Methyl Green-pyronin Research Articles

Staining of the Nucleolus with Protein and RNA Stains for Automatic Measurement of Nucleolar Size in Paraffin Sections

In this paper two staining methods for automatic assessment of nucleolar profile area in paraffin sections of breast carcinomas using the IBAS 2000 image analyzer are reported. In the first method, the nucleolar proteins are stained with phloxine B, in the second the nucleolar RNA is stained with methyl green-pyronin. Both methods are fully reproducible. Statistically, the profile areas observed for the same patient by the two methods were found not to differ significantly. Because of their rigidity and their size in relation to section thickness, nucleoli are particularly attractive cell structures for quantitation, for instance in studies of variables that might be useful in establishing the prognosis for carcinoma patients.

The ontogeny of gut‐associated lymphoid tissue in Oreochromis‐mossambicus

The ontogenesis of gut‐associated lymphoid tissue (GALT) was investigated using histological procedures. Samples were taken at various time intervals to include larval, first‐feeding and juvenile stages. Whole fish and tissue samples were embedded in either paraffin wax or metha‐crylate resin and sections stained with Giemsa, periodic acid‐Schiff (PAS), methyl green pyronin (MGP) and methylene blue. GALT was first observed as individual lymphocyte‐like and macrophage‐like cells in the lamina propria, and in later stages plasma cells and granulocytes were identified. Leucocytes began to infiltrate the epithelium just prior to first feeding and then rapidly once feeding had began. By day 50, GALT was equal to that observed in the adult gut.

Idiopathic retroperitoneal fibrosis (Sclerosing retroperitonitis)

Three cases of idiopathic retroperitoneal fibrosis, one of which was localized to the perirenal area, are presented. The predominance of plasma cells, which may be difficult to recognize because of distortion unless methyl green-pyronine staining is done, and the character of the fibrous tissue indicated the non-neoplastic nature of the processes. This diagnosis was confirmed by immunoperoxidase studies that demonstrated polyclonality of the lymphoplasmacytic component. Immunologic studies, which may be performed on paraffin-embedded tissue, are helpful in differentiating this lesion from the sclerosing lymphomas that also occur in the retroperitoneal area.

Morphology and functions of calyx fluid filaments in the reproductive tracts of endoparasitoid,Microplitis mediator [Hym.: Braconidae

Very long virus-like filaments were found in calyx region of the reproductive tracts of the braconid parasitoid,Microplitis mediator Haliday, which attacks the Noctuid,Pseudaletia (=Leucania) separata Walker. These filaments are nuclear in origin; Feulgen and methyl-green pyronin reactions revealed cytochemically the presence of DNA. Filaments are attached to the surface of the chorion of an egg until hatching and function to suppress the encapsulation reaction of the host. Furthermore, it was observed that serosal cells, 18-h and 24-h from oviposition, released substances possibly related to the inhibition of encapsulation.

Special Stains in the Microwave Oven

AbstractWe describe the modification of a number of stains for the microwave oven: Grocott's methenamine silver, acid fast bacteria, Giemsa, Lillie's melanin, methyl green pyronin, Masson's trichrome, and glycogen double digestion. Their adaptation has markedly reduced the time needed for staining. The staining procedures described here can be used on paraffin sections, frozen sections on coated slides, touch preparations, and cytology specimens.

Theileria infectivity of Hyalomma ticks in Haryana, India.

Theileria infection of Hyalomma ticks collected from three districts of Haryana was assessed in whole salivary glands by the methyl-green pyronin staining method. Of 1,662 ticks screened, 546 (32.8%) were found Theileria positive. Infection rate in 935 female ticks (36.9%) was more than that of 727 male ticks (27.6%). Density of Theileria infection (number of infected acini per infected tick) was also higher in female ticks. Theileria infection rate of ticks varied greatly in the three districts viz. only 12.0% in Rohtak, 25.8% in Hisar and 48.3% in Karnal. Per cent infection rate was high (63.7%) in ticks from indigenous cattle and low (18.6%) in those collected from buffaloes. However, the intensity of infection in infected ticks from cross-bred cattle was comparatively much higher. Frequency distribution of Theileria positive acini in infected ticks revealed a low density of infection per infected tick. This points to the largely stable endemic situation prevailing in Haryana. Only a single salivary acinus was found infected in 16.6% of the infected ticks, about 70% had up to 10 infected acini while only about 10% had over 25 infected acini per tick. The masses in acini presumed to be Theileria were confirmed by demonstrating parasitic masses on staining one of a pair of salivary glands and inoculating the suspension of the other half of the gland into two cross-bred cow calves which developed clinical signs and lesions typical of theileriosis.

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.

