A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

Effect of different periods of preconditioning with saliva on adhesion of C. albicans to a denture base resin by crystal violet staining and XTT assay.

Aim: The role of saliva on Candida adhesion to biomaterials has not been clearly defined. The present study investigates whether different periods of preconditioning with saliva would influence the adhesion of Candida albicans to a denture base resin. Methods: Ninety samples of acrylic resin with smooth surfaces were made and then divided into five groups: one control without saliva, and four experimental groups conditioned in saliva for periods of 30 min, 1, 3, or 12 h. Candida adhesion was evaluated by crystal violet staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-([phenylamino] carbonyl)-2H-tetrazolium-hydroxide assay. Results: The one-way analysis of variance revealed that there were no significant differences among the mean number of adherent cells or among the mean absorbance for all groups. No significant correlation was found between the two methods used for assessing Candida albicans adhesion. Conclusion: The different periods of preconditioning with saliva had no significant influence on the adhesion of Candida albicans to the denture base acrylic resin.

An Environmental Friendly Procedure for Photometric Determination of Hypochlorite in Tap Water Employing a Miniaturized Multicommuted Flow Analysis Setup

A photometric procedure for the determination of ClO(-) in tap water employing a miniaturized multicommuted flow analysis setup and an LED-based photometer is described. The analytical procedure was implemented using leucocrystal violet (LCV; 4,4', 4 ''-methylidynetris (N, N-dimethylaniline), C(25)H(31)N(3)) as a chromogenic reagent. Solenoid micropumps employed for solutions propelling were assembled together with the photometer in order to compose a compact unit of small dimensions. After control variables optimization, the system was applied for the determination of ClO(-) in samples of tap water, and aiming accuracy assessment samples were also analyzed using an independent method. Applying the paired t-test between results obtained using both methods, no significant difference at the 95% confidence level was observed. Other useful features include low reagent consumption, 2.4 mu g of LCV per determination, a linear response ranging from 0.02 up to 2.0 mg L(-1) ClO(-), a relative standard deviation of 1.0% (n = 11) for samples containing 0.2 mg L(-1) ClO(-), a detection limit of 6.0 mu g L(-1) ClO(-), a sampling throughput of 84 determinations per hour, and a waste generation of 432 mu L per determination.

High performance gold nanorods and silver nanocubes in surface-enhanced Raman spectroscopy of pesticides

The behavior of Au nanorods and Ag nanocubes as analytical sensors was evaluated for three different classes of herbicides. The use of such anisotropic nanoparticles in surface-enhanced Raman scattering (SERS) experiments allows the one to obtain the spectrum of crystal violet dye in the single molecule regime, as well as the pesticides dichlorophenoxyacetic acid (2,4-D), trichlorfon and ametryn. Such metallic substrates show high SERS performance at low analyte concentrations making them adequate for use as analytical sensors. Density functional theory (DFT) calculations of the geometries and vibrational wavenumbers of the adsorbates in the presence of silver or gold atoms were used to elucidate the nature of adsorbate-nanostructure bonding in each case and support the enhancement patterns observed in each SERS spectrum.

In vitro cytotoxic activity of Baccharis dracunculifolia and propolis against HEp-2 cells

Baccharis dracunculifolia is the most important vegetal source of propolis in southeast Brazil, and researchers have been investigating its biological properties. Propolis is a complex resinous hive product collected by bees from several plants, showing a very complex chemical composition. It has been employed since ancient times due to its therapeutic properties, such as antimicrobial, anti-inflammatory, antioxidant, immunomodulatory and antitumour activities, among others. The goal of this work was to compare the cytotoxic action of B. dracunculifolia, propolis and two isolated compounds (caffeic and cinnamic acids) on human laryngeal epidermoid carcinoma (HEp-2) cells in vitro. These cells were incubated with different concentrations of each variable, and cell viability was assessed by the crystal violet method. Lower concentrations of B. dracunculifolia (extract and essential oil), propolis, as well as caffeic and cinnamic acids, showed no cytotoxic activity against HEp-2 cells. On the other hand, elevated concentrations (50 and 100 mu g per 100 mu L) exerted a cytotoxic action, and propolis showed a more efficient action than its vegetal source and isolated compounds. Further investigation is still needed in order to explore the potential of these variables as antitumour agents and to understand their mechanisms of action.

Synthesis, antichagasic in vitro evaluation, cytotoxicity assays, molecular modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-acrylic acid benzylidene-carbohydrazide series

Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO(2), pIC(50) = 4.55 M) and 6l (X = F, Y = p-CN, pIC(50) = 4.27 M) as the most potent derivatives compared to crystal violet (pIC(50) = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising pro. le with low toxicity (0% of cell death). The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it. (C) 2008 Elsevier Ltd. All rights reserved.

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian Ginseng) on cultured human breast cancer cell line MCF-7

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line. as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated With butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations. (C) 2008 Elsevier GmbH. All rights reserved.

