Microplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. Since alamar blue has been recently identified as resazurin in cell cytotoxicity studies, we have standardized and evaluated a microplate method which uses the reduction of resazurin for dst to inh and rif in clinical isolates of m. International journal of mycobacteriology4 2015 306311 307. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. Microplate alamar blue assay for staphylococcus epidermidis bio. Validation of the alamarblue assay as a fast screening. Measuring cytotoxicity or proliferation alamarblue assay. Comparison between the cqs method and microplate reader quantification. Franzblaumicroplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium antimicrob. Microplate alamar blue assay versus bactec 460 system for. Adjust the cell count to 1 x 10 4 cellsml suggested cell density.
Pettit r, weber ca, kean mj, hoffmann h, pettit g, tan r et al. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. If the described method of microplate alamar blue biofilm. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window. The alamarblue hs and alamarblue cell viability reagents are readytouse resazurinbased reagents that function as cell health indicators by using the reducing power of living cells to quantitatively measure viability. The alamarblue assay is designed to measure quantitatively the proliferation of various human and animal. Stock solutions of all extracts at a concentration of 20 mgml were freshly prepared on the same day of the s1maba by resuspending 200 mg powder of each plant extract in 10 ml of sterile mq water. In the present study, a resazurin microplate method rmm using the fluorescent redox dye resazurin was developed. Living cells are metabolically active and are able to reduce via mitochondrial reductase, the nonfluorescent dye resazurin to the stronglyfluorescent dye resorufin fig. Alamarblue assay for cell proliferation bmg labtech. Microplate alamar blue assay for staphylococcus epidermidis biofilm susceptibility testing. The concentration of resazurin measured by the cqs was strongly correlated with the values measured by the elisa microplate reader y 206. We investigated a highthroughput method for determining the susceptibility of candida albicans biofilms that uses the oxidation reduction indicator alamar blue ab. L, relatively nontoxic and provided that it is carried out carefully, the same replicates can be followed over several time points.
In vitro antimycobacterial activity of six cameroonian. The microplate alamarblue assay maba is a sensitive, rapid, inexpensive, and nonradiometric method, which evaluates metabolic function and cellular health and offers the potential for screening large numbers of antimicrobial compounds. Biofilms from the tested candida albicans strains were markedly resistant to amphotericin b amb, nystatin nyt, fluconazole flc and 5fluorouracil 5fc, but susceptible to. Alamar blue redox dye is a type of resazurin that enters live, viable cells and then becomes reduced intracellularly. In this assay, the redox indicator alamar blue turns from blue to pink in the presence of mycobacterial growth. The rema plate method has proven to be, in recent experience, a reliable method for the detection of mdr tb 23. A simple method to measure cell viability in proliferation. Citeseerx microplate alamar blue assay for staphylococcus. Moreover, coban 2012 reported 100% agreement between the resazurin microplate assay for the rapid determination of mrsa and the cefoxitin mic determined by the reference broth microdilution method.
Method for measuring cytotoxicity or proliferation using alamarblue by fluorescence harvest cells which are in the log phase of growth and determine cell count. The main obstacle in antimycobacterial discovery is the extremely slow growth rates of pathogenic mycobacteria that lead to the long incubation times needed in antimycobacterial screening. Microplate reader assays for cell proliferation biocompare. The alamarblue hs and alamarblue cell viability reagents are readytouse resazurinbased reagents that function as cell health indicators by using the reducing power. The 96well microplate alamar blue assay maba allows for the quantitative. Resazurin is blue and nonfluorescent whereas resorufin is red and highly fluorescent. Pdf microplate alamarblue assay for paracoccidioides. The resazurin assay also known as alamar blue assay offers a simple, rapid, and sensitive measurement for the viability of mammalian cells and bacteria. Some in vitro testings has been developed and are currently available for antimycobacterial screening.
Dec 30, 20 another brothbased method, the microplate alamar blue assay, has been used for years with favorable results for mycobacterium tuberculosis complex isolates. Calcein am provides a simple, rapid, and accurate method to measure cell viability andor cytotoxicity. Maba is a microplate method that used as highthroughput screen antituberculosis activity of samples. Another brothbased method, the microplate alamar blue assay, has been used for years with favorable results for mycobacterium tuberculosis complex isolates. 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 multidrugresistant strains and the h37rv strain by using bacterial suspensions prepared directly from solid media. Alamar blue s stability and lack of toxicity allowed cfuml to be determined from the same wells as alamar blue absorbances. Simple resazurinbased microplate assay for measuring.