Standardized Methyl Green—Pyronin Y procedures using pure dyes

Fully standardized Methyl Green-Pyronin methods are presented. Pure Pyronin Y and purified Methyl Green or Ethyl Green are used either simultaneously in one dye bath or are used as a sequence of Pyronin Y and Ethyl or Methyl Green. Both methods, as shown by enzymatic pretreatment, give a reliable and reproducible staining in DNA with Ethyl or Methyl Green and of RNA with Pyronin Y on Carnoy fixed material. On formaldehyde fixed material it was found advantageous to use the sequential method as chromatin was hereby stained green instead of blue as seen with the simultaneous method.

Analysis of hemopoietic lineage of accessory cells in the developing thymus of Xenopus laevis.

The developmental history of accessory cells in the thymus was studied by grafting hemopoietic stem cells into cytogenetically distinct frog embryos (diploid-2N or triploid-3N) before the establishment of circulation and overt differentiation and colonization of the thymus. The DNA content of cortical thymocytes and circulating erythrocytes was quantified by staining with propidium iodide and measuring the amount of red fluorescence emitted by individual nuclei with the use of flow cytometry. Accessory cells from thymic medulla were separated by incubating for 2 hr on glass slides. For comparison, the developmental history of peritoneal macrophages was examined as representative, myeloid-derived phagocytic cells. DNA content of adherent cells was quantified by staining with the DNA-specific Feulgen reaction and measuring light absorption of individual nuclei by microdensitometry. Thymic accessory cells were subdivided into phagocytic and nonphagocytic phenotypes on the basis of latex bead ingestion. Phagocytic cells in the thymus were usually nonspecific esterase positive and phenotypically resembled peritoneal macrophages. Nonphagocytic cells from the thymus were usually esterase negative and had a dendritic morphology characterized by branched cytoplasmic extensions. Nonphagocytic cells were positive for cytoplasmic RNA based on staining with methyl green-pyronin Y. Phagocytic cells from both the thymus and the peritoneal cavity had no levels of cytoplasmic RNA detectable by this method. Analysis of the embryonic derivation of thymic accessory cells, based on the proportion of cells carrying the cytogenetic marker, demonstrated that thymic lymphocytes and thymic accessory cells were a concordant pair of cells, distinct from myeloid-derived erythrocytes and possibly macrophages. These experiments provide circumstantial evidence suggesting thymocytes and thymic accessory cells could arise from a bipotential precursor that diverges into these separate lineages after colonization of the epithelial thymic rudiment during early development.

Nucleic acid staining with the methyl green-pyronin method

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.

Intraocular Myeloma

An eighty-seven year old female presented with complaints of pain in the left eye. She had a history of chronic open angle glaucoma that was under control with medication. She had lost all vision, and the eye had become very painful and inflamed. She was admitted with a diagnosis of endopthalmitis. Enucleation was performed and inspection of the specimen revealed a firm globe with a white mass extending over the iris and across the pupil in the anterior chamber. Microscopic evaluation with the hematoxylin and eosin stain revealed a plasma cell infiltrate involving the ciliary process, extending into the posterior chamber, occlusion of the retinal artery, and extensive loss of nerve fibers within the optic nerve. An accumulation of a pinkish and somewhat fibrillar material within the tumor and optic nerve was also observed. Stains for myelin and amyloid and a methyl green pyronine were performed, and immunoperoxidase for IgG, IgA, IgM, kappa and lambda.

On the nucleoli of the dinoflagellate Prorocentrum

To date no nucleolus had been observed in Prorocentrum under the light microscope. The author failed to show the nucleoli of P. micans and P. cassubica with eosin in 70% alc or with methyl green-pyronin. But when these dinoflagellates were treated with an Ag-1 technique which had been improved for demonstrating NORs in unicellular organisms, nucleoli were stained dark brown or black, while all other parts showed no colour. When the materials were stained well, only the central part of the nucleolus was stained. Under the electron microscope, it was observed that all the silver grains were concentrated in the pars fibrosa of the nucleolus. P. cassubica had only one small oblate nucleolus attached to the nuclear envelope, with NOR usually in the shape of the letters O or C. P. micans had 1–7 nucleoli of various sizes and shapes with NORs in various complicated forms. The number of nucleoli bore a certain relationship to the living state of the dinoflagellate. One day after fresh medium was added, cells with 3 nucleoli were most common, and 28.5% of the individuals had 4–6 nucleoli. Cells having only one nucleolus accounted for 8.6%. 3 days after, cells with 2 nucleoli became dominant, and those with 4–6 decreased to 18.4%. After a month, cells with 1 nucleolus became most abundant, cells having 4 nucleoli decreased to 2.4%, and no cells had 5 or 6 nucleoli.

