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Christopher A Gray - One of the best experts on this subject based on the ideXlab platform.
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optimisation of the microplate Resazurin Assay for screening and bioAssay guided fractionation of phytochemical extracts against mycobacterium tuberculosis
Phytochemical Analysis, 2014Co-Authors: Taryn Oneill, Duncan Webster, John A Johnson, Haoxin Li, Caitlyn D Colquhoun, Christopher A GrayAbstract:Introduction Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate Resazurin Assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the Assay can be problematic and unreliable when screening methanolic phytochemical extracts. Objective To optimise the MRA for the screening and bioAssay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. Methods The effects of varying Assay duration, Resazurin solution composition, solvent (dimethyl sulphoxide – DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioAssay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. Results The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL Resazurin in 5% aqueous Tween 80 over 96 h incubation. Conclusion The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The Assay procedure has been used successfully for the screening and bioAssay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.
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Optimisation of the Microplate Resazurin Assay for Screening and BioAssay‐guided Fractionation of Phytochemical Extracts against Mycobacterium tuberculosis
Phytochemical analysis : PCA, 2014Co-Authors: Taryn O'neill, Duncan Webster, John A Johnson, Caitlyn D Colquhoun, Christopher A GrayAbstract:Introduction Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate Resazurin Assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the Assay can be problematic and unreliable when screening methanolic phytochemical extracts. Objective To optimise the MRA for the screening and bioAssay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. Methods The effects of varying Assay duration, Resazurin solution composition, solvent (dimethyl sulphoxide – DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioAssay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. Results The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL Resazurin in 5% aqueous Tween 80 over 96 h incubation. Conclusion The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The Assay procedure has been used successfully for the screening and bioAssay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.
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antimycobacterial screening of traditional medicinal plants using the microplate Resazurin Assay
Canadian Journal of Microbiology, 2010Co-Authors: Duncan Webster, Jill Moorej Moore, Tracy Manningt Manning, Dennis Kunimotod Kunimoto, Darren Leblancd Leblanc, John A Johnson, Christopher A GrayAbstract:Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette–Guerin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate Resazurin Assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 µg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT Assay also showed that the extracts were less toxic to 3 human cell lines than was ...
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Antimycobacterial screening of traditional medicinal plants using the microplate Resazurin Assay
Canadian journal of microbiology, 2010Co-Authors: Duncan Webster, Jill Moorej Moore, Tracy Manningt Manning, Dennis Kunimotod Kunimoto, Darren Leblancd Leblanc, John A Johnson, Timothy D.g. Lee, Christopher A GrayAbstract:Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette-Guerin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate Resazurin Assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 microg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT Assay also showed that the extracts were less toxic to 3 human cell lines than was the DMSO positive control. This study demonstrates that aqueous extracts of the roots of H. maximum and Acorus calamus possess strong in vitro antimycobacterial activity, validates traditional knowledge, and provides potential for the development of urgently needed novel antituberculous therapeutics.
Tom Coenye - One of the best experts on this subject based on the ideXlab platform.
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Taking the Silver Bullet Colloidal Silver Particles for the Topical Treatment of Biofilm-Related Infections.
ACS applied materials & interfaces, 2017Co-Authors: Katharina Richter, Ilse Vandecandelaere, Tom Coenye, Paula Facal, Nicky Thomas, Mahnaz Ramezanpour, Clare Cooksley, Clive A. Prestidge, Peter-john Wormald, Sarah VreugdeAbstract:Biofilms are aggregates of bacteria residing in a self-assembled matrix, which protects these sessile cells against external stress, including antibiotic therapies. In light of emerging multidrug-resistant bacteria, alternative strategies to antibiotics are emerging. The present study evaluated the activity of colloidal silver nanoparticles (AgNPs) of different shapes against biofilms formed by Staphylococcus aureus (SA), methicillin-resistant SA (MRSA), and Pseudomonas aeruginosa (PA). Colloidal quasi-spherical, cubic, and star-shaped AgNPs were synthesized, and their cytotoxicity on macrophages (THP-1) and bronchial epithelial cells (Nuli-1) was analyzed by the lactate dehydrogenase Assay. The antibiofilm activity was assessed in vitro by the Resazurin Assay and in an in vivo infection model in Caenorhabditis elegans. Cubic and star-shaped AgNPs induced cytotoxicity, while quasi-spherical AgNPs were not toxic. Quasi-spherical AgNPs showed substantial antibiofilm activity in vitro with 96% (±2%), 97% (±1...
