Methyl Green-pyronin Research Articles

Distribution and Metabolism of RNA in Entamoeba histolytica Grown in CLG Medium: Autoradiographic and Cytochemical Observations

Autoradiographic analysis of E. histolytica grown with Bacteroides which were prelabeled with uridine-5-H' (U5H') is described. Tritium was detected mostly in RNase-sensitive material in cytoplasm, and rarely the nuclei, of amebae at all stages of their 72-hr growth period. The most intense label was detected after the initial 24-hr period of growth. Since this was also previously observed in amebae propagated with exogenous U5H3, at least some of the activity reported in the previous study is due to labeled RNA derived from ingested bacteria. Some of the incorporated tritium was sensitive to DNase I (and the buffer in which the enzyme is dissolved). Of interest was the finding of a small amount of activity associated with compounds resistant to digestion with RNase plus DNase (RNase-resistant RNA?). Methyl green-pyronin demonstrated both diffuse and particulate pyronin-positive material in the cytoplasm of amebae. As was true of tritium incorporation, this was also more extensive after the initial 24-hr period of growth. RNase removed essentially all the diffuse stain. DNase also removed a small amount of such diffusely stained material. The particulate pyroninpositive material was only partially removed with RNase; it was also slightly DNase-sensitive and some of it was still retained after digestion with RNase followed by DNase or DNase followed by RNase. Trypsin (used before or after nuclease digestions) was without effect on the particulate pyronin-positive material. The loss of tritium from amebae which were prelabeled for 72 hr with U5H3 was also studied. As expected, activity which is more uniformly dispersed in these amebae, as compared to those propagated with prelabeled bacteria, does decline over a 72-hr period. Such label was more sensitive to RNase and DNase digestions, including buffers, than were cells grown with prelabeled Bacteroides. Autoradiographic evaluation of the uptake of tritium from exogenously supplied uridine5-H3 (U5H3) in RNase-sensitive material (RNA) in Entamoeba histolytica propagated in CLG medium with protoplasts of Bacteroides sp. has been reported (Sharma et al., 1969b). Such data provided information on the relative amounts of RNA synthesized in amebae at various stages of their growth cycle. In the present study, the distribution of tritium in RNase-sensitive material in amebae fed Bacteroides which were prelabeled with U5H3 was determined, and compared with the autoradiographic results previously obtained when exogenous U5H3 was used. Also included in this study are cytochemical data (viz., RNasesensitive pyronin-positive material) designed to determine the relative amounts and distribution of RNA in amebae at various stages of

Cytochemical and Autoradiographic Analyses on the Embryo Suspensor Cells ofPhaseolus Coccineos

SUMMARYSpecific staining methods (Feulgen, methyl green-pyronin, toluidine blue molybdate, acridine orange), digestion tests (DNase and RNase) and autoradiographic techniques (incorporation of tritiated thymidine, uridine, lysine, tryptophan; DNA detection by means of tritiated actinomycin D) have been used to study the embryo suspensor of Phaseolus coccineus. Most of material studied consisted of developing seeds in which the cotyledons of the embryo occupied one third to one half of the endospermatic cavity. In the typically club-shaped suspensor, both the « handle » portion (which contains cells with low to medium degree of endopolyploidy) and the « knob » portion (which contains polytene chromosome cells) have been analyzed.Indications have been found that the cells with low and medium degree of endopolyploidy undergo extra DNA synthesis (gene amplification) on many heterochromatic chromosome regions during early stages of embryogenesis. Later on, the extra DNA synthesized seems to be released, from t...