Immunogold staining: an alternative method for lymphocyte subset enumeration. Comparison with immunofluorescence microscopy and flow cytometry

An immunogold staining procedure for light microscopic enumeration of peripheral blood lymphocyte subsets defined by monoclonal antibodies (OKT3, OKT4, OKI8, OKIa 1, Leu 1, Leu 4, Leu 2a, Leu 3a, Leu 10, Leu 12, B1) is described. It uses colloidal gold-labeled goat anti-mouse Ig (GAM G40 and GAM G30) as second layer and a methyl-green pyronin counterstain. Performed on small volumes of blood without sophisticated laboratory equipment, this method allows accurate cell type recognition and permanent records, essential for longitudinal observations. By enumerating the gold particles on positively labeled cells, it was shown that the staining reactivity depended on the monoclonal antibody used. Lymphocytes reacting with OKT8 or OKIa 1 or B1 exhibited the strongest labeling whereas OKT4 + cells were weakly labeled. When compared with flow cytometry analysis in healthy subjects, the accuracy, precision and sensitivity of both methods were very similar. Similarly, a close correlation (97%) was found between immunogold staining and immunofluorescence microscopy in 35 patients with various diseases suggesting that immunogold staining may be useful in a clinical context.

The presence of Theileria sergenti in Haemaphysalis longicornis overwintering in pasture in Japan

Unfed Haemaphysalis longicornis ticks were collected from the soil of pasture during the winter and their salivary glands were examined by the methyl green pyronin staining method in order to detect overwintering of Theileria sergenti, the causative agent of bovine theileriosis in Japan. A transmission experiment was carried out using these ticks applied to a splenectomized calf. The protozoa which appeared in the peripheral blood were identified parasitologically and seroimmunologically as T. sergenti. The results suggested that T. sergenti in Japan might overwinter in H. longicornis which moulted after repletion on infected cattle in the autumn and hid in the soil during the winter. Moreover, it seemed possible that T. sergenti might develop to the infective stage in H. longicornis during the overwintering period.

A Rapid Method to Select Rhipicephalus appendiculatus Salivary Glands Infected with Theileria parva

Light microscopic studies on the detection and development of the salivary gland stages of Theileria parva in feeding adult ticks, Rhipicephalus appendiculatus, have involved the use of various stains such as Feulgen (Blewett and Branagan, 1973, Trop. Anim. Hlth Prod. 5:27-34), Giemsa (Pumell and Joyner, 1968, Parasitology 58: 725732) and Methyl Green Pyronin (MGP) (Walker et al., 1979, Trop. Anim. Hlth Prod. 11: 21-26; Irvin et al., 1981, Parasitology 82: 137-147). Although the method developed by Irvin et al. (1981, loc. cit.) significantly increased the speed with which parasites could be detected and examined, it still involved the dissection and teasing out of glands onto a microscope slide, followed by fixing, rinsing, staining, mounting in DPX under cover-slips and examination by a light microscope. We report here the development of a method which allows detection and selection of heavily infected salivary glands based on direct staining of a small part of 1 salivary gland in a drop of MGP. When adult ticks were immobilized in paraffin wax and the cuticle and gut removed, it was possible to fix and stain the salivary glands without removing them from the tick and thus detect T. parva-infected salivary gland acini using a dissection microscope at x 50 magnification. Carnoy's fixative (absolute ethanol 60%, chloroform 30%, glacial acetic acid 10%), was placed on each dissected tick for 5 min, followed by a rinse with 70% ethanol and one rinse with distilled water for 2 min each. Staining was carried out with a drop of MGP (aqueous Methyl Green 0.04%, aqueous Pyronin Y 0.02% in 0.5 M acetate buffer, pH 4.8) for 8 min. Nuclei of uninfected acinar cells stained blue whereas their cytoplasm containing RNA stained pink. The cytoplasm of infected cells containing parasite nuclear chromatin stained blue, allowing them to be distinguished from uninfected cells. In attempts to further simplify the method, it became clear that fixation prior to staining was not necessary for detection of the parasite. This observation then led to the development of a protocol for the selection of heavily infected salivary glands. Salivary glands infected with T. parva were obtained by feeding R. appendiculatus nymphs on cattle reacting to stabilate-induced infection (Cunningham et al., 1973, Int. J. Parasit. 3: 583587). Four-day fed adult female and male ticks were mounted in paraffin wax in a Petri dish and individual ticks numbered in the wax near the mouthparts. As many as 100 ticks could be mounted in 1 Petri dish. The dissection medium usually consisted of Dulbecco's phosphate buffered saline pH 7.4 (PBS), but other media such as Hanks' Balanced Salt Solution, Dulbecco's Minimum Essential Medium or Leibovitz's L1 5 could also be used. The dorsum and gut of the ticks were removed and the dissecting medium replaced by fresh PBS. A small piece of salivary gland was removed from every tick and deposited in a drop of PBS in a numbered well in the top row of a Wellcome PTFE Multispot slide (C. A. Hendley, Essex, UK), the numbers of the wells corresponding with the numbers of the ticks. The wells in the bottom row of the slide contained a drop of MGP. All salivary gland pieces were then transferred to the wells in the bottom row containing the MGP and left to stain for 8 min before transfer back to the original drops of PBS on the top row of the slide. Glands were examined and their infection level assessed. Salivary gland pieces from 15 to 20 ticks were routinely processed at a time. The numbers of the glands that contained many infected acini were recorded. When screening of ticks was completed and the numbers of all heavily infected ticks recorded, the remainder of the gland from which a sample was taken, together with the second gland, were removed for experimental use. Particular advantages of this method are its rapid process and minimal risk of losing or damaging the infected glands. This technique has been successfully applied for the selection of heavily infected tick salivary glands used for immunizing mice for the production of anti-sporozoite mon-

