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PubMed1979

A simple and rapid whole-mount staining method with methyl green-pyronin G applied to a molluscan brain preparation.

Subesophageal ganglia of molluscs have been stained as whole mounts with methyl green-pyronin G to display the relative location of individual neurons. Nuclei appear blue, perikarya red. Expose the ganglion cells by dissection of the connective tissue in snail Ringer. Transfer the ganglion to a fixative of 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.1 at 4 C for 12--24 hours, and wash in distilled water for 1 1/2 hours. Stain with methyl green-pyronin G for 1/2--1 hour and differentiate in 96% ethanol using many rapid changes. Transfer the ganglion to absolute ethanol for 2 1/2 hours and clear in xylene for 3 hours before embedding in Depex in a suitable dish. When the Depex has hardened, the preparation can be stored, and is readily available for subsequent examination. The method may be applicable to other invertebrate tissues, and may be useful in preparing objects for teaching purposes.

Animals↗

PubMed1979

Chromosomal banding patterns produced by methyl green-pyronin staining after trypsin treatment.

A method is described for producing banding patterns with methyl green-pyronin (MGP) stain in chromosomes of fibrosarcoma cells. 1) The stain was made by mixing equal volumes of 2% aqueous pyronin G, 2% aqueous methyl green, distilled water, and 0.1 M acetate buffer (pH 5.7). 2) Treatment with colcemide and hypotonic KCl (0.075 M) was performed as usual. 3) Metaphase chromosomes were prepared using the flame-drying technique and treated with 0.25% trypsin at 37 C for 45 to 90 seconds. Before staining, the slides were rinsed in PBS, in distilled water, and then were dipped in 0.05 M acetate buffer. 4) Chromosomes were stained for more than 20 minutes, rinsed in distilled water, and hot-air dried. Satisfactory results were obtained in uncontracted metaphase chromosomes. MGP stain has the advantage of permitting much longer trypsin treatment and staining time than the trypsin-Giemsa method while providing satisfactory banding patterns.

Animals↗

PubMed1979

[Use of a method of staining sections with methyl green-pyronine for histobacterioscopic purposes].

A method of staining of histological sections with methyl green-pyronine for histobacterioscopic detection of microflora in tissues is proposed. A bright red staining with pyronine of microbial agents and yeast-like fungi of the genus Candida due to a high content of RNA in them permits elective detection of their insignificant amounts in tissues even at low magnifications of the microscope.

Bacteriological Techniques↗

PubMed1971

Effects of coumarin, thiopurines, and pyronin Y on amplification of phleomycin-induced death and deoxyribonucleic acid breakdown in Escherichia coli.

Phleomycin (</=2 mug/ml) induces neither deoxyribonucleic acid (DNA) breakdown nor cell death in stationary-phase Escherichia coli B cells, but the addition of 8 mm caffeine immediately initiates these changes in the same way as increasing the phleomycin concentration 10-fold. This phenomenon is termed "amplification" (6). Pyronin Y, a number of nontoxic thio- and mercaptopurines (of which the most active were 6,7- and 6,9-dimethyl-2-methylthiopurine), and coumarin have been found to be considerably more efficient amplifiers of phleomycin activity than caffeine. Thus 2 mm 6,7- and 6,9-dimethyl-2-methylthiopurine, 0.16 mm pyronin, and 4 mm coumarin killed 10 to 100 times more phleomycin-treated bacteria within 2 hr than 8 mm caffeine. As with caffeine, amplification of cell death by these compounds was accompanied by degradation of DNA to acid-soluble fragments. A number of compounds including 2,6-dichloropurine, 6-hydroxy-2-methylthiopurine, alpha-naphthol, beta-naphthol, naphthionic acid, and alpha-naphthol-4,8-disulphonic acid inhibited the action of phleomycin, if they were present in the cell suspension during phleomycin treatment, but some caused amplification if added subsequent to the phleomycin. Although no mutants resistant to >/=10 mug of phleomycin per ml were observed among 10(11)E. coli B cells screened, such mutants occurred with a frequency of 10(-6) to 10(-7) among cultures resistant to 1 to 2 mug of phleomycin per ml. These double mutants were cross-resistant to phleomycin plus caffeine. The amplifying compounds, though structurally dissimilar, shared the common characteristic of binding selectively to denatured DNA as measured by equilibrium dialysis methods. The implications of these observations in supporting a model of phleomycin amplification proposed previously (6) and their utility in providing a logic for developing a new class of antibiotics are discussed.

