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Scott G Franzblau - One of the best experts on this subject based on the ideXlab platform.
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Microplate Alamar Blue Assay (MABA) and Low Oxygen Recovery Assay (LORA) for Mycobacterium tuberculosis
Mycobacteria Protocols, 2015Co-Authors: Sang Hyun Cho, Hyung Sup Lee, Scott G Franzblau, Scott FranzblauAbstract:Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplate-based susceptibility assays. The 96-well Microplate Alamar Blue Assay (MABA) allows for the quantitative determination of drug susceptibility against any strain of replicating Mycobacterium tuberculosis to be completed within a week at minimal cost. The Low-Oxygen Recovery Assay (LORA) uses a recombinant M. tuberculosis expressing luciferase and provides results of drug activity against non-replicating M. tuberculosis surviving under hypoxic conditions. Determining activity against non-replicating M. tuberculosis is an important factor when developing drug candidates against M. tuberculosis. Here we describe a step-by-step procedure for both the MABA and LORA.
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carbamidocyclophanes f and g with anti mycobacterium tuberculosis activity from the cultured freshwater cyanobacterium nostoc sp
ChemInform, 2014Co-Authors: Hahk Soo Kang, Scott G Franzblau, Aleksej Krunic, George E Chlipala, Wei Lun Chen, Steven M Swanson, Jimmy OrjalaAbstract:Carbamidocyclophane F (Ia) shows a potent anti-Mycobacterium tuberculosis activity in the microplate Alamar Blue assay and low-oxygen-recovery assay with MIC values of 0.8 and 5.4 μM, respectively.
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eucapsitrione an anti mycobacterium tuberculosis anthraquinone derivative from the cultured freshwater cyanobacterium eucapsis sp
Journal of Natural Products, 2010Co-Authors: Megan Sturdy, Sang Hyun Cho, Scott G Franzblau, Aleksej Krunic, Jimmy OrjalaAbstract:Eucapsitrione (1), an anthraquinone derivative with an indeno-anthracene-trione skeleton, was isolated from the cyanobacterium Eucapsis sp. (UTEX 1519) by bioassay-guided fractionation. The chemical structure was determined by analyzing MS and 1D and 2D NMR spectroscopic data. Eucapsitrione (1) showed anti-Mycobacterium tuberculosis activity in the microplate Alamar Blue assay and low-oxygen-recovery assay with MIC values of 3.1 and 6.4 μM, respectively.
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rapid low technology mic determination with clinical mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay
Journal of Clinical Microbiology, 1998Co-Authors: Scott G Franzblau, Richard S Witzig, J C Mclaughlin, Patricia Torres, Guillermo Madico, A Hernandez, Michelle T Degnan, Mary B Cook, Virginia K Quenzer, Robert M FergusonAbstract:A colorimetric, microplate-based Alamar Blue assay (MABA) method was used to determine the MICs of isoniazid (INH), rifampin, streptomycin (SM), and ethambutol (EMB) for 34 Peruvian Mycobacterium tuberculosis isolates (including both pansensitive and multidrug-resistant strains) and the H37Rv strain by using bacterial suspensions prepared directly from solid media. Results for all isolates were available within 8 days. Discordant results were observed on initial tests for 3 of 16 INH-susceptible isolates, 5 of 31 EMB-susceptible isolates, and 2 of 4 SM-resistant isolates (by the BACTEC 460 system). The overall agreements between the MICs obtained by MABA and the results obtained with the BACTEC 460 system were 87.9% for initial results and 93.6% after retesting 12 of 17 samples with discrepant results. Interpretation of MABA endpoints improved with technical experience. The MABA is a simple, rapid, low-cost, appropriate technology which does not require expensive instrumentation and which makes use of a nontoxic, temperature-stable reagent.
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microplate Alamar Blue assay versus bactec 460 system for high throughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: L A Collins, Scott G FranzblauAbstract:In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar Blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar Blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.
Robin K Pettit - One of the best experts on this subject based on the ideXlab platform.
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application of a high throughput Alamar Blue biofilm susceptibility assay to staphylococcus aureus biofilms
Annals of Clinical Microbiology and Antimicrobials, 2009Co-Authors: Robin K Pettit, Christine A Weber, George R PettitAbstract:Background: Staphylococcus aureus and S. epidermidis biofilms differ in structure, growth and regulation, and thus the high-throughput method of evaluating biofilm susceptibility that has been published for S. epidermidis cannot be applied to S. aureus without first evaluating the assay's reproducibility and reliability with S. aureus biofilms. Methods: Staphylococcus aureus biofilms were treated with eleven approved antibiotics, lysostaphin, or Conflikt®, exposed to the oxidation reduction indicator Alamar Blue, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration (MBIC) was defined as ≤ 50% Alamar Blue reduction and a purple/ Blue well 60 min after the addition of Alamar Blue. Because all of the approved antibiotics had MBICs >128 μg/ml (most >2048 μg/ml), lysostaphin and Conflikt®, with relatively low MBICs, were used to correlate Alamar Blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. aureus ATCC 29213 and three clinical isolates. Alamar Blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar Blue absorbances. Results: Overall, Alamar Blue reduction had excellent correlation with XTT reduction and with CFU/ml. For ATCC 29213 and two clinical isolates treated with lysostaphin or Conflikt ® , Alamar Blue reduction had excellent correlation with XTT reduction (r = 0.93-0.99) and with CFU/ml (r = 0.92-0.98). For one of the clinical isolates, the results were moderately correlated for Conflikt® (r = 0.76, Alamar Blue vs. XTT; r = 0.81, Alamar Blue vs. CFU/ml) and had excellent correlation for lysostaphin (r = 0.95, Alamar Blue vs. XTT; r = 0.97, Alamar Blue vs. CFU/ml). Conclusion: A reliable, reproducible method for evaluating biofilm susceptibility was successfully applied to S. aureus biofilms. The described method provides researchers with a simple, nontoxic, relatively inexpensive, high throughput measure of viability after drug treatment. A standardized biofilm Alamar Blue assay should greatly increase the rate of discovery of S. aureus biofilm specific agents.
