Bismuth Staining Research Articles

Assemblage of Bacterial Saccharic Microfibrils in Sheath Skeleton Formed by Cultured Leptothrix sp. Strain OUMS1

Mitsuaki Furutani, Tomoko Suzuki, Hiromichi Ishihara, Hideki Hashimoto, Hitoshi Kunoh and Jun Takada-Assemblage of Bacterial Saccharic Microfibrils in Sheath Skeleton Formed by Cultured <em>Leptothrix</em> sp. Strain OUMS1

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni.

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.

Immunocytochemistry, autoradiography, in situ hybridization, selective stains: complementary tools for ultrastructural study of structure-function relationships in the nucleus. Applications to adenovirus-infected cells.

A significant amount of new information on structure-function relationships in nuclei of adenovirus-infected cells has accumulated during the last decade as a result of the combined use of several new cytochemical techniques. Localization of viral DNA on ultrathin sections of infected cells has been investigated at the ultrastructural level by using specific DNA staining and immunocytochemistry with monoclonal anti-DNA antibodies. Both techniques, however, concomitantly visualize cellular and viral DNA. The specific stain for DNA reveals the configuration of the DNA molecules in the different nuclear substructures, whatever their synthetic activities. The immunodetection of DNA reveals that specific antibodies strongly bind to DNA of condensed host chromatin and to both encapsidated and nonencapsidated inactive viral genomes. However, the observation of an abnormally low level of labeling over the substructures in which synthetic activities of viral genomes are known to be intense demonstrates a serious limitation of this technique for the detection of active DNA. Postembedding in situ hybridization is the most useful method for identifying with certainty the structures containing defined nucleic acid sequences. By using a biotinylated viral DNA probe, in situ hybridization provides specific identification of structures containing either viral DNA or viral RNA molecules. In addition, with appropriate pretreatment of the sections, it is possible to reveal either all the viral DNA--that is, both double- and single-stranded DNA molecules (dsDNA, ssDNA)--or more specific species such as only ssDNA or only dsDNA molecules. The replicative and transcriptional activities of viral genomes are determined by high-resolution autoradiography. Autoradiography after a short pulse incorporation of appropriate radioactive precursors by infected cells reveals the sites of cellular and viral DNA replication or transcription. A short pulse followed by chase periods of different durations reveals the progressive migration of the cellular and viral synthesized products. The in situ distribution of the viral 72 kDa DNA-binding protein, a highly phosphorylated protein which protects the viral ssDNA, is revealed either by immunocytochemistry with specific antibodies or by the bismuth staining method which stains all highly phosphorylated proteins, including both cellular and viral proteins. The combined results of all these cytochemical procedures reveal the composition and functions of some of the structures induced by adenovirus infection. They demonstrate that viral genomes engaged in replication lead to the formation of the replicative foci in which two compartments rapidly develop, one of which results from the aggregation of single strands of viral DNA and their accompanying 72 kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructure of proteinase-secreting cells of Candida albicans studied by alkaline bismuth staining and immunocytochemistry

The ultrastructure of Candida albicans cells induced to secrete extracellular proteinase (EPR) has been studied. Electron microscopy employing alkaline bismuth staining, a method which stains polysaccharides, clearly revealed Golgi-like bodies and secretory vesicles in C. albicans cells. After EPR induction, there was no apparent increase in the number of these structures. Instead, many flocculent granules appeared at the periphery of induced cells. The granules were similar to secretory vesicles in size, but were more irregular in shape. Similar granules were observed in non-induced cells, though less frequently than in induced cells. Brefeldin A, a specific inhibitor of membrane transport in the secretory pathway, caused the accumulation of EPR and Golgi-like bodies in EPR-induced cells, but did not affect the accumulation of the granules. These results suggest that the granules are unrelated to EPR secretion. Electron microscope immunocytochemistry with affinity-purified anti-EPR antibodies showed that the granules in EPR-induced cells were recognized by the antibodies. This recognition was completely inhibited by the presence of glycogen, suggesting that antibodies cross-react with glycogen-like polysaccharides in the granules. Although the location of EPR within the cells remains unclear, the results suggest that EPR might be secreted via the constitutive secretory pathway, and that EPR is glycosylated to give a structure with some similarity to glycogen.

