Chemical sterilization in in vitro propagation of Arundina bambusifolia Lindl. and Epidendrum ibaguense Kunth

There is a great demand for simpler and less costly laboratory techniques and for more accessible procedures for orchid breeders who do not have the necessary theoretical basis to use the traditional seed and clone production methods of orchids in vitro. The aim of this study was to assess the use of sodium hypochlorite (NaClO) as a decontaminant in the process of inoculating adult orchid explants of Arundina bambusifolia and Epidendrum ibaguenses. Solutions of NaClO (1.200, 2.400, 3.600, 4.800 and 6.000 mg L-1 - equivalent to 50, 100, 150, 200 and 250 mL L-1 of commercial bleach - CB) were sprayed on the explants (1.0 mL) and the culture medium (GB5), in the presence or absence of activated charcoal (2 g L-1). The explants used were nodal segments of field-grown adult plants. The procedures for inoculating the explants were conducted outside the laminar flow chamber (LFC), except for the control treatment (autoclaved medium and explant inoculation inside the LFC). The best results for fresh weight yield, height and number of shoots were obtained using NaClO in solution at 1.200 mg L-1 (equivalent to 50 mL L-1 commercial bleach) with activated charcoal in the culture medium. Fresh weight figures were 1.10 g/jar for Arundina bambusifolia and 0.16 g/jar for Epidendrum ibaguenses. Spraying the NaClO solutions controls the contamination of the culture medium already inoculated with the explants.

Solubilization of diabase and phonolite dust by filamentous fungus

The objective of this study was to evaluate the effect of the fungus Aspergillus niger strain CCT4355 in the release of nutrients contained in two types of rock powder (diabase and phonolite) by means of in vitro solubilization trials. The experimental design was completely randomized in a 5 x 4 factorial design with three replications. It was evaluated five treatments (phonolite dust + culture medium; phonolite dust + fungus + culture medium; diabase powder + culture medium; diabase powder + fungus + culture medium and fungus + culture medium) and four sampling dates (0, 10, 20 and 30 days). Rock dust (0.4% w/v) was added to 125 mL Erlenmeyer flasks containing 50 mL of liquid culture medium adapted to A. niger. The flasks were incubated at 30°C for 30 days, and analysis of pH (in water), titratable acidity, and concentrations of soluble potassium, calcium, magnesium, zinc, iron and manganese were made. The fungus A. niger was able to produce organic acids that solubilized ions. This result indicates its potential to alter minerals contained in rock dust, with the ability to interact in different ways with the nutrients. A significant increase in the amount of K was found in the treatment with phonolite dust in the presence of the fungus. The strain CCT4355 of A. niger can solubilize minerals contained in these rocks dust.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Erythema induratum of Bazin and renal tuberculosis: report of an association

Erythema induratum of Bazin is a disease that usually affects women, in whom erythematous subcutaneous nodules and plaques appear on the posterior part of the lower extremities, some of which ulcerate. In many countries, tuberculosis is still the main etiologic factor. We report a case of a 40-year-old woman who presented a course of protracted and recurrent episodes over five years of cutaneous lesions on her legs. These tend to involute, but new crops appear at irregular intervals. It was painful, erythematous-violaceous nodules, some of which drained a reddish secretion. The histopathologic features of the lesions demonstrated inflammatory infiltration, with predominance of neutrophils in dermis and hypodermis, necrotizing vasculitis in the arterioles and septal fibrosis. There was no granuloma. The Ziehl-Neelsen stain did not revealed acid-fast bacilli, and the culture of biopsy specimen was negative. The tuberculin skin test was strongly positive (17 mm). The chest X-ray was normal. Few months later she presented adynamia and urinary complaints, such as polacyuria and dysuria. It has been done an urynalysis, which demonstrated acid pH urine, sterile pyuria and microscopic hematuria. It was then raised the diagnostic hypothesis of renal tuberculosis. The urine culture for M. tuberculosis was positive in two out of ten samples. The treatment was instituted with rifampin, isoniazid and pyrazinamide, with complete regression. This case illustrates a clear association between erythema induratum and renal tuberculosis, demonstrated by the remission of the cutaneous lesions after the treatment of the renal tuberculosis.

Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI) in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs) and one nucleic acid amplification test (NAAT) were evaluated against a cytotoxicity assay (CTA) and toxigenic culture (TC). Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2%) of 92 samples were positive according to the CTA, and 23 (25%) were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

Cultura cromogênica dum bacilo ácido-álcool resistente isolado de pus de lesão fechada de lepra humana

Last October 2nd the Author smeared nine tubes of Loewenstein medium with material obtained from closed pustulae of a seven years old boy, L2 case of leprosy. This material was very rich in Hansen bacilli in its different forms, inclusive globus, as is seen in the figures 2 and 3 of Plate 1. Part of this material obtained from pustulae opened by galvanocautery, was inoculated, at the same day, into white rats and guinea-pigs. November 26th a new biopsy gave more rich material, which was smeared again into Loewenstein fresh medium. December 15th three of the first and two of the second series of tubes of cultures showed germination of a yellow, dry and rough culture, covering almost the total surface of the medium. Microscopic examination of the culture showed that it was a pure culture of an acid-fast organism. Passages into glycerinated potatoes germinated well covering the surface of the same with a clear yellow granulated culture remaining the fluid (glycerinated water) quite limpid. The germination in glycerinated broth produced a yellow velum in the surface of the medium, as is seen in fig. 3 of Plate 2, without becoming turbid. The microorganism isolated twice from the same source of material was cocciforme (as Mycobacterium pulviforme of Marchoux), in the original culture, becoming more bacilliforme, always acid-fast, after passage into glycerinated media. The A. sent his culture to foreigner colleagues to study it and will inoculate it soon into laboratory animals.