Cutaneous Extramedullary Plasmacytomas

Extraosseous tumours of the skin were observed in three patients with multiple myeloma. Routine histopathologic study of paraffin-embedded specimens from these tumors suggested the diagnosis of lymphoma or reticulum-cell sarcoma. The cutaneous myeloma cells stained in a characteristic fashion with methyl green-pyronin and touch imprints of the tumors stained with Wright's stain demonstrated, however, typical myeloma cells. The myeloma serum proteins from two patients were characterized as IgG-L and BJP-K, respectively. These immunoglobulins or immunoglobulin fragments were not demonstrated in the cutaneous metastases by direct fluorescent staining. Touch imprints from one tumor revealed positive cytoplasmic staining for IgM-K and IgM-L in medium-sized lymphocytes, but not in tumor cells. In cryostat-sectioned specimens from this tumor, these fluorescent cells appeared to buttress the tumor masses in the skin. The possibility that these cells may reflect a host mechanism for rejecting tumor cells is suggested.

Methyl green-pyronin stain for the diagnosis of trachoma.

Methyl green-pyronin stain for the diagnosis of trachoma.

Effects of temperature, poststaining rinses and ethanol-butanol dehydrating mixtures on methyl green-pyronin staining.

Methyl green GA (Chroma) and pyronin GS (Chroma) were used. Procedure recommended: Stain for 1 hr at 37 C in a purified 0.5% aqueous methyl green, buffered to pH 4.1 with Walpoles acetate buffer, and containing 0.2% pyronin; rinse for 1-2 sec in ice-cold distilled water; blot sections evenly, and rinse with vigorous agitation in t-butanol; dehydrate in 2 changes of t-butanol for 5 min each; clear in xylene and mount. This technique results in a consistent staining pattern for qualitative nucleic acid differentiation, whereas older methods have been only partly satisfactory. Rinsing in ice-cold water is a critical step; t-butanol was superior to n-butanol and to ethanol-butanol mixtures for dehydration. Staining at 25-27 C is feasible hut less effective.

Refractile bodies in the developing and mature spermatozoa of Childia groenlandica (turbellaria: Acoela) and their possible significance.

1. Living and fixed mature spermatozoa of Childia groenlandica (Turbellaria: Acoela) have conspicuous refractile bodies present behind the oval nucleus, in a double longitudinal row in the tail; an undulating membrane is also evident, as well as (in some cases) a slender head filament which is not a flagellum but which may be the result of an acrosome reaction. The living spermatozoa are 50-60 microns long and moderately motile. In living and fixed specimens, the refractile bodies are found at least as early as the secondary spermatocyte stage and increase in numbers rapidly during the spermatid stages.2. The following results were obtained with cytochemical tests of the refractile bodies: (a) Feulgen, for DNA, negative; (b) methyl green-pyronin and azure B, for RNA, positive before and after RNase and PCA extraction; (c) alcian blue/ PAS for polysaccharides, positive before and after diastase digestion, for small accumulations of acid mucopolysaccharide (s) in centers, only, of refractile bodies; (d) Sud...

A new type of inclusion bodies in lymphocytes.

Inclusion bodies could be demonstrated in about 8 per cent of circulating lymphocytes from a female, age 70, suffering from chronic rheumatoid arthritis and leukopenia. By light microscopy the inclusions, which measured about 1 to 4 μ. in diameter, gave a reddish‐purple colour with May‐Grünwald Giemsa stain. They were faintly stained with Sudan black or PAS, but were not stained with Toluidine blue, Feulgen, or Methyl green pyronine. By electron microscopy bundles of tubules localized in cytoplasmic vacuoles or in the cytoplasmic matrix could be demonstrated. The outer diameter of each single tubule was approximately 420 Å, and the diameter of its surrounding membrane was 140 Å. The single tubule or bundles of tubules were often coiled. The mitochondria were sometimes arranged along the periphery of the inclusions.Similar inclusions were not found in other members of the patient's family, and it is suggested that the inclusions are related to the disease(s) and/or treatment of the patient.