Detection by bismuth staining of highly phosphorylated nucleo-proteins in plants. Determination of its specificity by X-ray microanalysis, SDS-PAGE and immunological analysis

Summary— Staining with bismuth salts after glutaraldehyde fixation is a very useful technique for preferential detection of phosphorylated nucleoproteins in mammalians and insects. In the present work we report an adaptation of this method for plant nuclei: staining with bismuth salts either in tissue blocks before embedding, or on thin sections of acrylic resin. Both procedures are highly reproducible and give the same pattern of staining in the nuclei in situ or isolated at the electron microscope. The specificity of bismuth binding to the dense nucleolar fibrillar component and interchromatin granules is proven by X‐ray microanalysis. The nuclear proteins which bind bismuth have been identified by bismuth and immunostains of blots from total nuclear proteins. This technique is a very useful and specific cytochemical tool for studying nuclear organization and functions in plants.

Identification and localization of a nucleolin homologue in onion nucleoli

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogensis.

Ultrastructure of the concentric membrane system in Arthrinium aureum.

An ultrastructural study was performed on Arthrinium aureum. The fungi were treated with glutaraldehyde and osmium tetroxide fixation. The hypha and conidia has a concentric membrane system which consisted of multiple membranes of a myelinoid appearance, and continued to the conidia and hypha plasma membrane. The fungi were also treated with periodic acid-alkaline bismuth (PABi) staining after glutaraldehyde and osmium tetroxide fixation. PABi positive materials were found on the marginal glycogen granules, the concentric membrane system and the conidia plasma membrane.

Ultrastructural, cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.

Simultaneous detection of highly phosphorylated proteins and viral major DNA binding protein distribution in nuclei of adenovirus type 5-infected HeLa cells.

Highly phosphorylated proteins in situ in sections of Lowicryl-embedded cells are preferentially stained by bismuth, provided that the reactivity of the amino groups is blocked by glutaraldehyde fixation. This study showed that bismuth staining can be preceded by indirect immunocytochemistry using gold particles as markers. As a result, both immunostained and bismuth-stained proteins can be detected concomitantly on the same section. This was also carried out on sections of formaldehyde-fixed cells which were immunolabeled, then post-fixed with glutaraldehyde, and finally exposed to bismuth stain. These procedures were applied to sections of adenovirus Type 5-infected HeLa cells. Bismuth ions and viral anti-72 KD antibody bound concomitantly to intranuclear virus-induced single-stranded DNA (ssDNA) accumulation sites, structures in which viral replicative activity is intermittent, and also to the fibrillogranular peripheral replicative zones which surround the ssDNA accumulation sites and in which replication of viral genomes is continuous. The delicate fibrillar network enclosed within virus-induced compact rings of unknown function is slightly bismuth stained and binds few antibodies to viral 72 KD protein. Three intranuclear structures were stained exclusively with bismuth: the fibrillar component of the nucleolus, which is involved in ribosome formation; the interchromatin granules; and the virus-induced "fibrillar spots" of unknown significance. Thus, not all highly phosphorylated proteins in adenovirus-infected cells are viral 72 KD protein. In glutaraldehyde-fixed Miller spreads of nucleic acid molecules from adenovirus-infected cells, bismuth deposits occurred over unique thick filaments, the only portion of the viral deoxyribonucleoprotein molecules shown to be associated with viral 72 KD protein. In vitro studies revealed that the latter protein, known to be multiply phosphorylated, concomitantly binds anti-72 KD antibody and bismuth ions. These data have broadened the scope of the use of bismuth staining. Taken together, they indicate that in adenovirus infection highly phosphorylated proteins accumulate over intranuclear structures related to both replication of viral genomes and alteration of ribosomal metabolism.

