Methyl Green-pyronin Research Articles

Purity of commercial non-certified European samples of Pyronin Y.

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.

The effect of primary and secondary immunization on the lymphoid tissues of the carp, Cyprinus carpio L.

Mirror carp immunized with human gamma globulin (HGG) in Freund's complete adjuvant (FCA) show a proliferative response involving cells whose cytoplasm stains deep red with methyl green-pyronin (pyroninophilic cells). This response occurs particularly in the haemopoietic parenchyma of the pronephros and mesonephros. It peaks at week 3, with the formation of clusters of pyroninophilic cells in the pronephros. Immunization with Aeromonas salmonicida elicited a less intense pyroninophilic response but caused a larger increase in pigment-containing cells. After a secondary immunization with HGG in FCA, a distinct response was observed in the spleen: Pyroninophilic cells collected within the ellipsoid sheaths in large numbers and formed nodules. The reticulum of such nodules acquired spherical proportions and resembled the white pulp reticulum of the tetrapod spleen. The roles of such pyroninophilic cells and the possibility that aggregations of them may be functionally analogous to homoiotherm germinal centres are discussed.

Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus ticks.

A rapid method is described for preparing and staining salivary glands of Rhipicephalus appendiculatus ticks infected with Theileria parva. The technique, involving the use of a modified methyl green pyronin stained minimizes the risk of losing material and allows examination of stained glands within minutes of preparation. The technique was applied in a series of studies in which ticks were either infected with T. parva under different conditions, or maturation of parasites in adult ticks was stimulated by different means. When nymphal ticks were fed on the ears of cattle the subsequent infection rate of the adult ticks showed no correlation with the parasitaemia of the cattle at the time of nymphal engorgement. There was no difference in infection rates between adult ticks in which parasite maturation had been stimulated either by incubation at 37 degree C or by feeding on rabbits. However, parasite maturation took about 1 day longer in incubated ticks than in rabbit-fed ticks. Female ticks were consistently more highly infected than males, both in terms of the percentage of ticks infected and the mean number of infected acini/tick. Ticks were infected with T. parva by injection of nymphs with parasitaemic bovine blood, but the resultant adult infection was lower than that in ticks which had been infected naturally by feeding on cattle.

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.

Immune response to first mating in the female rat

The iliac and popliteal lymph nodes were removed from 110 Sprague Dawley female rats taken from a closed colony. All were killed at age 12–13 wk in groups of 10 animals; one group of virgin controls, the others at 2-day intervals during the first 10 days of either inbred or allogeneic pregnancy. Every tenth section of the lymph nodes was stained with methyl green pyronin and counts were made of the large pyroninophilic cells in the thymus-dependent area of the cortex for evidence of T cell proliferation and of the plasma cells in the medulla for evidence of B cell proliferation. After allogeneic mating significant T cell proliferation was evident in both the iliac and popliteal lymph nodes by the second post-coital day and this proliferative response was sustained until the sixth post-coital day and recurred on the tenth post-coital day. After inbred mating transient T cell proliferation was observed on the fourth post-coital day in the iliac lymph nodes. Significant B cell proliferation occurred in the iliac lymph nodes on the second day of allogeneic pregnancy and on the fourth day of inbred pregnancy, being sustained in both until the end of the observation period. These results suggest that allogeneic mating and possibly semi-allogeneic implantation induce an immune response in the female rat.

Staining and histochemistry of undecalcified bone embedded in a water-miscible plastic.

Undecalcified bone fixed in a variety of fixatives and embedded in a new formulation of 2-hydroxypropyl methacrylate at 4 c has been sectioned at 1 to 5 microns. The embedding mixture contains 2-butoxyethanol as plasticizer and triethyleneglycol dimethacrylate as cross-linker. The accelerator was benzoyl peroxide and the catalyst was N,N-dimethylaniline. With proper embedding and care in sectioning it is possible to obtain sections with relatively little bone compression, excellent preservation of cellular elements, and a minimum of wrinkling. A wide variety of stains have been used for these sections and those reported here are Gill's hematoxylin-eosin, Nocht's azure-eosin, Feulgen, Hoechst 33258 (bisbenzimid H 33258), methyl green-pyronin, PAS, alizarin red, and von Kossa silver stain. There was excellent preservation of acid and alkaline phosphatase activities. A new method of prestaining immunofluorescent labeling was also applied to bone and examples of staining with anticollagen I and antifibronectin are presented.