The maturation of Theileria annulata in Hyalomma anatolicum anatolicum stimulated by incubation or feeding to produce sporozoites

Adult Hyalomma anatolicum anatolicum ticks infected with Theileria annulata (Hissar strain) were incubated at 36°C or fed on rabbits. Tick salivary glands were stained whole with methyl green pyronin or ground up and deposited on microscope slides and stained with Giemsa's solution. Separate batches of ticks from both treatments were ground up, centrifuged and filtered to produce sporozoite suspensions. The suspensions were examined as deposits on microscope slides stained with Giemsa's solution. The Theileria in the salivary glands of the fed ticks matured more completely and rapidly than in the incubated ticks. The peak numbers of sporozoites from the fed ticks was greater by at least tenfold than the peak from the incubated ticks. This peak was on the third day of feeding or on the fourth day of incubation. It was confirmed that fed ticks will be more suitable for sporozoite production for infection of cattle and production of stabilates.

Natural infection rates of Hyalomma anatolicum anatolicum with theileria in Sudan

Hyalomma anatolicum anatolicum ticks were collected around cattle pens at two locations close to Khartoum. They were assessed for theileria infection by four methods. Salivary glands were stained whole with methyl green pyronin and examined for parasite masses. Adult ticks were partially fed on rabbits, ground up in medium, and the suspensions were examined in Giemsa stained smears and by inoculation into bovine lymphocyte cultures. Ticks were fully fed on calves which were monitored for developing theileriosis. H a anatolicum were found infected with parasite masses similar to those seen in experimental infections with T annulata. At one site 38 per cent of 102 ticks were infected and the mean number of parasite masses per tick for the whole sample was 37. At the other site 86 per cent of 156 ticks were similarly infected and the mean parasite masses per tick was 19.5. Suspensions of sporozoites contained sporozoites typical of those found in experimental preparations of T annulata. Sporozoites harvested from ticks from both locations infected and transformed normal bovine lymphocyte cultures. H a anatolicum ticks from both locations produced fatal theileria infection in susceptible calves.

Methyl Green-Pyronin staining with pure Pyronin Y.

Methyl Green-Pyronin staining with pure Pyronin Y.

C-banded chromosomes stained with methyl green-pyronin.

C-banded chromosomes stained with methyl green-pyronin.

Morphology and transcriptional activity of mouse oocyte chromosomes.

Lampbrush chromosomes of growing amphibian oocytes carry thousands of lateral loops each of which consists of a chromatin fiber heavily encrusted with nascent ribonucleoprotein fibrils. These are believed to be responsible for the accumulation and maintenance of RNA transcripts found stored in the egg. In the case of mammalian oocytes, lampbrush chromosomes are most likely to occur during the major growth phase and also possibly during pachytene-early diplotene stages of meiosis. We have examined pachytene and early diplotene mouse oocyte chromosomes through the light microscope using sections of plastic-embedded material and air dried spreads stained with either silver nitrate or methyl green pyronin. Our results indicates that the projections radiating from the chromosomal axis are bundles of chromatin fibers rather than single fibers covered with an ribonucleoprotein matrix. These bundles may represent partially unfolded chromomeres. The axis itself could be partially dispersed revealing threads surrounding a fine linear element. -- Little is known about chromosome structure in growing mammalian oocytes, the stage when transcriptional activity is likely to be most rapid. In our preparations chromosomes at this stage appear as partially condensed fuzzy threads of relatively uniform width. In some cases, the fuzzy thread is seen to contain a dense linear core in the center. Thus, during the growth phase, the chromosomes retain a relatively condensed axis, a characteristic of meiotic chromosomes in general. RNA-containing material is found diffusely spread within the nucleus but not specifically associated with the chromosomes. Electron microscopic analysis of spread chromatin from growing oocytes demonstrates that most transcription units possess only one or two nascent ribonucleoprotein fibrils while a few have more. These and other published data indicate that mouse oocytes do not have true lampbrush chromosomes at any stage of their development.