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Clonagem e análise da expressão de genes de proteínas de mamão papaia com atividade inibitória sobre poligalacturonases fúngicas; Cloning and expression analysis of papaya genes encoding proteins with inhibitory activity against fungal polygalacturonases

Broetto, Sabrina Garcia
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 10/07/2013 PT
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As proteínas inibidoras de poligalacturonases (PGIPs) presentes na parede celular são capazes de limitar o potencial destrutivo da poligalacturonase (PG) fúngica e, assim, constituem um tipo importante dentre os diversos sistemas de defesa do tecido vegetal frente à infecção fúngica. No mamão, o ataque fitopatogênico é o principal causador de danos pós-colheita, e sua alta susceptibilidade pode estar relacionada com a baixa eficácia ou pouca abundância dos meios de defesa anti-fitopatogênica. Uma vez que isso pode estar relacionado com as PGIPs e nada se conhece sobre o papel dessas proteínas nesse fruto, o objetivo do trabalho foi clonar os genes das PGIPs de mamoeiro e definir seu padrão de expressão em diferentes órgãos e tecidos e ao longo do amadurecimento. Para tanto, foram identificadas no genoma do mamoeiro, a partir de critérios que definem a identidade de uma PGIP, duas prováveis sequências dentre 13 candidatas iniciais. Ambas foram clonadas a partir das sequências genômicas e de cDNA, sequenciadas e sua identidade confirmada, sendo denominadas Cppgip4 e Cppgip6. As análises de expressão relativa em diversos tecidos e idades fisiológicas do mamoeiro demonstraram que os dois genes apresentaram diminuição da expressão com o desenvolvimento dos frutos...

Bioprospecção de atividade antioxidante e antimicrobiana da casca de Stryphnodendron adstringens (Mart.) Coville (Leguminosae-Mimosoidae)

De Souza, Tatiana Maria; Severi, J. A.; Silva, V. Y. A.; Santos, E.; Pietro, R. C. L. R.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 221-226
POR
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The aim of this work was to evaluate the class of secondary metabolites responsible for the antioxidant and antimicrobial activities of bark extracts of Stryphnodendron adstringens (Mart.) Coville (Leguminosae-Mimosoidae), a plant widely used in folk medicine in Brazil. Extracts of the bark were prepared with 50% ethanol, 70% ethanol, acetone:water (7:3, v/v) and chloroform. Antioxidant activity was prospected by spraying thin-layer chromatographs of the extracts with 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and measuring the DPPH radical scavenging capacity by spectrophotometry. Antibacterial activity was revealed by the agar diffusion method and bioautography. TLC spots assigned to tannins in the polar extracts showed antioxidant activity by DPPH radical scavenging and the chloroform extract showed the least scavenging activity. Antimicrobial activity was indicated by the bacterial growth inhibition haloes around polar extracts and bioautography showed activity in the TLC spots assigned to tannins. It was concluded that polar extracts of the bark of S. adstringens possessed antioxidant and antimicrobial activities which were due to secondary metabolite derived from the tannin class, which are the main constituent of these bark extracts...

In vitro antimicrobial activity of AH Plus, EndoREZ and Epiphany against microorganisms

Maekawa, Lilian; Nassri, Maria Renata; Ishikawa, Camila; Martins, Carolina; Chung, Adriana; Koga-Ito, Cristiane
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 469-472
ENG
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Objective : The aim of the present study was to evaluate the antimicrobial activity of endodontic sealers against microorganisms. Materials and Methods : The agar diffusion method was used. A double base layer of Mueller Hinton agar was done. The microorganisms used were: Candida albicans, Enterococcus faecalis, Escherichia coli and Staphylococcus aureus. The wells were obtained by removing a standardized portion of the agar. After the distribution of the sealers, Petri plates were incubated for 24 h. Inhibition halos formed around the wells were measured. Results : Epiphany did not show any antimicrobial activity on the tested microorganisms (without inhibition halo). The AH Plus showed the greatest inhibition halo on C. albicans followed by EndoREZ on S. aureus. EndoREZ also showed greater inhibition halo in comparison to AH Plus on E. faecalis and E. coli. Conclusion : It could be concluded that AH Plus and EndoREZ showed antimicrobial activity against all the tested microorganisms. No antimicrobial activity was observed for Epiphany.