A Simple, Modified Methyl Green-Pyronin Y Stain for DNA and RNA in Formalin-Fixed Tissues

A Simple, Modified Methyl Green-Pyronin Y Stain for DNA and RNA in Formalin-Fixed Tissues

A Simple and Rapid Wholemount 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.

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.

Localization of androgen- and estrogen-concentrating neurons in the diencephalon and telencephalon of the mouse.

Adult male mutant mice and their normal littermates (+/y) were castrated (at least 96 h before the experiment) and injected iv with 1 μg/100 g BW either 5α-dihydro-[1,2,4,5,6,7(N)-3H]testosterone (107 Ci/mM) or 17β-[2,4,6,7,-3H(N)]estradiol (115 Ci/ mM) dissolved in 30% ethanol-saline. The animals were killed 2 h later and the brains were removed, sliced, and mounted on tissue holders. The mounted tissues were then frozen in liquified propane and stored in liquid nitrogen until processing. Four-micrometer frozen sections were cut in a cryostat and mounted for autoradiography on emulsion-coated slides, according to the dry mount procedure. The slides were then exposed at —15 C for 2 or 6 months, then photographically processed and stained with methyl green-pyronin. In autoradiograms of the brains of normal male mice injected with [3H]dihydrotestosterone, radioactivity was found concentrated in nuclei of neurons of the following areas: septal-preoptic region, many nuclei within the hypothalamus, thalamus, a...

Characterization Of The Dna-Synthesizing Cells In Rheumatoid Synovial Tissue

DNA-synthesizing cells from the rheumatoid arthritis synovial tissue of 7 patients and from 5 patients with traumatic and degenerative joint lesions were studied by incubating fresh synovial tissue samples with tritiated thymidine. Labelled cells were identified using autoradiography. The tissue sections were stained with methyl green pyronine. It was found that 0.66% (range 0.38-1.4) of the cells were labelled with 3H-thymidine whereas in control materials the percentage was 0.17 (range 0-0.38). Approximately half of the labelled subsynovial cells in rheumatoid patients (range 37-67%) were small lymphocytes, according to morphological criteria. About one-tenth of the labelled cells (range 0-17%) looked like medium-sized lymphocytes, while the rest had the characteristics of fibroblasts or were unidentifiable.

Drug-induced limb dysplasias in fetal rabbits.

Pregnant New Zealand White rabbits were treated on gestation day 12 with 19.2 mg/kg methotrexate (MTX), 750 mg/kg hydroxyurea (HU), or 1,500 mg/kg acetazolamide. Rabbits were killed either 2-32 hours posttreatment for histological analysis of embryos or at day 29 for gross and skeletal examination of fetuses. tmtx produced cleft palate, hydrocephalus, and fore- and hindlimb reduction defects. Histological analysis revealed pyknosis and edema in mesenchymal tissues at four to eight hours following treatment. The apical ectodermal ridges (AER) of treated embryos permanently lost their characteristic pseudostratified organization. By 32 hours the limb buds had regained their normal appearance except for the AER. HU affected all fetuses with skull and facial anomalies as well as severe reduction deformities of all limbs. Histologically HU-treated embryos had numerous, basophilic, intercellular granules (presumably cell debris) which appeared within two to four hours in the limb bud mesenchyme, neural tube, and dorsal root ganglion. The architecture of the AER was unchanged. Acetazolamide produced bilateral retarded ossification or possible aplasia of the first metacarpal and talus in nearly 80% of fetuses. Microscopic examination disclosed no apparent alterations in limb-bud morphology. Methyl green-pyronin Y staining called attention to green intracellular droplets within the endoderm of the trachea and bronchi at two hours posttreatment. It was concluded that the three drugs do not produce limb dysplasias by a common teratogenic mechanism.