Plant tissue culture techniques

Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus) or organized tissues or organs put in culture, under controlled sterile conditions.

Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain rreaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.

Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Leishmania (Leishmania) chagasi

The role of Proechimys semispinosus as reservoir of Leishmania (Viannia) panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID) in the snout or feet with 10(7) promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7) promastigotes) or ID in the ear (10(8) promastigotes). PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i.) and became more resistant upon reinfection. Infectivity to sand flies was low (1/20-1/48 infected/fed flies) and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.

Sensitivity and specificity of polymerase chain reaction in Giemsa-stained slides for diagnosis of visceral leishmaniasis in children

The aim of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) in the detection of Leishmania DNA in archived Giemsa-stained bone marrow slides for diagnosis of visceral leishmaniasis (VL), and to compare PCR with conventional diagnostic techniques, like direct microscopy and parasite culture. Specimens of archived Giemsa-stained bone marrow slides from 91 patients with VL and from 79 controls with other diseases or conditions were studied. PCR showed the highest sensitivity (92.3%) and had good specificity (97.5%). Direct examination detected 79.1% and culture 59% of positive samples. In addition, PCR was able to detect VL in 16 of 19 patients (84.2%) with negative microscopy. PCR in Giemsa-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may be useful in the diagnosis of difficult cases. Slide smears are easily stored, do not require special storage conditions such as low temperatures, and can be easily mailed to centers where PCR is available, making it an excellent option for diagnosis in the field.

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

Embryo rescue from interspecific crosses in apple rootstocks

The objetive of this work was to rescue immature embryos of apple rootstocks Malus prunifolia (Marubakaido) and Malus pumila (M9) after 40-60 days of pollination and to put them into MS culture media supplemented with agar (6 g L-1) and casein hydrolysate (500 mg L-1). Embryos originated from interspecific crosses and open pollination showed differences in the in vitro responses, depending on the female parent, the developmental stage of the embryo, and the culture medium composition. Embryos of the M. pumila rootstock, rescued within 40 days after pollination and put in culture medium supplemented with indolacetic acid (IAA), gibberellic acid (GA3), kinetin and maltose, resulted in a normal development of plantlets. However, embryos originating from hand-pollination, cultivated in medium supplemented with 14 µM IAA, 5 µM kinetin and 1.5 µM Ga3 (MS1), mainly those of M. prunifolia x M. pumila, showed a high percentage of rusted embryos (96.2%). Embryos from open pollination of M. prunifolia and M. pumila formed calluses. It was possible to identify the influence of the female parent by the enhanced development of M. pumila shoots derived from open or hand-pollination. The crossing of responsive species and the use of the technique of embryo culture provided a rapid and uniform germination and, consequently, the development of fully normal seedlings.

Milk flow, teat morphology and subclinical mastitis prevalence in Gir cows

The aim of this work was to evaluate the association between milk flow, teat morphological measurements and subclinical mastitis prevalence in Gir cows. Eighty cows in the 2nd and 3rd lactations, with 90 to 200 days of lactation, were divided according to milk flow during milking into fast or slow groups. Teat morphometry was assessed by ultrasound scanning of the right anterior teat and external measurements. Milk samples were collected for somatic cells count (SCC) and microbiological culture. The effect of milk flow during milking was evaluated by analysis of variance of milk yield, SCC, morphometry and external measurements. The association of morphometry and external measurements of the teats with the SCC and microorganisms found in milk were analysed. Milk flow was significantly correlated to milk production. Gir cows with slower milk flow had longer teat canal and greater milk yield, in comparison to cows with fast milk flow. Teat-end to floor distance influenced SCC of Gir cows. Prevalence of subclinical mastitis and the type of mastitis-causing pathogens were not affected by milk flow during milking

In vitro conservation of Piper aduncum and Piper hispidinervum under slow-growth conditions

The objective of this work was to evaluate in vitro storage of Piper aduncum and P. hispidinervum under slow-growth conditions. Shoots were stored at low temperatures (10, 20 and 25°C), and the culture medium was supplemented with osmotic agents (sucrose and mannitol - at 1, 2 and 3%) and abiscisic acid - ABA (0, 0.5, 1.0, 2.0 and 3.0 mg L-1). After six-months of storage, shoots were evaluated for survival and regrowth. Low temperature at 20ºC was effective for the in vitro conservation of P. aduncum and P. hispidinervum shoots. In vitro cultures maintained at 20ºC on MS medium showed 100% survival with slow-growth shoots. The presence of mannitol or ABA, in the culture medium, negatively affected shoot growth, which is evidenced by the low rate of recovered shoots.