Distribution of Immunoglobulins in Human Rectal Mucosa

Summary Eight normal subjects were studied utilizing a simplified technique for determining the number of pyroninophilic and immunoglobulin-containing cells per unit area of interstitial tissue in the rectal mucosa. Histologically normal proctoscopic biopsy sections were stained with methyl greenpyronin and fluorescein-labeled monospecific antisera against various human immunoglobulins. Cell density indices for pyroninophilic cells exceeded the sum of the cell density indices for immunoglobulin cells in each subject. IgA-containing cells predominated over IgM, IgG, and IgD cells. These quantitative histological and immunohistochemical data provide normal values for continuing studies of local immunological processes in the normal and diseased gastrointestinal tract. In addition, IgA repeatedly was identified within the apical portion of the cytoplasm of epithelial cells of rectal mucosal glands. The predominance of IgA in gastrointestinal tissues and secretions and the finding of intraepithelial cell IgA support the concept of a unique type of local IgA system, the biological importance of which remains unknown.

Distribution of Immunoglobulins in Human Rectal Mucosa

Summary This study endeavored to identify and quantitate the major classes of immunoglobulins in rectal mucosal biopsies from 9 adults with quiescent or mildly active ulcerative colitis and 7 abnormal mucosal control subjects. Serial cryostat-cut tissue sections were stained with hematoxylin-eosin, methyl green-pyronin, and fluorescein-conjugated monospecific antisera to human serum immunoglobulins. As in normal and abnormal control subjects, IgA was demonstrated within the apical portion of the cytoplasm of rectal mucosal epithelial cells in all ulcerative colitis tissues. Although IgA cells predominated over IgM-, IgG-, and IgD-containing cells, the population density of IgA cells in the lamina propria of the rectal mucosa in ulcerative colitis was lower than in normal rectal tissues. Unlike control subjects, in ulcerative colitis IgA commonly was present in extracellular interstitial mucosal sites and IgA-containing cells were distributed irregularly and some fields showed very few IgA cells. The mean number of IgG cells per unit area of mucosal interstitium also was reduced in ulcerative colitis rectal tissues. The ulcerative colitis patients and the two control groups showed no staining differences for IgM and IgD.

アオミドロ葉緑体の細胞化学的研究I

Chloroplasts of four species of Spirogyra are examined by cytochemical methods for the possible presence of DNA. Both the Feulgen reaction and methyl green-pyronin staining indicate the presence within the chloroplast of many DNA-containing bodies.These bodies are spherical or somewhat ellipsoidal in form, approximately one micron in diameter. They are located around the pyrenoid or linearly arranged in the central zone of the chloroplast. The size and location of these bodies are somewhat different in each species.These bodies resemble the DNA-containing bodies in the chloroplast of Chlamy-domonas which were previously reported by Ris & Plaut.

The effect of CO, KCN and NaN 3 on the nucleic acid content of Ehrlich ascites tumor cells

Ehrlich ascites cells were treated in vivo with a 2% aqueous solution of carbon monoxide or KCN or NaN 3. On the ninth day after transplantation the quantity of ascitic fluid, the number and average size of cells and the total packed cell volume (TPCV) were determined. Nucleic acid fractions of the cells were isolated and measured on the basis of their phosphorous content. Smears of the cells were stained with methyl-green pyronin and by the Feulgen method. It was found that after treatment with carbon monoxide the nucleic acid content of the cells increased similarly to that already shown for yeast cells. After treatment with KCN or NaN 3 the nucleic acid content of the cells decreased.

Experimental study of morphogenesis in chick embryo using azaguanine.

1. Differentiation in early chick embryo has been studied experimentally using 8-azaguanine. The chemical applied during definitive streak to head process leads to considerable malformations in the developing neural tube and other organs. When it is administered for 3 hours before the definitive streak stage similar abnormalities develop in the embryos. A 6 hours' treatment before the formation of definitive streak leads to almost complete inhibition of differentiation. 2. Azaguanine is not toxic and even in extremely malformed embryos there is no cellular damage as revealed by sections stained with methyl green-pyronine. 3. The effects of azaguanine cannot be reversed by a subsequent treatment with guanosine monophosphate or triphosphate. 4. Incorporation of labelled phenylalanine following treatment with azaguanine is considerably inhibited. 5. In the light of these results it is concluded that azaguanine is incorporated into newly synthesised ribonucleic acid and makes it functionally abnormal.

On the mechanism of the methyl green-pyronin stain for nucleic acids.

