Comparative effect of osmium tetroxide and ruthenium tetroxide on Lacazia loboi ultrastructure.

Lobomycosis is a skin infection produced by the fungus Lacazia loboi, which mainly affects some indigenous and afro-descendant populations in Tropical America. We previously reported the comparative effect of osmium tetroxide (OsO4 ) and ruthenium tetroxide (RuO4 ) in the electron microscopy (EM) of other related microorganisms. The objective of this study is to compare the effect of postfixation with OsO4 and RuO4 in the ultrastructure of L. loboi yeasts. Skin biopsies on patients diagnosed with lobomycosis were fixed in glutaraldehyde at 3% and postfixed in the following solutions: (a) 1% OsO4 , (b) 0.2% RuO4 , and (c) OsO4 at 1% followed by RuO4 at 0.2%. They were then processed using the conventional method for EM. Unlike OsO4, the treatment with RuO4 revealed different shades of gray and electron dense bands in the cell wall and other cell components of L. loboi. The most notable finding was the presence of radial filamentous structures around the yeast, which made the image look like the sun. Postfixation with RuO4 revealed ultrastructural details that had not been previously reported for L loboi. The combined use of OsO4 and RuO4 in EM of microorganisms with cell walls can be useful to evaluate the effect of microbicide substances.

Use of scanning electron microscopy in the cochlea of guinea pigs / Microscopia eletrônica de varredura em cócleas de cobaias

Abstract Introduction: The use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol. Objective: To verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy. Methods: Three groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5 mg/kg, D1-D5-D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea. Results: The organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar. Conclusion: The proposed modifications resulted in images of similar quality as those observed using the traditional methodology.

Resumo Introdução: O emprego da microscopia eletrônica no estudo da orelha interna permitiu observar detalhes minuciosos das células ciliadas especialmente em estudos de ototoxicidade. Entretanto, o preparo desse material é trabalhoso e delicado. Para simplificar a manipulação desses materiais, testou-se o uso de dois agentes, azul de toluidina e ácido etilenodiamino tetra-acético, além da retirada do tetróxido de ósmio na preparação de cócleas de cobaias albinas. Testamos também a aplicabilidade dessas metodologias em um protocolo de ototoxicidade. Objetivo: Verificar a qualidade das imagens obtidas com e sem o uso de ácido etilenodiamino tetra-acético, azul de toluidina e tetróxido de ósmio na preparação de cócleas de cobaias albinas para a microscopia eletrônica de varredura. Método: Foram utilizados três grupos de cócleas. No Grupo 1 preparou-se 10 cócleas com a metodologia usual, dissecando a cápsula ótica sem descalcificac¸ão e utilizando tetróxido de ósmio como pós-fixador. No Grupo 2 preparamos 10 cócleas descalcificadas com ácido etilenodiamino tetra-acético, injetando azul de toluidina no espac¸o endolinfático para facilitar a identificação do órgão de Corti. No Grupo 3 utilizamos 4 cócleas de cobaias que receberam 3 doses de cisplatina (7,5 mg/kg, D1-D5-D6), duas preparadas com a metodologia do Grupo 1 e duas com a do Grupo 2. Foram obtidas imagens da microscopia eletrônica de varredura da região do órgão de Corti do giro basal de cada cóclea. Resultados: O órgão de Corti foi mais facilmente identificado com o azul de touidina. A dissecção da cóclea foi mais precisa nas cócleas descalcificadas A qualidade das imagens e a preservac¸ão do órgão de Corti obtidas com as duas metodologias foi similar. Conclusão: As modificações propostas resultaram em imagens de qualidade similar as observadas com o uso da metodologia tradicional.

Use of scanning electron microscopy in the cochlea of guinea pigs.

INTRODUCTION: The use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol. OBJECTIVE: To verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy. METHODS: Three groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5mg/kg, D1-D5-D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea. RESULTS: The organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar. CONCLUSION: The proposed modifications resulted in images of similar quality as those observed using the traditional methodology.

Enantiocomplementary access to carba-analogs of C-nucleoside derivatives by recombinant Baeyer-Villiger monooxygenases.

A novel and stereoselective synthetic route towards carba-C-nucleosides was investigated applying an enantiodivergent biooxidation strategy by two different Baeyer-Villiger monooxygenases. Within only three chemo-enzymatic steps it was possible to introduce four chiral centers starting from commercially available non-chiral starting material.

The "single-section" Golgi method adapted for formalin-fixed human brain and light microscopy.

The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The "single-section" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, "sandwiched" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the "single-section" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results.

Glândula submandibular de ratos com envelhecimento: observações ao microscópio eletrônico de varredura de alta resolução / Submandibular gland of rats with ageing: observations with high resolution scanning electron microscopy

As características tridimensionais dos componentes intracelulares de células acinares e de ductos foram reveladas usando o método ósmio-DMSO-ósmio. As amostras foram maceradas em solução de tetróxido de ósmio diluído após a fratura na solução de dimetil sulfoxido. As lamelas do retículo endoplasmático granular são reveladas entremeadas por várias mitocôndrias. As lamelas do retículo endoplasmático granular são localizados ao redor dos núcleos na porção basal e estas estruturas são observadas em imagens tridimensionais de microscopia eletrônica de alta resolução.

