Complete staining of nerve fiber and myoneural junctions with acetylthiocholine and silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.

Infecção experimental pelo Encephalitozoon cuniculi em camundongos imunossuprimidos com dexametasona

Objective Microsporidian Encephalitozoon cuniculi has been recognized as an opportunistic pathogen in immunosuppressed individuals, such as AIDS patients. The objective of the study was to develop pharmacologically immunosuppressed animals as a model of the natural occurring E. cuniculi infection.Methods Distint groups of adult Balb-C mice were immunosuppressed with different doses of dexamethasone (Dx, 3 or 5 mg/kg/day, intraperitoneal route - IP) and inoculated with E. cuniculi spores by IP route intraperitoneally. Control groups (inoculated animals but non-immunosuppressed and non-inoculated animals but immunosuppressed) were also used. The spores of E. cuniculi were previously cultivated in MDCK cells. The animals were sacrificed and necropsied at 7, 14, 21, 28 and 35 days post-inoculation. Tissue fragments were collected and processed for light microscopy studies, using Gram-chromotrope and hematoxylin-eosin staining techniques.Results In all immunosupressed and inoculated inoculated immunosuppressed mice,specially in those that received 5 mg/kg/day of dexamethasone, the most prominent necropsy findings were hepatomegaly and splenomegaly. The experimental inoculation resulted in a disseminated non-lethal infection, characterized by granulomatous lesions in several organs (liver lungs, kidneys, gut and brain) but notably in the hepatic tissue. Spores of E. cuniculi were only seen in few animals treated with 5 mg/kg/day of Dx at 35 days post-infection.Conclusions Microsporidiosis in Dx-immunosuppressed mice provides a useful model for studies of the microsporidial infection, resembling that one naturally occurring in immunodeficient individuals with AIDS.

Chromosomal studies on five species of the genus Leptodactylus Fitzinger, 1826 (Amphibia, Anura) using differential staining

Cytogenetic studies were carried out on five species of Leptodactylus, namely L. fuscus, L, notoaktites, L. labyrinthicus, L. ocellatus, and L. podicipinus, after standard staining, Ag-NOR and C-banding as well as BrdU incorporation for three of them. The species had 2n = 22 chromosomes and two basic karyotype patterns. Chromosome 8 was a marker bearing a secondary constriction. In all species, this secondary constriction corresponded to the Ag-NOR site. The species had centromeric C-bands in all chromosomes of the complement, but some interstitial or telomeric bands seemed to differentiate some karyotypes, either at the species or the population level. In L. ocellatus, the C-banding pattern confirmed the occurrence of a heteromorphic pericentric inversion in chromosome 8 in specimens from one of the populations. The BrdU incorporation technique showed no detectable difference in the replication patterns of the major bands in the chromosomes of L. noroaktites, L. labyrinthicus, and L. ocellatus.

Evaluation of the Possibility of Removing Staining by Repolishing Composite Resins Submitted to Artificial Aging

This in vitro research verified the possibility of eliminating staining caused by coffee and red wine in five composite resins, after being submitted to thermal cycling. Thirty-six specimens were prepared and immersed in water at 37 degrees C for 24 hours. After polishing, specimen color was measured in a spectrophotometer Cintra 10 UV (Visible Spectrometer, GBC, Braeside, VIC, Australia). All specimens were submitted to thermal cycling at temperatures of 5 and 55 degrees C with a dwell time of 1 minute, for 1,000 cycles in a 75% ethanol/water solution. After thermal cycling, the specimens were immersed in water at 37 degrees C until 7 days had elapsed from the time the specimens were prepared. All specimens were then taken to the spectrophotometer for color measurement. The specimens were divided into three groups (N = 12): distilled water (control), coffee, and red wine. For the staining process to occur on only one surface, all the sides, except one, of the surfaces were isolated with white wax. The specimens were immersed in one of the solutions at 37 degrees C for 14 days. The specimens were dried and taken to the spectrophotometer for color measurement. After this, the specimens were submitted to 20 mu m wear three times, and the color was measured after each one of the wear procedures. Calculation of the color difference was made using CIEDE2000 formula. According to the methodology used in this research, it was concluded that the staining caused by coffee and red wine was superficial and one wear of 20 mu m was sufficient to remove the discoloration.