Development and validation of a successful microbiological agar assay for determination of ceftriaxone sodium in powder for injectable solution

Aléssio, Patrícia V.; Salgado, Hérida R. N.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 334-342
ENG
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Ceftriaxone sodium is a cephalosporin with broad-spectrum antimicrobial activity and belongs to the third generation of cephalosporins. Regarding the quality control of medicines, a validated microbiological assay for the determination of ceftriaxone sodium in powder for injectable solution has not been reported yet. This paper reports the development and validation of a simple, accurate and reproducible agar diffusion method to quantify ceftriaxone sodium in powder for injectable solution. The assay is based on the inhibitory effect of ceftriaxone sodium on the strain of Bacillus subtilis ATCC 9371 IAL 1027 used as test microorganism. The results were treated statistically by analysis of variance and were found to be linear (r = 0.999) in the selected range of 15.0-60.0 μg/mL, precise with a relative standard deviation (RSD) of repeatability intraday = 1.40%, accurate (100.46%) and robust with a RSD lower than 1.28%. The results demonstrated the validity of the proposed bioassay, which allows reliable ceftriaxone sodium quantitation in pharmaceutical samples and therefore can be used as a useful alternative methodology for the routine quality control of this medicine. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution

Fiorentino, Flávia Angélica Másquio; Corrêa, Marcos Antonio; Salgado, Hérida Regina Nunes
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 351-358
ENG
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Chlorhexidine (CHX) is a broad-spectrum antiseptic that is used in many topical pharmaceutical formulations. Because there is no official microbiological assay reported in the literature that is used to quantify CHX, this paper reports the development and validation of a simple, sensitive, accurate and reproducible agar diffusion method for the dosage of chlorhexidine digluconate (CHX-D) in an aqueous solution. The assay is based on the inhibitory effect of CHX-D upon the strain of Staphylococcus aureus ATCC 25923, which is used as the test microorganism. The design 3x3 parallel-line model was used. The results were treated statistically by analysis of variance (ANOVA), and they were excellent in terms of linearity (r = 0.9999), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 0.5 to 4.5%. The results obtained were precise, having relative standard deviations (RSD) for intra-day and inter-day precision of 2.03% and 2.94%, respectively. The accuracy was 99.03%. The method proved to be very useful and appropriate for the microbiological dosage of CHX-D in pharmaceutical formulations; it might also be used for routine drug analysis during quality control in pharmaceutical industries.

Evaluation of Brazilian medical devices using agar diffusion cytotoxicity assay

Vidal,Mirian N. P.; Aiub,Claudia; Abrantes,Shirley; Zamith,Helena P. S.
Fonte: Associação Brasileira de Hematologia e Hemoterapia e Terapia Celular Publicador: Associação Brasileira de Hematologia e Hemoterapia e Terapia Celular
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2009 EN
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Over the last decades, governmental actions and mechanisms created to protect consumer rights have been linked to a growing effort to guarantee the quality and reliability of products. Samples of condoms (latex), medical devices and blood bags (PVC - polyvinyl chloride) have been tested using the agar diffusion assay. This assay evaluates the cytotoxicity induced by biomaterials by measuring the biological reactivity of mammalian cell cultures in contact with these materials. PVC is used in the production of medical devices because of its specific properties, such as flexibility, obtained after the addition of plasticizers (phthalates), which can cause toxicity even at low doses. Latex is a natural elastomer used for surgical gloves and condoms with a formulation that includes dispersion of liquid latex and chemicals, such as antioxidants and a vulcanizing accelerator, both of which are able to induce cytotoxicity. Samples were analyzed by the National Institute of Quality Control in Health - INCQS of the Oswaldo Cruz Foundation (Fiocruz) in accordance with the governmental sanitary surveillance actions on respect to the quality control. We observed an increase in the quality of the products in relation to the results of the agar diffusion assay during the period between 2000 and 2007. This situation...