A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels. Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays. Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in Vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter. Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. in this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation. (C) 2009 Elsevier Ltd. All rights reserved.

Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells

Background: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor Cell proliferation and, following clonal expansion of these cells. promotion of differentiation along the osteogenic lineage. Objectives: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Method: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogcnic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin. osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP. osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

Quantification of Listeria spp. contamination on shell and flesh of cooked black tiger prawns (Penaeus monodon)

Aims: To quantify Listeria levels on the shell and flesh of artificially contaminated cooked prawns after peeling, and determine the efficacy of Listeria innocua as a model for L. monocytogenes in this system. Methods and Results: A L. monocytogenes and L. innocua strain were inoculated separately onto cooked black tiger prawns using two protocols ( immersion or swabbing with incubation). Prawns were peeled by two methods ( gloved hand or scalpel and forceps) and numbers of Listeria on shells, flesh and whole prawn controls were determined. Prawns were exposed to crystal violet dye to assess the penetration of liquids. Regardless of preparation method or bacterial strain there were ca 1log(10) CFU more Listeria per shell than per peeled prawn. Dye was able to penetrate to the flesh in all cases. Conclusions: Shell-on prawns may be only slightly safer than shell-off prawns. Listeria innocua is an acceptable model for L. monocytogenes in this system. Significance and Impact of the Study: Reduced risk from L. monocytogenes on prawns can only be assured by adequate hygiene or heating.

Cytotoxicity in L929 fibroblasts and inhibition of herpes simplex virus type 1 Kupka by estuarine cyanobacteria extracts

The cyanobacteria are known to be a rich source of metabolites with a variety of biological activities in different biological systems. In the present work, the bioactivity of aqueous and organic (methanolic and hexane) crude extracts of cyanobacteria isolated from estuarine ecosystems was studied using different bioassays. The assessment of DNA damage on the SOS gene repair region of mutant PQ37 strain of Escherichia coli was performed. Antiviral activity was evaluated against influenza virus, HRV-2, CVB3 and HSV-1 viruses using crystal violet dye uptake on HeLa, MDCK and GMK cell lines. Cytotoxicity evaluation was performed with L929 fibroblasts by MTT assay. Of a total of 18 cyanobacterial isolates studied, only the crude methanolic extract of LEGE 06078 proved to be genotoxic (IF > 1.5) in a dose-dependent manner and other four were putative candidates to induce DNA damage. Furthermore, the crude aqueous extract of LEGE 07085 showed anti- herpes type 1 activity (IC50 = 174.10 μg dry extract mL−1) while not presenting any cytotoxic activity against GMK cell lines. Of the 54 cyanobacterial extracts tested, only the crude methanolic and hexane ones showed impair on metabolic activity of L929 fibroblasts after long exposure (48–72 h). The inhibition of HSV-1 and the strong cytotoxicity against L929 cells observed emphasizes the importance of evaluating the impact of those estuarine cyanobacteria on aquatic ecosystem and on human health. The data also point out their potential application in HSV-1 treatment and pharmacological interest.

Data quality in biofilm high-throughput routine analysis: intralaboratory protocol adaptation and experiment reproducibility

Biofilm research is growing more diverse and dependent on high-throughput technologies and the large-scale production of results aggravates data substantiation. In particular, it is often the case that experimental protocols are adapted to meet the needs of a particular laboratory and no statistical validation of the modified method is provided. This paper discusses the impact of intra-laboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development, considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the XTT assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non-rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intra-laboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories.

Candida tropicalis biofilms: biomass, metabolic activity and secreted aspartyl proteinase production

According to epidemiological data, Candida tropicalis has been related to urinary tract infections and haematological malignancy. Several virulence factors seem to be responsible for C. tropicalis infections, for example: their ability to adhere and to form biofilms onto different indwelling medical devices; their capacity to adhere, invade and damage host human tissues due to enzymes production such as proteinases. The main aim of this work was to study the behaviour of C. tropicalis biofilms of different ages (24120 h) formed in artificial urine (AU) and their ability to express aspartyl proteinase (SAPT) genes. The reference strain C. tropicalis ATCC 750 and two C. tropicalis isolates from urine were used. Biofilms were evaluated in terms of culturable cells by colony-forming units enumeration; total biofilm biomass was evaluated using the crystal violet staining method; metabolic activity was evaluated by XTT assay; and SAPT gene expression was determined by real-time PCR. All strains of C. tropicalis were able to form biofilms in AU, although with differences between strains. Candida tropicalis biofilms showed a decrease in terms of the number of culturable cells from 48 to 72 h. Generally, SAPT3 was highly expressed. C. tropicalis strains assayed were able to form biofilms in the presence of AU although in a strain- and time-dependent way, and SAPT genes are expressed during C. tropicalis biofilm formation.

Characterization of microbial populations in a wastewater treatment plant focusing on Staphylococcus aureus and Pseudomonas aeruginosa

Dissertação de mestrado em Bioengenharia