The combination of pyronin, methyl green, malachite green, crystal violet, and Alcian Blue with a large number of polynucleotides and acidic polysaccharides has been investigated. The “critical electrolyte concentration” approach has been used to provide a measure of affinity between dye and substrate. The interaction of methyl green with DNA, RNA and heparin has been examined spectroscopically. Previously published results are re-examined, and with the new experiments permit consistent interpretations of the specificities of dye binding in terms of modern ideas of nucleic acid and dye structure. All dyestuffs except Alcian Blue bind more strongly to polynucleotides than would be expected if solely electrostatic bonds were present. Pyronin and planar monovalent cationic dyes interact best with polynucleotides in which purine and pyrimidine bases are freely accessible, as in single stranded molecules without extensive secondary structure, such as RNA, denatured DNA, etc. Non-planar triphenylmethane dyes, e.g. methyl green, malachite green etc. bind less strongly to such substrates, but because of their shape they fit well into the secondary structure of native DNA. Tumour RNA and DNA did not differ from “normal” RNA and DNA. By varying the electrolyte concentration, pyronin-methyl green selectivity e.g. for DNA or RNA, can be controlled, and non-nucleotide staining suppressed. The relevance of the new interpretation to the ribonuclease-pyronin technique is discussed.

麻疹並びにジステンパーウイルス感染細胞の細胞化学的研究

The morphological changes observed in dog kidney cultured cells infected with distemper virus (Hokkaido strain) were compared cytochemically with those of measlea virus (Edmonston strain). The results were as follows. 1) Twelve to fourteen after inoculation the CPE was first observed in dog kidney cells infected with distemper virus. The appearanoe of the CPE was seven to ten days later than that of measles virus. 2) The CPE of the distemper virus infected cells was the same multinucleated giant cells, cytoplasmic inclusion bodies, and intranuclear inolusion bodies as those observed in the measles virus infected cell. 3) Both the inclusion bodies in the distemper virus infected cells also showed Feuglen negative, red staining with methylgreen pyronine, RNase resistent, and the cytoplasmic inclusion bodies blue to bluish green, otherwise the intranuclear inclusion bodies unstainable with acridine orange. This indicates that as with measles virus infection the cytoplasmic inclusion bodies have RNA, acid mucopolysacchrides and basic proteins, the intranuclear inclusion bodies no nucleic acids. 4) In the tissue culture cells inoculated with distemper virus the intranuclear inclusion bodies appeared at the same time or a few days earlier than the cytoplasmic inclusion bodies when stained with Giemsa or acridine orange. 5) The red particles in the cytoplasma of the measles virus infected cells were not detected in the cells inoculated with distemper virus when stained with acridine orange. 6) With acridine orange staining there were no differences of largeness and brightness of the nuclei and nucleoli between the distemper virus infected cells and the control cells. Then it seemed seldom to be an increased metabolism of nucleic acids. 7) Summing up, the morphorogical changes in the tissue culture cells infected with measles virus and distemper virus were quite the same except for the appearance of the CPE after virus inoculation.

Inflorescence Initiation In Lolium Temulentum l. VIII. Histochemical Changes At the Shoot Apex During Induction

Shoot apices of Lotium temulentum plants exposed to 1 long day were harvested at the beginning of the long day, and on each of the five following days. Longitudinal sections were stained for DNA (Feulgen), DNA and RNA (methyl greenpyronin or acridine orange fluorescence), or basic nuclear proteins (ammoniacal silver nitrate, fast green, or bromophenol blue, with or without acetylation or deamination).

Methyl green-pyronin as a stain for autoradiographs of plant material.