Screening methods to determine antibacterial activity of natural products

Valgas,Cleidson; Souza,Simone Machado de; Smânia,Elza F A; Smânia Jr.,Artur
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2007 EN
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The emergence of new infectious diseases, the resurgence of several infections that appeared to have been controlled and the increase in bacterial resistance have created the necessity for studies directed towards the development of new antimicrobials. Considering the failure to acquire new molecules with antimicrobial properties from microorganisms, the optimization for screening methods used for the identification of antimicrobials from other natural sources is of great importance. The objective of this study was to evaluate technical variants used in screening methods to determine antibacterial activity of natural products. Thus, a varied range of natural products of plant, fungi and lichen origin were tested against two bacterial species, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, by two variants of the agar diffusion method (well and disc), two variants of the bioautographic method (direct and indirect) and by microdilution assay. We concluded that the well-variant of the diffusion method was more sensitive than the disc-variant, whilst the direct-variant of the bioautographic method exhibited a greater sensitivity if compared to indirect-variant. Bioautographic and diffusion techniques were found to have similar sensitivity; however the latter technique provided more suitable conditions for the microbial growth. In this study...

Development of agar diffusion method for dosage of gramicidin

Solano,Ana Gabriela Reis; Pereira,Larissa de Melo Campos Sousa; Leonel,Míriam de Fátima Vianna; Nunan,Elzíria de Aguiar
Fonte: Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Publicador: Universidade de São Paulo, Faculdade de Ciências Farmacêuticas
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2011 EN
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Gramicidin, an antimicrobial peptide active against Gram positive bacteria, is commonly used in pharmaceutical preparations for topical use. Considering that only the turbidimetric method has been described in the literature, the present study sought to develop and validate an agar diffusion method for the dosage of gramicidin. The method was developed and validated using the Kocuria rhizophila ATCC 9341 as a test microorganism. Two designs were used: a 3x3 parallel-line model, and a 5x1 standard curve. The validation demonstrated that the method follows the linear model (r²= 0.994), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 5 to 25.3 µg/mL. The results obtained for both designs were precise, having a relative standard deviation (R.S.D.) for intra-day precision of 0.81 for the 3x3 assay and 1.90 for the 5x1 assay. For the inter-day precision, the R.S.D. was 1.35 for the 3x3 and 2.64 for the 5x1. The accuracy was verified and results confirmed to be accurate, having a tolerance interval of 95%, which lay within permitted limits and appropriate trueness. In addition, the method was considered selective, with limit of detection and upper and lower limits of quantification of 2.00...

Comparison of Agar Diffusion Methodologies for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

Burns, Jane L.; Saiman, Lisa; Whittier, Susan; Larone, Davise; Krzewinski, Jay; Liu, Zhenling; Marshall, Steven A.; Jones, Ronald N.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2000 EN
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Pseudomonas aeruginosa is the most common pathogen infecting the lungs of patients with cystic fibrosis (CF). Improved antimicrobial chemotherapy has significantly increased the life expectancy of these patients. However, accurate susceptibility testing of P. aeruginosa isolates from CF sputum may be difficult because the organisms are often mucoid and slow growing. This study of 597 CF isolates of P. aeruginosa examined the correlation of disk diffusion and Etest (AB BIODISK, Solna, Sweden) results with a reference broth microdilution method. The rates of interpretive errors for 12 commonly used antipseudomonal antimicrobials were determined. The disk diffusion method correlated well (zone diameter versus MIC) for all of the agents tested. However, for mucoid isolates, correlation coefficients (r values) for piperacillin, piperacillin-tazobactam, and meropenem were <0.80. The Etest correlation with reference broth microdilution results (MIC versus MIC) was acceptable for all of the agents tested, for both mucoid and nonmucoid isolates. Category interpretation errors were similar for the disk diffusion and Etest methods with 0.4 and 0.1%, respectively, very major errors (false susceptibility) and 1.1 and 2.2% major errors (false resistance). Overall...

Error rates associated with the use of recently proposed breakpoints for testing Pseudomonas aeruginosa versus gentamicin, tobramycin, and amikacin by the standardized disk agar diffusion test.