Characteristics of gram-stained cervical smear from patients with Chlamydia trachomatis infection.

Cervical discharges from 142 women attending the Public Gynecologic Service of Araraqura (SESA), Brazil were cultured for Chlamydia trachomatis. Gram-smears and plating on semiquantitative sheep blood agar and chocolate agar were also carried out. An isolation rate of 18% was reported. The presence of purulent cervical secretion was observed in 8 (32%) out of 25 women. It was also observed that a substantial proportion of culture-positive women had no symptoms. Our data demonstrate that screening tests should be based on specific diagnostic techniques for Chlamydia trachomatis since the majority of infected women we examined were asymptomatic.

Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xyp sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques

The mitotic and meiotic chromosomes of the beetles Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) were analysed using standard staining, C-banding and silver impregnation techniques. We determine the diploid and haploid chromosome numbers, the sex determination system and describe the chromosomal morphology, the C-banding pattern and the chromosome(s) bearing NORs (nucleolar organizer regions). Both species shown 2n = 20 chromosomes, the chromosomal meioformula 9 + Xyp, and regular chromosome segregation during anaphases I and II. The chromosomes of E. atomaria are basically metacentric or submetacentric and P. dermestoides chromosomes are submetacentric or subtelocentric. In both beetles the constitutive heterochromatin is located in the pericentromeric region in all autosomes and in the Xp chromosome; additional C-bands were observed in telomeric region of the short arm in some autosomes in P. dermestoides. The yp chromosome did not show typical C-bands in these species. As for the synaptonemal complex, the nucleolar material is associated to the 7th bivalent in E. atomaria and 3rd and 7th bivalents in P. dermestoides. Strong silver impregnated material was observed in association with Xyp in light and electron microscopy preparations in these species and this material was interpreted to be related to nucleolar material.

Comparação entre o sistema Autoścan-4 e métodos convencionais na identificação de bacilos Gram-negativos não fermentadores, isolados de materiais clínicos de pacientes em Presidente Prudente - SP

Ninety eight strains of glucose-nonfermenting Gram-negative bacilli were analyzed and isolated from several clinical materials of 95 patients admitted at the Dr. Domingos Leonardo Cerávolo University Hospital and three from outpatients. All of them were assisted in the Laboratory of Clinical Analysis of Unoeste University, Presidente Prudente, SP, from the period of October of 1999 to April of 2001. In this work, the level of agreement between the semi-automated commercial system AutoScan-4 and the conventional system for the identification of those bacteria were studied comparatively. There was agreement in 81 (82.7%), showing that both methodologies are useful for identification; partial agreement in six strains (6.1%) and disagreement in 11 (11.2%). The comercial system did not identify nine (9.2%) of the strains and reported them as very rare biotypes.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Padrão de sensibilidade aos antimicrobianos de bacilos gram-negativos não fermentadores isolados de materiais clínicos de pacientes em Presidente Prudente - SP

The aim of this study was the assessment of isolation frequency and antimicrobial susceptibility pattern of nonfermenting Gram-negative bacilli. Ninety eight strains of nonfermenting Gram-negative bacilli, isolated from several clinical materials of patients admited at the Dr. Domingos Leonardo Cerávolo University Hospital and at Dr. Odilo Antunes Siqueira State Hospital, as well as from every outpatient; assisted at Laboratory of Clinical Analysis of Unoeste University, Presidente Prudente, São Paulo, in the period of October 1999 to April 2001 were analyzed. The most frequent species were Pseudomonas aeruginosa (65.3%) and Acinetobacter baumannii (23.5%). The frequency of the other isolated species was smaller than 2.5%. In the antimicrobial susceptibility tests, the two species more prevalent showed high resistance. The antibiotic most active in vitro was the imipenem, with 79.6% in microdiluition method, and 76.6% in diffusion method, for Pseudomonas aeruginosa strains and 100.0% in both microdiluition and diffusion methods, for Acinetobacter baumannii. The cephalosporins of third generation, the ciprofloxacin and the aminoglycosides, presented percentage of susceptibility varying from 22.4 to 69.7%. These results bring implications to the emergency use of the antimicrobial agents in the treatment of patients with severe infection.