Perichromatin Granule-like Granular Structures in the Nuclei of Antral-Follicular Oocytes of the Chinese Hamster

In the nuclei of antral-follicular oocytes of the Chinese hamster (Cricetulus griseus), numerous granular structures ranging from 60 nm to 250 nm in diameter were present in the nucleolar area, and a few were observed on the surface of heterochromatin materials and within the interchromatin space. When treated with bismuth staining en bloc after glutaraldehyde fixation (GA-Bi staining), these granular structures were shown to be composed of fine fibrils intensely contrasted with bismuth, indicating that these may be regarded as one type of perichromatin granule.

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosomephase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late capphase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.

Activation of carbohydrate-containing golgi vesicles in susceptible leaf cells of Japanese pear responding with AK-toxin.

AK-toxin from the Japanese pear pathotype of Alternaria alternata caused plasma membrane modifications in cells of susceptible Japanese pear leaves 1hr after toxin exposure, but not in cells of resistant leaves even after 10hr of toxin exposure. In susceptible cells, modifications were characterized as invagination and fragmentation of plasma membrane, accumulation of amorphous materials exterior to plasma membrane, and occurrence of tubular structures from plasmodesmata. Additionally, Golgi vesicles increased in number within 6hr after toxin exposure, but markedly decreased at 10hr in susceptible cells. An alkaline bismuth stain for carbohydrates well reacted with both Golgi vesicles and the materials inside them. Golgi vesicles which were observed in peripheral cytoplasm beneath invaginated plasma membrane appeared to fuse with the plasma membrane and to secrete bismuth-reactive amorphous materials containing carbohydrates. Ultrastructural changes were not observed in cell wall near modified plasma membrane of toxin-treated susceptible cells. Activation of Golgi activity which was observed in toxin-treated susceptible cells did not occur in toxin-treated resistant cells. These ultrastructural results suggested that the bismuth-reacted amorphous materials were not degenerated products from cell wall, but rather were formed by secretory products from Golgi vesicles.

Appearance of Perichromatin Granules in Decondensing Sperm Nuclei in Fertilized Mouse Eggs

A number of granular structures measuring about 35-45 nm in diameter appeared in the dispersing chromatin materials of the decondensing sperm nuclei in fertilized mouse eggs. These granules were contrasted by the bismuth staining en bloc following either formaldehyde or glutaraldehyde fixation, and were encircled by an electron-lucent "halo." The facts indicate that these are perichromatin granules.

The effect of adenosine analogue (DRB) on a major nucleolar phosphoprotein nucleolin

Nucleolin, a phosphorylated nucleolar protein, of 100 kDa selectively stained with bismuth tartrate and silver nitrate, is implicated in the transcription and maturation of pre-ribosomal RNA. Nucleolin also fulfills a structural function in nucleolar organization. Using immunocytochemistry the action of 5-6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of hn/RNA synthesis known to modify the organization of the nucleolus, was studied for its effects on the distribution and the amount of nucleolin present. After DRB treatment, the morphology of the nucleolus was rapidly disturbed, but the distribution of the nucleolin remained unchanged: the dense fibrillar and the granular components were always positively immunostained. Thirty min after incubation with the drug, a strong increase of the amount of nucleolin occurred. Prolonged treatment led to a marked loss of label. Silver and bismuth staining showed that DRB does not seem to significantly affect the phosphorylation of nucleolin.

Interchromatin granules during phytohemagglutinin stimulation of human lymphocytes

We studied the formation of interchromatin granules (IGs) in phytohemagglutinin (PHA)-stimulated lymphocytes. The bismuth staining method was used for the visualization of IGs, and we also applied high-resolution autoradiography after incubating cells in the presence of 3H-leucine during different stages of lymphocyte activation. The disaggregation of chromatin and the enlargement of interchromatinic areas in stimulated lymphocytes were found to be accompanied by an increase in the number of IGs, and it was shown that IGs were formed during all of the investigated stages of lymphocyte stimulation.

Golgi vesicles fused with invaginated plasma membranes in host cells treated with a host-specific toxin from the Japanese pear pathotype of Alternaria alternata.