Woolfrey, B F; Fox, J M; Quall, C O; Lally, R T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1983 EN
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Two hundred fifteen Pseudomonas aeruginosa isolates were tested in parallel by the disk agar diffusion test, using a standardized agar preparation, and by a microbroth test, using dilutions differing by small arithmetic increments. For gentamicin, recently proposed breakpoints of resistance (R) less than or equal to 12 mm and susceptibility (S) greater than or equal to 16 mm produced error rates of 20 and 6.8%, respectively. Limiting the error rate for susceptible interpretations to less than or equal to 2% produced a widening of the intermediate zone to include 67.4% of the isolates tested. For tobramycin, the recently proposed breakpoints of R less than or equal to 12 mm and S greater than or equal to 15 mm were associated with error rates of 66.7 and 1.4%, respectively. Breakpoints of R less than or equal to 12 mm and S greater than or equal to 13 mm were demonstrated to be equally effective when the error rate for susceptible interpretations was limited to less than or equal to 2% by error rate-bound analysis. For amikacin, proposed breakpoints of R less than or equal to 14 mm and S greater than or equal to 17 mm were associated with error rates of 27.3 and 3.2%, respectively. Limiting the error rates for susceptible interpretations to less than or equal to 2% required breakpoints of R less than or equal to 14 mm and S greater than or equal to 18 mm. The ability to establish effective susceptibility breakpoints for tobramycin and amikacin appeared not to be related to the disk agar diffusion test process itself but rather to the high degree of susceptibility of the P. aeruginosa population. These findings severely limit the usefulness of the disk agar diffusion procedure for testing P. aeruginosa versus the aminoglycosides. For this purpose...

Non-Immunological Precipitation of Serum by Sodium Dodecyl Sulfate in Agar Diffusion

Cho, C. T.; Feng, K. K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1974 EN
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Marmoset serum or serum of other species of animal may react with sodium dodecyl sulfate and forms nonspecific precipitin lines in agar diffusion. The protein detergent complexes are not readily dialyzable. Therefore precipitin lines derived from studies that use sodium dodecyl sulfate-treated antigens in agar diffusion must be interpreted with caution.

Disk Agar Diffusion Susceptibility Testing of Yeasts

Saubolle, Michael A.; Hoeprich, Paul D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1978 EN
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A disk agar diffusion method was developed for testing the susceptibility of rapidly growing yeasts in vitro. A totally defined, completely synthetic agar culture medium (synthetic amino acid medium, fungal) and clinical isolates of Candida spp. and Torulopsis glabrata were used. Turbidimetric adjustment of cell suspensions resulted in standard, reproducible inocula, which gave sharp, clear zones of inhibition when applied by an agar overlay method. Optimal disk loads were determined for amphotericin B, amphotericin B methyl ester, 5-fluorocytosine, clotrimazole, and miconazole. Disk potencies were stable over a 2-month period when stored in a vacuum desiccator at −30°C. Using an error ratebounded classification, the zones of inhibition were correlated with both broth dilution and agar dilution minimum inhibitory concentrations (MICs). With amphotericin B and amphotericin B methyl ester, all isolates were susceptible, yielding zone diameters which clustered within 5 mm. Overall correlations between zone diameters and broth dilution MICs with 5-fluorocytosine, miconazole, and clotrimazole were 97, 96, and 82% (excluding T. glabrata), respectively; correlations of zone diameters with agar dilution MICs were 96, 92, and 88%, respectively. Disk diffusion susceptibility testing of yeasts appears to be generally applicable. However...

Metachromatic Agar-Diffusion Methods for Detecting Staphylococcal Nuclease Activity

Lachica, R. V. F.; Genigeorgis, C.; Hoeprich, P. D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1971 EN
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Based on the metachromatic property of Toluidine Blue O, three, convenient agar-diffusion methods have been developed that enable detection of the nuclease of Staphylococcus aureus at concentrations as low as 0.005 μg/ml in agar and broth cultures. The interactions of agar and deoxyribonucleic acid with Toluidine Blue O are discussed.