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A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Ilse Vandecandelaere, Heleen Van Acker, Tom CoenyeAbstract:We describe a 96-well microtiter plate-based system as an in vitro model for biofilm formation and quantification. Although in vitro Assays are artificial systems and thus significantly differ from in vivo conditions, they represent an important tool to evaluate biofilm formation and the effect of compounds on biofilms. Stainings to evaluate the amount of biomass (crystal violet staining) and the number of metabolically active cells (Resazurin Assay) are discussed and specific attention is paid to the use of this model to quantify persisters in sessile populations.
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comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates
Journal of Microbiological Methods, 2008Co-Authors: Elke Peeters, Hans J Nelis, Tom CoenyeAbstract:In the present study six Assays for the quantification of biofilms formed in 96-well microtiter plates were optimised and evaluated: the crystal violet (CV) Assay, the Syto9 Assay, the fluorescein diacetate (FDA) Assay, the Resazurin Assay, the XTT Assay and the dimethyl methylene blue (DMMB) Assay. Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, Propionibacterium acnes and Candida albicans were used as test organisms. In general, these Assays showed a broad applicability and a high repeatability for most isolates. In addition, the estimated numbers of CFUs present in the biofilms show limited variations between the different Assays. Nevertheless, our data show that some Assays are less suitable for the quantification of biofilms of particular isolates (e.g. the CV Assay for P. aeruginosa).
Duncan Webster - One of the best experts on this subject based on the ideXlab platform.
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optimisation of the microplate Resazurin Assay for screening and bioAssay guided fractionation of phytochemical extracts against mycobacterium tuberculosis
Phytochemical Analysis, 2014Co-Authors: Taryn Oneill, Duncan Webster, John A Johnson, Haoxin Li, Caitlyn D Colquhoun, Christopher A GrayAbstract:Introduction Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate Resazurin Assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the Assay can be problematic and unreliable when screening methanolic phytochemical extracts. Objective To optimise the MRA for the screening and bioAssay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. Methods The effects of varying Assay duration, Resazurin solution composition, solvent (dimethyl sulphoxide – DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioAssay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. Results The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL Resazurin in 5% aqueous Tween 80 over 96 h incubation. Conclusion The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The Assay procedure has been used successfully for the screening and bioAssay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.
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Optimisation of the Microplate Resazurin Assay for Screening and BioAssay‐guided Fractionation of Phytochemical Extracts against Mycobacterium tuberculosis
Phytochemical analysis : PCA, 2014Co-Authors: Taryn O'neill, Duncan Webster, John A Johnson, Caitlyn D Colquhoun, Christopher A GrayAbstract:Introduction Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate Resazurin Assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the Assay can be problematic and unreliable when screening methanolic phytochemical extracts. Objective To optimise the MRA for the screening and bioAssay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. Methods The effects of varying Assay duration, Resazurin solution composition, solvent (dimethyl sulphoxide – DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioAssay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. Results The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL Resazurin in 5% aqueous Tween 80 over 96 h incubation. Conclusion The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The Assay procedure has been used successfully for the screening and bioAssay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.
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antimycobacterial screening of traditional medicinal plants using the microplate Resazurin Assay
Canadian Journal of Microbiology, 2010Co-Authors: Duncan Webster, Jill Moorej Moore, Tracy Manningt Manning, Dennis Kunimotod Kunimoto, Darren Leblancd Leblanc, John A Johnson, Christopher A GrayAbstract:Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette–Guerin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate Resazurin Assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 µg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT Assay also showed that the extracts were less toxic to 3 human cell lines than was ...
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Antimycobacterial screening of traditional medicinal plants using the microplate Resazurin Assay
Canadian journal of microbiology, 2010Co-Authors: Duncan Webster, Jill Moorej Moore, Tracy Manningt Manning, Dennis Kunimotod Kunimoto, Darren Leblancd Leblanc, John A Johnson, Timothy D.g. Lee, Christopher A GrayAbstract:Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette-Guerin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate Resazurin Assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 microg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT Assay also showed that the extracts were less toxic to 3 human cell lines than was the DMSO positive control. This study demonstrates that aqueous extracts of the roots of H. maximum and Acorus calamus possess strong in vitro antimycobacterial activity, validates traditional knowledge, and provides potential for the development of urgently needed novel antituberculous therapeutics.
John A Johnson - One of the best experts on this subject based on the ideXlab platform.