Methyl green-pyronin staining of autoradiographs successfully followed photographic processing. Stripping film was used, and staining was postponed until after film exposure to avoid extraction of labeled cellular constituents during the staining process. The transparency of the stain facilitates the localization of silver grains. Prior to application of the film, sections may be treated with nucleases. However, mild acid treatments, such as optimum hydrolysis for the Feulgen reaction, greatly reduce the intensity of the methyl green nuclear stain without removing a major DNA fraction. This result indicates that methyl green staining of DNA loses its specificity rather easily. With adequate controls, however, both stains can indicate the relative abundance of each nucleic acid. The staining procedure is: Autoradiographs are taken to water either from a dry state by means of a graded alcohol series or directly from the last photographic wash. Slides are transferred to 2 changes of dilute McIlvaine's buffer (1:10) at pH 4.2 for 5 min each, then stained 15 min in a solution consisting of 80 ml of the diluted buffer and 20 ml of a 2% aqueous solution of methyl green (Nat. Aniline), previously extracted 3-4 times with an equal volume of chloroform; dissolved in this solution is 0.5 gm of pyronin Y (G. T. Gurr) Slides are rinsed quickly in 2 changes of water, blotted and allowed to air dry. Typical results are: nuclei and chromosomes—blue; cytoplasm—pink; nucleoli—deep pink-red; and cell walls—pale blue.

SYNOVIAL LINING CELLS AND PLASMA CELLS IN THE SYNOVIAL MEMBRANE IN CASES OF CLASSIC RHEUMATOID ARTHRITIS. A STUDY BASED PARTLY ON HISTOLOGICAL SECTIONS STAINED BY THE METHYL GREEN-PYRONINE METHOD.

ZusammenfassungDer Verfasser hat eine Beziehung zwischen der Intensitat der Plasmazellen in der Synovialmembran und der Seropositivitat festgestellt. Die grosste Anzahl von Plasmazellen wurde nachgewiesen wo die Gammaglobulin-Konzentration in der Gelenkflussigkeit am grossten war, eine Tatsache, die Annahme bestatigt, dass eine zum Teil ortliche Bildung von Gammaglobulin in der Synovia stattfindet.Wenn man die Plasmazellen mit den Synovialmesenchymzellen in der rheumatischen Synovialmembran vergleicht, findet man — wenn auch diese Befunde statistisch ohne Bedeutung sind — dass die Plasmazellen fur die Bildung des (1) Gammaglobulins in der Gelenkflussigkeit und (2) den Rheumatoidfaktor von besonderer Wichtigkeit zu sein scheinen, wahrend die synovialen Mesenchymzellen den Ausgangspunkt fur die Bildung von (1) RNA (Ribonucleinsaure) darstellen, welche in RA Fallen mit zahlreichen Kriterien als Ausdruck fur eine intensive Proteinbildung in diesen Zellen zunimmt. Ausserdem konnen die synovialen Mesenchymzelle...

The nucleoli in mitotic divisions of mammalian cells in vitro.

In a number of mammalian cell strains nucleoli persisted through mitosis. This phenomenon was especially pronounced in several cell lines derived from Chinese hamster tissues. All the methods employed, including radioautography with tritiated uridine, cytochemical stains (methyl green-pyronin and azure B), fluorescent microscopy (coriphosphine O), ribonuclease digestion, and electron microscopy, demonstrated that the bodies identified as persistent nucleoli in the mitotic stages had the same characteristics as did the nucleoli in the interphase. Persistent nucleoli may attach to the chromosomes or may be free in the cytoplasm. In cells where no persistent nucleoli as such were noted, nucleolar material was observed to attach to the chromosomes in shapeless masses which moved with the chromosomes during anaphase. At least a portion of the nucleolar material was included in the daughter nuclei, presumably for immediate use for protein synthesis after cell division.

USE OF A FLUORESCEIN-LABELED SONICALLY DISRUPTED BACTERIAL ANTIGEN TO DEMONSTRATE ANTIBODY-PRODUCING CELLS.