Methyl green-pyronin with hematoxylin and orange G for the identification of inflammatory cells in tissue sections.

Methyl green-pyronin is a notoriously difficult stain to reproduce. Although very useful in detecting cells containing substantial amounts of RNA, it is of limited use in broader problems of cell identification. By careful standardization of the proportions of methyl green to pyronin and combination of these stains with hematoxylin to enhance nuclear contrast and with orange G to improve connective tissue staining, it was possible to produce a consistently reliable staining preparation in which it is possible to identify all the component cells of a mixed inflammatory infiltrate in routine paraffin sections.

Histochemical observation of the mycetomes of Idiocerus clypealis Leth

Histochemical tests on the mycetomes and mycetocytes of Idiocerus clypealis show positive PAS and Congo red reactions for the presence of glycogen and glycoprotein (amyloid). The mycetocytes failed to take alcian blue and methyl green pyronin Y stains showing the absence of mucopolysaccharides and RNA respectively. Positive millions and bromophenol blue reactions prove that they contain tyrosine and general proteins. The presence of lipids and lipoprotein is shown by a positive stain with sudan black B.

Studies On The Lungfluke, Paragonimus Iloktsuenensis: VI. Effect Of Prednisolone Injection On The Immune Responses Of Albino Rats

In order to understand the effect of prednisolone injection on the immune responses of albino rat against Paragonimus iloktsuenensis infection, the differential leucocyte counts, appearance of immunoblast (large pyroninophilic cell, LPC) in the spleen and lungs in various experimental groups were observed in relation with the growth, maturation and migration sites of this rodent lungfluke. Rats of 3 experimental groups (A series), each group consisted of 5 rats, were infected with 20 metacercariae of P. iloktsuenensis, and they were kept for 3 days(Group I), 3 weeks(Group II), and 4 weeks (Group III) of infection period. The same number of experimental groups, each group of rats received 10 mg/kg dose of prednisolone injection every other day, were also kept and examined in comparison with the former groups. Preparation of peripheral blood smear and collection of worms were completed immediately after the end of infection period, and they were stained with Giemsa or Semichon's acetocarmine. Paraffin sections of the spleen and the lung tissues were stained with hematoxylin-eosin and methyl green pyronin (MGP). Those materials from A and B series of experimental groups were examined under the light microscope, and the results obtained were as follows: 1. On observing differential leucocyte counts of peripheral blood smear, lymphocyte counts were consistently higher than those of uninfected controls in A series of infected groups, while those of B series were consistently low. On the other hand, neutrophil counts of A series showed lower counts than those of B series. In general, fluctuation patterns of both A and B series of experimental groups were almost the same, although lymphocyte and neutrophil counts showed reciprocal relation. The eosinophil counts of both series were negligible, especially in the groups of B series. 2. The counts of LPC in the periarterial lymphatic sheath of the spleen were rapidly increased in the groups of A series, while those of B series were much less than those of A series, and the appearance of considerable LPC in the spleen was also delayed in B series. Furthermore, LPC of peribronchial lymphatic tissue in A series started to increase after the invasion of lungflukes into the lungs, while those of B series were much less due to the inhibited migration of lymphocytes into the lesions. 3. Number, size and maturity of collected worms showed no significant differences between the groups of A and B series, but migration speed of the lungflukes was somewhat accelerated in B series than in A series. In this connection, it was considered that the immune responses of albino rats did not contribute for the complete protection against P. iloktsuenensis, but inhibited the migration of this lungfluke to some extent.

Studies on the lungfluke, Paragonimus iloktsuenensis: V. Host tissue reactions in albino rats