The secretory activity of Golgi bodies in Japanese pear leaves treated with AK-toxin was examined with an electron microscope by staining sections with alkaline bismuth stain as a tracer of Golgi vesicles to discern the viability of the toxin-treated susceptible cells. The toxin caused the invaginations of plasma membranes and the loss of abundant membranous fragments from the invaginated plasma membranes in the susceptible cells. In the susceptible cells treated with the toxin for 1, 3, and 6hr, many Golgi vesicles which were positive to alkaline bismuth stain were present at the peripheral cytoplasm beneath invaginated plasma membranes and some of them fused with the invaginated plasma membranes. The accumulation and fusion of the Golgi vesicles were rarely found in cells of the necrotic susceptible leaves treated with the toxin for 10hr. However, the phenomena were not observed in the toxin-treated resistant cells and the water-treated susceptible ones. The susceptible cells were viable within 6hr after the toxin treatment because the cells had the high activity of Golgi bodies and the high ability of plasma membranes to fuse with many Golgi vesicles. On the contrary, the susceptible cells appeared not to be viable 10hr after the treatment because they had both the low activity of Golgi bodies and the poor ability of plasma membranes. The results indicate that the enhanced production of Golgi vesicles may be associated with the viability of cells in the toxin-treated susceptible leaves, and that the produced Golgi vesicles may be supplied for repairing the damaged plasma membranes.

Alkaline Bismuth Solution as anEn BlocStain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.

Electron microscopic study on the bismuth staining of nucleoli in growing mouse oocytes.

The electron microscopic bismuth staining method of Locke and Huie (16) was applied to the nucleoli of dictyate-stage growing mouse oocytes. After bismuth staining following glutaraldehyde fixation (GA-Bi staining), bismuth was largely localized in the fibrillar centers and the adjoining zones of dense fibrillar components not only in the nucleoli of growing oocytes during the unilaminar, bilaminar and plurilaminar follicle stages, but also in the nucleoli of mature antral-follicular oocytes. These results suggest that the materials stained with the GA-Bi method in the nucleoli of mouse oocytes may have no direct correlation with the transcriptional activity of ribosomal DNA.In the nucleoli of antral follicular oocytes, the large spherical bodies, consisting of condensed fibrillar components, were unstained with GA-Bi staining but densely stained with bismuth following formaldehyde fixation (FA-Bi staining). This fact suggested the presence of nuclear basic proteins such as histones and protamines in these nucleolar bodies.

Alkaline Bismuth Stain as A Tracer for Golgi Vesicles of Plant Cells

An alkaline solution of bismuth subnitrate reacts well with carbohydrate-rich components of Golgi bodies in sections prepared from plant leaves fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. The metal deposits formed are so fine that the stain is appropriate to ultrastructural observation at high magnification. The Golgi vesicles show polarity with respect to the localization of the reactive deposits. Golgi vesicles that had migrated farther from the Golgi cisternae showed greater reactive deposits and higher membrane contrast than those close to the Golgi cisternae. These results indicate that the alkaline bismuth stain is an excellent tracer for Golgi bodies of plant cells.

Localization of highly phosphorylated proteins in cells altered by Herpes simplex virus infection

Highly phosphorylated proteins detectable by their ability to bind bismuth ions were localized in rabbit fibroblasts before and during infection with Herpes simplex viruses type 1 and type 2. The bismuth tartrate procedure of Locke and Huie applied to glutaraldehyde-fixed cells revealed a low level of bismuth binding in a restricted portion of the normal nucleolus in non-infected cells. From 2.5-17 hr post-infection during virus development and maturation, the phosphorylated proteins were more widespread and the intensity of reaction was augmented. Bismuth deposits were then associated with virus-modified pre-existing structures including all of the nucleolar fibrils, the more abundant interchromatin granules, reduplications of some areas of the inner nuclear membrane and the Golgi apparatus. Virus-induced structures which were stained included nuclear dense bodies, the teguments of enveloped virions and the contents of extranuclear enveloped structures devoid of capsids. Following detergent-induced destruction of membranes, staining was lost from the nuclear envelope and cytoplasmic virions, which demonstrated that the highly phosphorylated proteins were tightly bound to nuclear and viral membranes. Bismuth staining of nitrocellulose sheets containing proteins extracted from whole cells revealed no reaction in normal cells but three positive bands were found in infected cells.