Antibiotics, Antineoplastic↗

PubMed1981

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.

Acridine Orange↗

PubMed1982

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green.

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3- to 5-fold reduction in the fluorescence intensity of intracellular MG-PY. Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.

Animals↗

PubMed1987

Interactions of pyronin Y(G) with nucleic acids.

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.

DNA↗

PubMed1987

Application of pyronin Y(G) in cytochemistry of nucleic acids.

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.

Animals↗

PubMed1984

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.

Iron↗

PubMed1979

Chromatin fluorescence by pyronin staining.

Human, chicken and mouse cells from different tissues show a bright red-orange fluorescence of the chromatin after staining with pyronin Y. The possibility that intercalation of the dye into double helical nucleic acids accounts for this fluorescence pattern is briefly discussed.

Animals↗

PubMed1988

Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells.

Cellular RNA in Chinese hamster ovary (CHO) cells synchronized in mitosis (M) or G2 phase, as well as in interphase cells subjected to hyperthermia (42 degrees C, 10 min), was stained with acridine orange (AO), ethidium bromide (EB), or pyronin Y (PY) and the resultant fluorescence was measured by flow cytometry. Total RNA content detected after staining with AO increased in M as compared to G2-phase cells, consistent with continued RNA synthesis during G2 phase. The content of double-stranded RNA, stained with EB (after DNase treatment), was also somewhat higher in M cells. In contrast, the stainability of RNA with PY decreased by 27% in M- compared to G2-phase cells. Furthermore, a decrease in stainability of RNA with PY was observed in G2 cells compared to cells in G1 phase. In separate experiments, RNA stainability with AO or EB was generally unaffected when interphase CHO cells were exposed to 42 degrees C for 10 min, though this same treatment resulted in a 26% decrease in RNA stainability with PY. The decreased PY stainability of cellular RNA in M or heat-treated cells was observed at a relatively narrow range of dye concentration (1.0-2.0 micrograms/ml). The observed hypochromicity of RNA coincides with dissociation of polyribosomes into single ribosomes known to occur during mitosis and following exposure to hyperthermia. It is presumed that the phenomenon involves selective denaturation and condensation of ribosomal (r) RNA by PY in single ribosomes which does not occur in polyribosomes. While the molecular mechanisms responsible for stabilization of rRNA in polyribosomes preventing its denaturation and condensation by PY are unknown, PY appears to be a sensitive probe that can be used to detect and study these changes in rRNA confirmation in situ.

Animals↗

PubMed1995

Simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation using 7-amino-actinomycin D and pyronin Y.