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microplate Alamar Blue assay for susceptibility testing of candida albicans biofilms
Medical Mycology, 2007Co-Authors: Kimberly K Repp, Scott Menor, Robin K PettitAbstract:Although biofilm-based fungal infections are an important cause of morbidity and mortality in patients, there is no standardized method for the in vitro evaluation of the drug susceptibility of biofilms. We investigated a high-throughput method for determining the susceptibility of Candida albicans biofilms that uses the oxidation reduction indicator Alamar Blue (AB). Biofilms from the tested Candida albicans strains were markedly resistant to amphotericin B (AMB), nystatin (NYT), fluconazole (FLC) and 5-fluorouracil (5FC), but susceptible to Conflikt disinfectant. The latter was used in comparative studies of AB reduction with two other methods for assessing in vitro drug susceptibility i.e., 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and enumeration of viable colony counts (CFU/ml). AB results correlated well with XTT (r=0.88-0.93) and CFU/ml (r=0.93-0.99) for all four C. albicans test strains. This simple, reproducible method for determining in vitro drug susceptibility should facilitate discovery of antifungals active against Candida biofilms.
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microplate Alamar Blue assay for staphylococcus epidermidis biofilm susceptibility testing
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Robin K Pettit, Christine A Weber, Melissa J Kean, Holger Hoffmann, George R Pettit, Kelly S Franks, Marilyn L HortonAbstract:Biofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility. Staphylococcus epidermidis ATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar Blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as ≤50% reduction and a purplish well 60 min after the addition of Alamar Blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 μg/ml (most >4,096 μg/ml), and four of the experimental compounds had MBICs of ≤128 μg/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar Blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. epidermidis ATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar Blue results correlated well with XTT (r = 0.83 to 0.97) and with CFU/ml results (r = 0.85 to 0.94). Alamar Blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar Blue absorbances. If the described method of microplate Alamar Blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.
Kristin Schirmer - One of the best experts on this subject based on the ideXlab platform.
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application of Alamar Blue 5 carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout
Analytical Biochemistry, 2005Co-Authors: Anja Schreer, Cheryl Tinson, James P Sherry, Kristin SchirmerAbstract:We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes--Alamar Blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity-are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10(-5) M 17beta-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar Blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.
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application of Alamar Blue 5 carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout
Analytical Biochemistry, 2005Co-Authors: Anja Schreer, Cheryl Tinson, James P Sherry, Kristin SchirmerAbstract:Abstract We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes—Alamar Blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity—are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10−5 M 17β-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar Blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.
L A Collins - One of the best experts on this subject based on the ideXlab platform.
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microplate Alamar Blue assay versus bactec 460 system for high throughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: L A Collins, Scott G FranzblauAbstract:In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar Blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar Blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.
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microplate Alamar Blue assay versus bactec 460 system for high throughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: L A Collins, Scott G FranzblauAbstract:In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar Blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar Blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was <1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria. With the increased need for drugs to combat multidrugresistant tuberculosis (TB), there is an urgent need for rapid, low-cost, high-throughput assays for screening new drug candidates. Ideally, such assays would also be useful for assessing activity against Mycobacterium avium, an opportunistic pathogen which is resistant to most existing antimicrobials. Due to the slow growth of Mycobacterium tuberculosis, M. avium, and related species, incubation times for drug susceptibility assays which rely on the development of colonies or turbidity are excessively long. Susceptibility can be determined more rapidly with the radiometric BACTEC 460 system, the clinical drug susceptibility system of choice for most of the last decade (9, 10), but the high cost, high volume, lack of high-throughput format, and requirement for radioisotope disposal all limit its usefulness for mass screening. Recently, there have been a number of new mycobacterial drug susceptibility assays described (2, 4, 5, 12, 16). Most of these assays, however, lack one or more of the desired attributes of a mass screening assay: rapidity, high throughput, and low cost of supplies and equipment. The Alamar Blue oxidation-reduction dye is a general indicator of cellular growth and/or viability; the Blue, nonfluores
Anja Schreer - One of the best experts on this subject based on the ideXlab platform.
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application of Alamar Blue 5 carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout
Analytical Biochemistry, 2005Co-Authors: Anja Schreer, Cheryl Tinson, James P Sherry, Kristin SchirmerAbstract:We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes--Alamar Blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity-are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10(-5) M 17beta-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar Blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.
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application of Alamar Blue 5 carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout
Analytical Biochemistry, 2005Co-Authors: Anja Schreer, Cheryl Tinson, James P Sherry, Kristin SchirmerAbstract:Abstract We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes—Alamar Blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity—are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10−5 M 17β-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar Blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.