The three-dimensional characteristics of the intracellular components of acinar and ductal cells were revealed using the osmium-DMSO-osmium method. The samples were macerated in diluted osmium after fractured in DMSO solution. The stacks of the rough endoplasmic reticulum are revealed intermingling by several mitochondria. The lamellae of the rough endoplasmic reticulum are located around the nuclei at basal portion and these structures are shown in three-dimensional HRSEM images.

Glândula submandibular de ratos com envelhecimento: observações ao microscópio eletrônico de varredura de alta resolução / Submandibular gland of rats with ageing: observations with high resolution scanning electron microscopy

As características tridimensionais dos componentes intracelulares de células acinares e de ductos foram reveladas usando o método ósmio-DMSO-ósmio. As amostras foram maceradas em solução de tetróxido de ósmio diluído após a fratura na solução de dimetil sulfoxido. As lamelas do retículo endoplasmático granular são reveladas entremeadas por várias mitocôndrias. As lamelas do retículo endoplasmático granular são localizados ao redor dos núcleos na porção basal e estas estruturas são observadas em imagens tridimensionais de microscopia eletrônica de alta resolução.(AU)

The three-dimensional characteristics of the intracellular components of acinar and ductal cells were revealed using the osmium-DMSO-osmium method. The samples were macerated in diluted osmium after fractured in DMSO solution. The stacks of the rough endoplasmic reticulum are revealed intermingling by several mitochondria. The lamellae of the rough endoplasmic reticulum are located around the nuclei at basal portion and these structures are shown in three-dimensional HRSEM images.(AU)

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae).

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.

Modulation of smooth muscle cell function: morphological evidence for a contractile to synthetic transition in the rat ventral prostate after castration.

In this study, we evaluated the involvement of rat ventral prostate smooth muscle cells (SMC) in secretory activity and whether this function is modulated after castration. Cell morphology was examined at both light and electron microscopy levels and the organelles involved in secretory function were labeled by the zinc-iodide-osmium (ZIO) method at the ultrastructural level and their volume density was determined by stereology. Castration resulted in marked changes of the SMC, which adopted a spinous aspect and abandoned the layered arrangement observed in the prostates of non-castrated rats. The volume density of ZIO reactive organelles increased progressively after castration, reaching significantly higher levels 21 days after castration. Since previous studies have demonstrated that SMC express SMC markers (even 21 days after castration) and are able to respond to adrenergic stimulation, we concluded that differentiated SMC are able to shift from a predominantly contractile to a more synthetic phenotype without changing their differentiation status.

Zinc Iodide-Osmium Tetroxide (ZIO) Reactive Golgi Apparatus in the Principal Cells of Dog Epididymal Epithelium / El Tetróxido de Zinc Iodide-Osmio (ZIO) Reactiva el Aparato de Golgi en las Células Principales del Epitelio Epididimal del Perro

The cisternae of the Golgi apparatus of dog epididymal principal cells were labeled by the zinc iodide-osmium tetroxide method (ZIO). These cisternae were observed in the supranuclear region of the cytoplasm of the epididymal principal cells. Abundant endoplasmic reticulum cisternae, multivesicular bodies, mitochondria, lysosomes and vesicular elements of variable size were also found in this region, all associated with the sacks of the well-developed Golgi apparatus. The use of the ZIO method facilitates the observation and identification of the cisternae of the Golgi apparatus, thus permitting a correlation between structure and function in the so-called Golgi area. These ultrastructural characteristics support the secretory role of epididymal principal cells in the dog.

La cisterna del aparato de Golgi de las células principales del epidídimo del perro, fueron tratados con el método de tetróxido de zinc iodide-osmium (ZIO). Estas cisternas fueron observadas en la región supranuclear del citoplasma de las células principales del epidídimo. Abundante cisternas del retículo endoplasmático, cuerpos multivesiculares, mitocondrias, lisosomas y elementos vesiculares de tamaño variable, fueron encontrados en esta región, todos asociados con los sacos del aparato de Golgi maduro. El uso del método de ZIO facilita la observación e identificación del aparato de Golgi, permitiendo efectuar una correlación entre estructura y función en el área de Golgi. Estas características estructurales suponen el rol secretorio de las células epididimarias principales en el perro.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

Comparative effect of osmium tetroxide and ruthenium tetroxide on Penicillium sp. hyphae and Saccharomyces cerevisiae fungal cell wall ultrastructure.