We developed an improved technique that permits simultaneous DNA and RNA quantitation by a flow cytofluorometry using 7-amino-actinomycin D (7AAD) and pyronin Y (PY), respectively. Detailed cell cycle analyses based upon the cellular DNA/RNA levels were performed using cells suspended in a buffer containing 0.004% saponin. This method preserved the light scattering properties of human peripheral blood cells, thus lymphocyte, monocyte and granulocyte populations could be evaluated. In addition, since 7AAD and PY exhibit red (> 650 nm) and orange fluorescence (570 nm) respectively, the green fluorescence channel of the flow cytometer was reserved for surface phenotyping using FITC-conjugated antibodies. The 7AAD/PY method is applicable to the simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation when combined with FITC-conjugated surface markers in heterogeneous samples. To demonstrate the three-color analysis, PHA-activated human peripheral blood lymphocytes were stained for cell surface markers with monoclonal antibodies. The cells were suspended in buffer containing 0.004% saponin, then stained with 7AAD and PY. The DNA and RNA were analyzed in indivisual CD4+, CD8+ and CD20+ cells, and the characteristic cell cycle status was found. Cell activation was further analyzed using antibodies against interleukin-2 (IL-2) receptors (CD25), transferrin receptors (CD71) or HLA-DR molecules. Transferrin receptors were expressed in late G1 phase (G1B) just before the initiation of DNA synthesis, whereas IL-2 receptors and HLA-DR were expressed very early in the G1 phase (G1T). Since this technique preserves both light scatter properties as well as cell surface proteins, it is ideally suited for detailed cell cycle analyses of heterogeneous samples such as peripheral blood or bone marrow cells.

Antigens, Differentiation↗

PubMed1986

Pyronin G as a fluorescent probe for quantitative determination of the membrane potential of mitochondria.

Added to mitochondrial suspension, pyronin G changes the intensity of its fluorescence depending on the membrane potential (energy state) of the mitochondria. The mechanism of this effect is studied and a dependence is obtained between the membrane potential and the fluorescence intensity. This permits quantitative determination of the membrane potential by the changes in the fluorescence of the suspension. A method is proposed for measuring the membrane potential of vesicles in the -120 to -220 mV interval.

Animals↗

PubMedDate not supplied

Human spermatozoa stained with toluidine blue-pyronine. A rapid method for differentiation.

Spermatozoa stained with toluidine blue-pyronine can be differentiated very rapid within one hour after liquefaction of the ejaculate. In contrast to the method of Testsimplets the slides can be stored for record purpose. Furthermore its quality is comparable with the common staining techniques of spermatozoa. Therefore it can be recommended in the routine practice.

Humans↗

PubMed1980

One-step staining of Neisseria gonorrhoeae in urethral discharge by methyl green-pyronin.

Methyl green-pyronin (MGP) was used in a one-step procedure to stain smears of urethral discharge from 169 men. Duplicate smears were stained by Gram's method and discharge was cultured for Neisseria gonorrhoeae. The organisms were isolated from 67 specimens and intracellular diplococci were seen in 74 smears after Gram staining and in 77 after staining by MGP. Furthermore, more extracellular and intracellular diplococci were seen in smears stained by MGP than by Gram's method and the proportion of polymorphonuclear leucocytes found to contain the organisms was greater after staining with MGP. Staining with MGP is simple, rapid, inexpensive, and easily automated.

Humans↗

PubMed1986

Methyl green-pyronin stain distinguishes proliferating from differentiated nonproliferating cell nuclei after acid denaturation of DNA.

We applied methyl green-pyronin (MG-P) stain, which is usually used for the selective staining of DNA and RNA, to frozen sections of rat jejunal and esophageal mucosa, following digestion with RNase and treatment with various concentrations of HCl. The pyroninophilia of the nuclei increased with increasing strength of the acid, but the susceptibility of the nuclei to acid differed among cell populations. In the jejunal epithelium, at an appropriate acid strength the nuclei in the crypts of Lieberkuhn were less acid-sensitive and remained blue-green, whereas those in the villi were more pyroninophilic and stained lavender. Under the same conditions, the nuclei in the basal layer of the esophageal epithelium were blue-green and those in the spinous and granular layers were increasingly lavender. These results suggest that in cell-renewal systems the differentiated, nonproliferating cells are more sensitive to acid denaturation of DNA than the undifferentiated, actively proliferating cells. MG-P stain, which is able to distinguish double-stranded from single-stranded DNA, may be used as a tool to stain proliferating and nonproliferating cell nuclei differentially in tissue sections.

Animals↗

PubMed1986

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain.

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.

Brain↗

PubMed1993

Effects of mounting media on fading of toluidine blue and pyronin G staining in epoxy sections.

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.

Animals↗