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optimisation of the microplate Resazurin Assay for screening and bioAssay guided fractionation of phytochemical extracts against mycobacterium tuberculosis
Phytochemical Analysis, 2014Co-Authors: Taryn Oneill, Duncan Webster, John A Johnson, Haoxin Li, Caitlyn D Colquhoun, Christopher A GrayAbstract:Introduction Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate Resazurin Assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the Assay can be problematic and unreliable when screening methanolic phytochemical extracts. Objective To optimise the MRA for the screening and bioAssay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. Methods The effects of varying Assay duration, Resazurin solution composition, solvent (dimethyl sulphoxide – DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioAssay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. Results The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL Resazurin in 5% aqueous Tween 80 over 96 h incubation. Conclusion The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The Assay procedure has been used successfully for the screening and bioAssay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.
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Optimisation of the Microplate Resazurin Assay for Screening and BioAssay‐guided Fractionation of Phytochemical Extracts against Mycobacterium tuberculosis
Phytochemical analysis : PCA, 2014Co-Authors: Taryn O'neill, Duncan Webster, John A Johnson, Caitlyn D Colquhoun, Christopher A GrayAbstract:Introduction Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate Resazurin Assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the Assay can be problematic and unreliable when screening methanolic phytochemical extracts. Objective To optimise the MRA for the screening and bioAssay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. Methods The effects of varying Assay duration, Resazurin solution composition, solvent (dimethyl sulphoxide – DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioAssay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. Results The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL Resazurin in 5% aqueous Tween 80 over 96 h incubation. Conclusion The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The Assay procedure has been used successfully for the screening and bioAssay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.
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antimycobacterial screening of traditional medicinal plants using the microplate Resazurin Assay
Canadian Journal of Microbiology, 2010Co-Authors: Duncan Webster, Jill Moorej Moore, Tracy Manningt Manning, Dennis Kunimotod Kunimoto, Darren Leblancd Leblanc, John A Johnson, Christopher A GrayAbstract:Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette–Guerin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate Resazurin Assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 µg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT Assay also showed that the extracts were less toxic to 3 human cell lines than was ...
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Antimycobacterial screening of traditional medicinal plants using the microplate Resazurin Assay
Canadian journal of microbiology, 2010Co-Authors: Duncan Webster, Jill Moorej Moore, Tracy Manningt Manning, Dennis Kunimotod Kunimoto, Darren Leblancd Leblanc, John A Johnson, Timothy D.g. Lee, Christopher A GrayAbstract:Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette-Guerin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate Resazurin Assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 microg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT Assay also showed that the extracts were less toxic to 3 human cell lines than was the DMSO positive control. This study demonstrates that aqueous extracts of the roots of H. maximum and Acorus calamus possess strong in vitro antimycobacterial activity, validates traditional knowledge, and provides potential for the development of urgently needed novel antituberculous therapeutics.
Ilse Vandecandelaere - One of the best experts on this subject based on the ideXlab platform.
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Taking the Silver Bullet Colloidal Silver Particles for the Topical Treatment of Biofilm-Related Infections.
ACS applied materials & interfaces, 2017Co-Authors: Katharina Richter, Ilse Vandecandelaere, Tom Coenye, Paula Facal, Nicky Thomas, Mahnaz Ramezanpour, Clare Cooksley, Clive A. Prestidge, Peter-john Wormald, Sarah VreugdeAbstract:Biofilms are aggregates of bacteria residing in a self-assembled matrix, which protects these sessile cells against external stress, including antibiotic therapies. In light of emerging multidrug-resistant bacteria, alternative strategies to antibiotics are emerging. The present study evaluated the activity of colloidal silver nanoparticles (AgNPs) of different shapes against biofilms formed by Staphylococcus aureus (SA), methicillin-resistant SA (MRSA), and Pseudomonas aeruginosa (PA). Colloidal quasi-spherical, cubic, and star-shaped AgNPs were synthesized, and their cytotoxicity on macrophages (THP-1) and bronchial epithelial cells (Nuli-1) was analyzed by the lactate dehydrogenase Assay. The antibiofilm activity was assessed in vitro by the Resazurin Assay and in an in vivo infection model in Caenorhabditis elegans. Cubic and star-shaped AgNPs induced cytotoxicity, while quasi-spherical AgNPs were not toxic. Quasi-spherical AgNPs showed substantial antibiofilm activity in vitro with 96% (±2%), 97% (±1...
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A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Ilse Vandecandelaere, Heleen Van Acker, Tom CoenyeAbstract:We describe a 96-well microtiter plate-based system as an in vitro model for biofilm formation and quantification. Although in vitro Assays are artificial systems and thus significantly differ from in vivo conditions, they represent an important tool to evaluate biofilm formation and the effect of compounds on biofilms. Stainings to evaluate the amount of biomass (crystal violet staining) and the number of metabolically active cells (Resazurin Assay) are discussed and specific attention is paid to the use of this model to quantify persisters in sessile populations.