Histomicrobiologic aspects of the root canal system and periapical lesions in dogs' teeth after rotary instrumentation and intracanal dressing with Ca(OH)2 pastes

OBJECTIVE: The purpose of this study was to evaluate the distribution of microorganisms in the root canal system (RCS) and periapical lesions of dogs' teeth after rotary instrumentation and placement of different calcium hydroxide [Ca(OH)2]-based intracanal dressings. MATERIALS AND METHODS: Chronic periapical lesions were experimentally induced in 80 premolar roots of four dogs. Instrumentation was undertaken using the ProFile rotary system and irrigation with 5.25% sodium hypochlorite. The following Ca(OH) 2-based pastes were applied for 21 days: group 1 - Calen (n=18); group 2 - Calen+CPMC (n=20); group 3 - Ca(OH)2 p.a. + anaesthetic solution (n=16) and group 4 - Ca(OH)2 p.a.+ 2% chlorhexidine digluconate (n=18). Eight root canals without endodontic treatment constituted the control group. Histological sections were obtained and stained with Brown & Brenn staining technique to evaluate the presence of microorganisms in the main root canal, ramifications of the apical delta and secondary canals, apical cementoplasts, dentinal tubules, areas of cemental resorption and periapical lesions. The results were analyzed statistically by the Mann-Whitney U test (p<0.05). RESULTS: The control group showed the highest prevalence of microorganisms in all sites evaluated. Gram-positive cocci, bacilli and filaments were the most frequent morphotypes. Similar microbial distribution patterns in the RCS and areas of cementum resorption were observed in all groups (p>0.05). The percentage of RCS sites containing microorganisms in groups 1, 2, 3, 4 and control were: 67.6%, 62.5%, 78.2%, 62.0% and 87.6%, respectively. CONCLUSION: In conclusion, the histomicrobiological analysis showed that the rotary instrumentation and the different calcium hydroxide pastes employed did not effectively eliminate the infection from the RCS and periapical lesions. However, several bacteria seen in the histological sections were probably dead or were inactivated by the biomechanical preparation and calcium hydroxide-based intracanal dressing.

Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization

A comparative study of holocentric chromosomes in the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans was carried out in order to characterize heterochromatin, rDNA active sites and nucleolar proteins. Cytological preparations of seminiferous tubules were stained by silver impregnation, C banding, fluorochromes CMA 3/DA and DAPI/DA, and fluorescent in situ hybridization (FISH) with Drosophila melanogaster 28S rDNA probe. Our results showed interesting aspects of the organization of chromatin and chromosomes in the meiotic cells of these insects. In R. pallescens, sex chromosomes (X, Y) were distinct from autosomes, when submitted to silver impregnation, C banding, CMA 3 staining, and FISH, confirming that these chromosomes bear nucleolar organizer regions (NORs). In P. megistus, two of the three sex chromosomes were CMA 3/DAPI-; at early meiotic prophase and at diakinesis, silver impregnation corresponded with FISH signals, indicating that in this species, two chromosomes (probably a sex chromosome and an autosome) bear NORs. In T. infestans, silver nitrate and FISH also stained corresponding areas on meiotic chromosomes. Our data suggest that in triatomines, in general, the number and location of NORs are species-specific. These regions may be considered important chromosome markers for comparative studies to improve the understanding of evolutionary mechanisms in these hematophagous insects. ©FUNPEC-RP.