The fungal cell wall viewed through the electron microscope appears transparent when fixed by the conventional osmium tetroxide method. However, ruthenium tetroxide post-fixing has revealed new details in the ultrastructure of Penicillium sp. hyphae and Saccharomyces cerevisiae yeast. Most significant was the demonstration of two or three opaque diverse electron dense layers on the cell wall of each species. Two additional features were detected. Penicillium septa presented a three-layered appearance and budding S. cerevisiae yeast cell walls showed inner filiform cell wall protrusions into the cytoplasm. The combined use of osmium tetroxide and ruthenium tetroxide is recommended for post-fixing in electron microscopy studies of fungi.

Zone between plaque and attached periodontal tissues on chronic periodontitis-affected teeth: an SEM study.

The zone between plaque and attached periodontal tissues on chronic periodontitis-affected teeth was examined by a combined macroscopic and scanning electron microscopy (SEM) approach. Examined were 27 teeth with chronic periodontitis (chronic periodontitis-affected group) and three healthy teeth with no evidence of periodontal disease (control group). Both groups were collected immediately after extraction, fixed in 2% glutaraldehyde, and post-fixed in 1% osmium tetroxide. Then, teeth were macroscopically examined to identify their stained zones. Teeth were dehydrated, critical-point dried, gold coated, and examined in an SEM. Both healthy and chronic periodontitis-affected teeth showed a very similar staining pattern on their surfaces. An unstained band with a belt-like appearance was observed around the teeth, delimited by two osmium-stained zones. Some weakly stained areas were frequently observed inside the unstained band. The SEM examination showed four different regions in both groups. These regions appeared in the following coronoapical sequence: dental plaque, plaque-free zone, junctional epithelium, and attached periodontal tissues. A dental cuticle covering the cementum surface from the plaque border to the junctional epithelium was detected on chronic periodontitis-affected teeth. Some aspects of this particular zone may be involved in the pathogenesis of inflammatory periodontal disease and therefore may have some influence on treatment for chronic periodontitis-affected teeth.

Comparative effect of osmium tetroxide and ruthenium tetroxide on Penicillium sp. hyphae and Saccharomyces cerevisiae fungual cell wall ultrastructure / Efecto comparativo del tetróxido de osmio y del tetróxido de rutenio como fijadores sobre la ultraestructura de la pared celular de hongos

The fungal cell wall viewed through the electron microscope appears transparent when fixed by the conventional osmium tetroxide method. However, ruthenium tetroxide post-fixing has revealed new details in the ultrastructure of Penicillium sp. hyphae and Saccharomyces cerevisiae yeast. Most significant was the demonstration of two or three opaque diverse electron dense layers on the cell wall of each species. Two additional features were detected. Penicillium septa presented a three-layered appearance and budding S. cerevisiae yeast cell walls showed inner filiform cell wall protrusions into the cytoplasm. The combined use of osmium tetroxide and ruthenium tetroxide is recommended for post-fixing in electron microscopy studies of fungi.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts.

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

Zinc iodide-osmium tetroxide (ZIO) reactive sites in the extrafloral nectary of Citharexylum mirianthum Cham. (Verbenaceae).

This paper reports on a study of the zinc iodide-osmium tetroxide method (ZIO) applicability to formaldehyde-glutaraldehyde prefixed extrafloral nectary tissues of Citharexylum mirianthum Cham. (Verbenaceae). The ZIO solution impregnates the dictyosome stacks and adjacent vesicles, smooth endoplasmic reticulum, nuclear envelope, multivesicular bodies, and peroxisomes. The use of this method greatly facilitates the observation and recognition of organelles in each nectary region. It also allows the correlation between structure and function in nectariferous cells.

Treatment of acute and chronic synovitis by non-surgical means.

This paper reviews personal experience in the treatment of recurrent haemarthrosis and chronic synovitis by non-surgical means. Experience with synoviorthesis with rifampicine and radioactive colloids is analyzed, and a multiple chromosomal study to demonstrate safety of radioactive injections is described. The results obtained are so very satisfactory as to recommend non-aggressive synoviorthesis as the treatment of choice to prevent recurrence of bleeding. Long experience in the treatment of chronic arthropathy with intrarticular corticosteroids and hyaluronic acid has shown very promising results.

A cytochemistry study of the inner membrane complex of the pellicle of tachyzoites of Toxoplasma gondii.

Confocal laser scanning microscopy and transmission electron microscopy were used to study the inner membrane complex of tachyzoites of Toxoplasma gondii. DiOC6, a lipophilic cationic fluorescent dye used to visualize the endoplasmic reticulum of eukaryotic cells, labeled cytoplasmic structures in a reticulated pattern and the periphery of the nucleus of the host cell. Intracellular and extracellular tachyzoites were stained. Observation of several focal planes showed labeling of the most peripheral region of the protozoan. Reaction product was observed in the outer nuclear membrane, in profiles of the endoplasmic reticulum, and in the inner membrane complex of tachyzoites subjected to the KI-OsO4 technique. Taken together, these observations suggest that the inner membrane complex may represent a specialized region of the endoplasmic reticulum of tachyzoites of T. gondii.