In order to understand the tissue responses of albino rat host against Paragonimus iloktsuenensis infection, the histopathological changes of the spleen and the lungs in 6 experimental groups of rats were observed in relation with the growth, maturation and migration of this lung fluke. Rats of the experimental groups, each group consisted of 5 rats, were infected with the metacercariae of P. iloktsuenensis which were isolated from brackish water crab, Sesarma dehaani, and were kept for 3 days, 3 weeks, 4 weeks, 7 weeks, 10 weeks and 14 weeks of infection period. Peripheral blood smear slides for the differential leukocyte count were prepared and also worm collection was completed immediately after the infection period. Paraffin sections of the spleen and the lung tissue were stained with hematoxylin-eosin and methyl-green-pyronin (MGP) stain. Those materials from the experimental groups were examined in comparison with the materials obtained from control group, with special reference to immunologic aspects of host response. The results obtained were as follows: 1. The counts of large pyroninophilic cell (LPC) in the periarterial sheath of spleen were rapidly increased in earlier period of infection, and those of peribronchial lymphatic tissue started to increase after the penetration of lungfluke into the lungs. 2. The LPC counts of both the spleen and the lungs were on the decrease in conjunction with the necrosis of the lung fluke in 14th week of infection. 3. On observing differential leukocyte count of peripheral blood smear, the fluctuation of lymphocyte count was proportional to that of LPC count, and the lymphocyte count was consistently higher than that of normal rats. On the other hand, neutrophil count of experimental group showed reciprocal relation to the LPC counts. 4. The nature and characteristics of pulmonary lesion produced by the P. iloktsuenensis were just the same as those produced by P. westermani. The lesions were represented by thick and fibrosclerotic cavern, granuloma due to eggs, pneumonic process and cellular infiltrations.

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or trichloracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde, a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green—pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.

Methyl Green-Pyronin Staining in Germinated Fruit Tree Pollen: Methods to Enhance Stainability of the Nuclei and Chromosomes

Brachet's method for the identification of tissue DNA and RNA is very specific because the nature of the stained material is confirmed by the enzymatic extraction. However, the small sized nuclei of pollen of fruit trees (Malus, Persica, Corylus) lose their affinity for methyl green after heating for 2 hours at 37°C with RNase or distilled water (as control). RNA cannot be extracted with cold perchloric acid, because of the low specificity of this extraction.In contrast, treatment with glacial acetic acid or acetic anhydride for 18 hrs, following proper alcoholic dehydration, enhances the nuclear stainability. The recommended fixative is Navashin's, with increased concentration of acetic acid up to 30% (to remove mitochondria), for 3 hrs. After thorough washing and bleaching with mixture of equal parts saturated aqueous ammonium oxalate and 3% H2O2 the slides are incubated with either enzyme or distilled water at 37°C for 2 hrs and treated with glacial acetic acid or acetic anhydride. Staining with methyl green (B. D. H.) 0.3% (w/v) and pyronin (Merck) 0.25% (w/v) in acetate buffer at pH 4.7, is performed at 37°C for 2 hrs. Differentiation is achieved by dipping in ice cold distilled water followed by a 5-40 min rinse in two changes of t-butyl alcohol.The slides are mounted in Euparal. The acetic anhydride treatment was shown to block the amino groups of basic proteins which, otherwise, would interfere with the DNA staining by methyl green.

Application of 1 2 5I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response

An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 1 2 5I-labelled IgG 2 anti CSA (for demonstration of antigen) or with 1 2 5I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection.

Histochemistry of the keratohyalin granules in human oral leukoplakia.

The keratohyalin granules from 25 human oral leukoplakias, showing benign hyperorthokeratosis histologically, were examined employing a series of histochemical techniques. The tissues were fixed in 10% neutral buffered formalin, 80% methanol, or Carnoy's fluid. The keratohyalin granules stained intensely with Pauly's reagent, Congo red and Harris hematoxylin, indicating the presence of proteins. This was confirmed by abolishing the staining reaction by pretreatment with proteolytic enzymes. The keratohyalin granules also reacted with methyl green-pyronin by staining pink at their peripheries; this staining was abolished by pretreatment with ribonuclease, indicating the presence of ribonucleotides. The keratohyalin granules partially stained with toluidine blue and colloidal iron, indicating the presence of acid polysaccharides. The keratohyalin granules did not react with the Feulgen reagent, suggesting the absence of DNA. Our studies indicate that the keratohyalin granules in human oral leukoplakia are primarily protein(s) complexed with polyribonucleotides. The presence of a carbohydrate moiety suggests the possibility of a protein-polysaccharide component in the granules.