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera

Hexamerins are insect storage proteins abundantly secreted by the larval fat body into the haemolymph. The canonical role of hexamerins consists of serving as an amino acid reserve for development toward the adult stage. However, in Apis mellifera, immunofluorescence assays coupled to confocal laser-scanning microscopy, and high-throughput sequencing, have recently shown the presence of hexamerins in other organs than the fat body. These findings have led us to study these proteins with the expectation of uncovering additional functions in insect development. We show here that a honeybee hexamerin, HEX 110, localizes in the cytoplasm and nucleus of ovarian cells. In the nucleus of somatic and germline cells, HEX 110 colocalized with a nucleolar protein, fibrillarin, suggesting a structural or even regulatory function in the nucleolus. RNase A provoked the loss of HEX 110 signals in the ovarioles, indicating that the subcellular localization depends on RNA. This was reinforced by incubating ovaries with pyronin Y, a RNA-specific dye. Together, the colocalization with fibrillarin and pyronin Y, and the sensitivity to RNase, highlight unprecedented roles for HEX110 in the nucleolus, the nuclear structure harbouring the gene cluster involved in ribosomal RNA production. However, the similar patterns of HEX 110 foci distribution in the active and inactive ovaries of queens and workers preclude its association with the functional status of these organs.     

Purity of commercial non-certified European samples of Pyronin Y

Summary The purity of six European non-certified samples of Pyronin Y was compared with that of two American samples certified by the Biological Stain Commission. The methods used were spectrophotometry and a Methyl Green-Pyronin staining test (both as applied by the Biological Stain Commission), thin layer chromatography, mass spectrometry, determination of pH, and content of some electrolytes. It was found that none of the European batches of Pyronin Y passed the complete test as prescribed by the Biological Stain Commission. Their dye content was uniformly low (between 5 and 19%). Furthermore, thin layer chromatography and mass spectrometry revealed that two of the dye samples contained no Pyronin Y or only traces.It is concluded that assessment of an unknown sample of a dye labelled Pyronin Y should be initiated with thin layer chromatography. The pH and content of electrolytes in an aqueous solution of the dye should also be determined in order to obtain reproducible staining results. Finally, the value of the work performed by the Biological Stain Commission is underlined, although more sophisticated methods are necessary for testing the purity of dyestuffs.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time,dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.