Nonspecific Precipitation of Serum Proteins by Sodium Lauryl Sulfate in Agar Diffusion and Immunoelectrophoresis

Palmer, E. L.; Martin, M. L.; Hierholzer, J. C.; Ziegler, D. W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1971 EN
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The anionic detergent sodium lauryl sulfate (SLS), in a final concentration of 0.1% and greater, reacted with whole serum in agar diffusion and immunoelectrophoresis to form artifactual precipitin lines. These lines occurred when either Ionagar or agarose was used as the supporting gel and were not affected by the presence of urea and 2-mercaptoethanol. Analytic chemical tests confirmed that the precipitating agent is SLS, and staining techniques showed that the detergent precipitates both protein and lipoprotein components of whole serum. Multiple artifactual precipitin lines occurred with a wide variety of animal sera, and a single line formed with human 7S immunoglobulin. Hence, in agar diffusion studies in which SLS is present in the test system, these artifactual lines may be easily misinterpreted as true antigen-antibody precipitin reactions.

Agar Diffusion Method for the Assay of Colicins

Richardson, H.; Emslie-Smith, A. H.; Senior, B. W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1968 EN
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An agar diffusion method for the assay of colicins A, B, D, E2, E3, and K is described. The assays were performed in large, square pyrex dishes that contained an agar layer seeded with an indicator organism sensitive to the colicin. The samples were applied to the agar in steatite beads positioned in a randomized sequence. The plates were stored at 4 C for 24 hr to allow the colicins to diffuse into the agar. After incubation at 37 C, the activity of each colicin preparation was estimated by measuring the diameter of the zone of inhibition of the growth of the indicator strain around each bead. The results of each assay were subjected to a statistical analysis, which included an analysis of variance and calculation of the theoretical regression and the confidence interval of the assay. The size of the inhibition zones, the form of the regression, and the slope of the regression of the responses were affected by the type and concentration of the agar, the depth of the agar layer, the indicator organism, the indicator inoculum density, and the time allowed for prediffusion of the colicins. Optimal conditions for the assay of each colicin were determined. Using a four-point assay design, the relative colicin concentration of unknown preparations was estimated in terms of a standard preparation of the same colicin. The experimental error of these assays (95% confidence interval) was about ± 10%.

Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin▿

Espinel-Ingroff, A.; Arthington-Skaggs, B.; Iqbal, N.; Ellis, D.; Pfaller, M. A.; Messer, S.; Rinaldi, M.; Fothergill, A.; Gibbs, D. L.; Wang, A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
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The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera, Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera, Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus, Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 μg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-μg disks, voriconazole 1- and 10-μg disks, two sources of caspofungin 5-μg disks [BBL and Oxoid], and posaconazole 5-μg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R, 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results...

Multicenter evaluation of a new disk agar diffusion method for susceptibility testing of filamentous fungi with voriconazole, posaconazole, itraconazole, amphotericin B, and caspofungin

Espinel-Ingroff, A.; Arthington-Skaggs, B.; Iqbal, N.; Ellis, D.; Pfaller, M.; Messer, S.; Rinaldi, M.; Fothergill, A.; Gibbs, D.; Wang, A.
Fonte: Amer Soc Microbiology Publicador: Amer Soc Microbiology
Tipo: Artigo de Revista Científica
Publicado em //2007 EN
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The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera, Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera, Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus, Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 µg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-µg disks, voriconazole 1- and 10-µg disks, two sources of caspofungin 5-µg disks [BBL and Oxoid], and posaconazole 5-µg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R, 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results...

Modeling development of inhibition zones in an agar diffusion bioassay

Chandrasekar, Vaishnavi; Knabel, Stephen J; Anantheswaran, Ramaswamy C
Fonte: John Wiley & Sons, Ltd Publicador: John Wiley & Sons, Ltd
Tipo: Artigo de Revista Científica
EN
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A two-temperature agar diffusion bioassay is commonly used to quantify the concentration of nisin using Micrococcus luteus as the indicator microorganism. A finite element computational model based on Fick's second law of diffusion was used to predict the radius of the inhibition zone in this diffusion bioassay. The model developed was used to calculate nisin concentration profiles as a function of time and position within the agar. The minimum inhibitory concentration (MIC) of nisin against M. luteus was determined experimentally. The critical time (Tc) for growth of M. luteus within the agar diffusion bioassay was experimentally determined using incubation studies with nisin. The radius of the inhibition zone was predicted from the computational model as the location where the predicted nisin concentration at Tc was equal to MIC. The MIC was experimentally determined to be 0.156 μg mL−1, and Tc was determined to be 7 h. Good agreement (R2 = 0.984) was obtained between model-predicted and experimentally determined inhibition zone radii.

Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution

Fiorentino, Flávia Angélica Másquio; Corrêa, Marcos Antonio; Salgado, Hérida Regina Nunes
Fonte: Universidade de São Paulo. Faculdade de Ciências Farmacêuticas Publicador: Universidade de São Paulo. Faculdade de Ciências Farmacêuticas
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; ; ; Formato: application/pdf
Publicado em 01/06/2013 ENG
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Clorexidina (CHX) é um antisséptico com amplo espectro de ação utilizada em muitos tipos de preparações farmacêuticas para uso tópico. Uma vez que não há na literatura ensaio microbiológico oficial para quantificar a clorexidina, este trabalho objetivou o desenvolvimento e validação de um ensaio microbiológico simples, sensível, exato e reprodutível, por difusão em ágar, para doseamento de digliconato de clorexidina (CHX-D) em solução aquosa. O ensaio é baseado no efeito da inibição de Staphylococcus aureus ATCC 25923, utilizado como microorganismo teste, pela CHX-D. Utilizou-se o delineamento 3x3. Os resultados foram verificados estatisticamente pela análise de variância (ANOVA) e apresentaram excelente linearidade (r = 0,9999), demonstrando que o método segue o modelo linear com regressão significativa entre o diâmetro da zona de inibição e o lagaritmo da concentração no intervalo de 0,5 a 4,5%. Os resultados obtidos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 2,03% e DPR para precisão inter-dias de 2,94%. A exatidão foi 99,03%. O método provou ser muito útil e apropriado para doseamento microbiológico da CHX-D em formas farmacêuticas e pode ser empregado para análise desta substância no controle de qualidade em indústrias farmacêuticas.; Chlorhexidine (CHX) is a broad-spectrum antiseptic that is used in many topical pharmaceutical formulations. Because there is no official microbiological assay reported in the literature that is used to quantify CHX...

Development of agar diffusion method for dosage of gramicidin

Solano, Ana Gabriela Reis; Pereira, Larissa de Melo Campos Sousa; Leonel, Míriam de Fátima Vianna; Nunan, Elzíria de Aguiar
Fonte: Universidade de São Paulo. Faculdade de Ciências Farmacêuticas Publicador: Universidade de São Paulo. Faculdade de Ciências Farmacêuticas
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; ; ; Formato: application/pdf
Publicado em 01/09/2011 ENG
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692.8867%
A gramicidina, um peptídeo antimicrobiano ativo contra bactérias Gram positivo, é utilizada em preparações farmacêuticas de uso tópico. Neste trabalho procurou-se desenvolver e validar outro método para o doseamento de gramicidina tendo em vista que somente o método turbidimétrico é descrito. O método de difusão em ágar foi desenvolvido e validado utilizando como microrganismo teste Kocuria rhizophila ATCC 9341. Foram utilizados dois delineamentos: retas paralelas 3x3 e curva padrão 5x1. A validação demonstrou que o método segue o modelo linear (r²= 0,994) havendo regressão significativa entre o diâmetro dos halos de inibição e o logaritmo da concentração na faixa de 5,00 a 25,3 µg/mL. Os resultados obtidos por ambos os delineamentos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 0,81 para ensaio 3x3 e de 1,90 para ensaio 5x1. Para a precisão inter-dias o DPR foi de 1,35 para 3x3 e de 2,64 para 5x1. A exatidão foi verificada e os resultados foram exatos apresentando intervalo de tolerância a 95% dentro dos limites permitidos e veracidade adequada. O método foi seletivo com limites de detecção e quantificação inferior e superior iguais a 2,00, 5,00 e 25,3 µg/mL...