Eveland, Warren C. (University of Michigan, Ann Arbor). Use of a fluorescein-labeled sonically disrupted bacterial antigen to demonstrate antibody-producing cells. J. Bacteriol. 88:1476-1481. 1964.-Cells obtained by primary tissue culture of the spleens of chickens immunized with sonically disrupted Escherichia coli O111 organisms were stained with a fluorescein-labeled homologous antigen by use of direct immunofluorescent methods. Brilliant staining of the cytoplasm in cells from immunized birds appeared to be diffuse in certain cells and rather globular in others. In contrast, cells from nonimmunized birds showed no staining at all. The cells involved in the specific reaction appeared to be those of the lymphocyte-monocyte-plasma cell types, as shown when stained by the May-Grunwald-Giemsa method. Preparations stained by the methyl green-pyronin technique revealed an increase in the pyrinophilic cells in the preparations from the immunized birds, thus demonstrating increased amounts of ribonucleic acid in these cells, which in turn is consistent with the presence of antibody globulin. Specificity of the reaction was confirmed also by (i) staining antibody-coated E. coli O111 organisms with the conjugate, (ii) precipitin reaction with specific antibody, and (iii) specific agglutination with circulating antibody from the immunized birds.

Cytochemical Observations on the Interphase Nucleus of the Parasitic Ameba Hydramoeba hydroxena (Entz)

The study was undertaken to determine the distribution of DNA, RNA, and histone in the nucleus, and thereby to clarify the nature of the peripheral layer. Whereas the endosome contains granules and a network which are definitely Feulgen-positive, the peripheral layer is only faintly so. Nevertheless, it contains histone and stains intensely red with methyl green-pyronin, and blue with toluidine blue. Ribonuclease removes nearly all its basophilic material, which is clearly RNA. Thus, the peripheral layer consists largely of chromatin. The original description of Hydramoeba hydroxena included an account of the general morphology of the nucleus (Entz, 1912). Further observations were made by Wermel (1925), who reported that the interphase nucleus is Feulgen-negative. A more thorough study was made by Reynolds and Threlkeld (1929), who interpreted the characteristic intranuclear mass as a compound karyosome. The peripheral karyosomal layer, in which discrete blocks are usually visible, was designated as ectokaryosomal chromatin and the central body as an endosome. The terminology is inconsistent in that it presents the paradoxical condition of an endosome within a karyosome, whereas these terms are generally used as synonyms (Mackinnon and Hawes, 1961, p. 12). For convenience, I shall retain the term endosome, but karyosome will not be used. Actually, the results of Reynolds and Threlkeld do not show conclusively that the peripheral layer contains chromatin. These authors did not use the Feulgen reaction, but based their conclusion on stainability in iron hematoxylin. Wermel's negative results are likewise inconclusive, since he counterstained all his specimens in light green. The counterstaining of Feulgen preparations in light green or fast green is not always advisable; if the reaction is weak, these dyes may obscure the characteristic reddish-purple color and lead to erroneous interpretations. It is evident that the distribution of chromatin in the nucleus of Hydramoeba hydroxena is not entirely decided. In the present study, several cytochemical methods, rather than conventional hematoxylin staining, were used in order to determine the distribution of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the nucleus, and thereby to clarify the nature of the peripheral layer. MATERIALS AND METHODS The host was the false brown hydra, Hydra pseudoligactis Hyman, which has relatively little resistance to Hydramoeba infections (Stiven, 1962). Heavily infected hydras were kindly supplied by my colleague, Dr. Alan E. Stiven. Since hydramebae attach firmly to hosts of low resistance, the infected hydras and their amebae were fixed simultaneously and subsequently sectioned in paraffin at 4 p. The ameba nuclei were studied in such sections. The following methods were used: Feulgen reaction without counterstaining; fixation, Carnoy (with chloroform) or Schaudinn (with acetic acid) (Pearse, 1960, p. 823). Methyl green-pyronin Y; fixation, Carnoy (Pearse, loc. cit., p. 826). Toluidine blue for basophilia; fixation, Carnoy; 5 to 10 min in a 0.1% aqueous solution of the dye, followed by dehydration in tertiary butyl alcohol. Ribonuclease extraction (Pearse, loc. cit., p. 916). Fast green for nuclear histone; fixation, 10% neutral formalin (Conn et al., 1960, p. 219). Received for publication 6 May 1964.