Pdf the use of the microplate alamar blue assay maba to. As examples, there are those that monitor dna synthesis by the use of either a radioactive label 3 hthymidine uptake or immunofluorescence antibrdu monoclonal antibody. Antimycobacterialactivity tests the antimycobacterial activity of the plant crude extracts was tested using the microplate alamarblue assay 18,19. Microplate based alamar blue assay maba is a practical, sensitive and inexpensive assay method of cell viability in which fluorescence reduction assay of alamar blue, a resazurin a dark blue dye and nonfluoroscent in oxidized form, available as a sterile and liquid reagent commercially, is used in a microplate format. The subsequent cell signals can be detected using microplate readers. A similar reagent, known as water soluble tetrazolium salts wsts, are tetrazolium dyes that do not enter cells but become reduced extracellularly by. It is a sensitive assay if working with higher than 5x10 3 cells per 100.
The assay was conducted according to clsi m27a3 method 2008, using an inoculum of 2. Microplate alamar blue assay for staphylococcus epidermidis biofilm susceptibility testing robin k. Inter and intraassay reproducibility of microplate alamar. The optimum cell density may vary between cell types. Antimicrobial susceptibility of standard strains of. Long hansens disease center, baton rouge, louisiana 70894, usa. A simple method to measure cell viability in proliferation and cytotoxicity assays 256 braz oral res 2009. If the described method of microplate alamar blue biofilm susceptibility testing, which is simple, reproducible, cost. Tetrazolium microplate assay as a rapid and inexpensive. Resazurin has been identified as the main component of alamar blue 22. Microplate alamarblue assay maba for the determination of antimicrobial activity of each plant extract, mabas were performed 19, 23, 24. Rapid, lowtechnology mic determination with clinical. In this study, we used the microplate alamar blue assay to test the activities of 15 drugs against 24 standard ntm strains in china.
Plate cells and expose to test agent as determined by researcher. The results were compared to those of the proportion method pm on lj. Cell lines are often cultivated in contact with the target materials, and after a variable period. Resazurin microtiter assay plate method for detection of. A colorimetric, microplatebased alamar blue assay maba method was used to determine the mics of isoniazid inh, rifampin, streptomycin sm, and. Alamar blues stability and lack of toxicity allowed cfuml to be determined from the same wells as alamar blue absorbances.
Here we describe a stepbystep procedure for both the maba and lora. The 96well 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. Microplate alamar blue assay maba and low oxygen recovery. The inexpensive microplate alamar blue assay maba is an indirect colorimetric dst method for determining the mics of tb drugs for strains of mycobacterium tuberculosis. Trek diagnostic systems, in comparison with these other methods of susceptibility testing, may have many potential advantages. The inexpensive microplate alamar blue assay maba is an indirect colorimetric dst method for determining the mics of tb drugs for strains of mycobacterium tuberculosis 10. This is a trusted and established reagent which has been available since 1993. Microplate alamar blue assay for susceptibility testing of candida. A simple method for the isolation and purification of total lipides from animal tissues.
The use of the microplate alamar blue assay maba to. This method, the microplate alamar blue assay maba, is based on the detection of colorimetric changes caused by the oxidation and reduction capabilities of alamar blue dye. L collins and s g franzblau pharmacology research department, gillis w. Microplate alamar blue assay for staphylococcus epidermidis. The aim of the study was to compare resazurin microplate assay rema and crystal violet decolorization.
Resazurin changes from blue nonfluorescent to pinkhighlyfluorescent testing format. For example, ab results can be read visually, spectrofluorometrically or spectrophotometrically, its reduction is dependent on active metabolism, it is amenable to highthroughput, its use. Original article resazurin microtiter assay for detection of. Horton5 cancer research institute,1 department of chemistry and biochemistry,2 and school of life sciences,3.
If the described method of microplate alamar blue biofilm susceptibility testing, which is simple, reproducible, costeffective, nontoxic, and amenable to high throughput, is applicable to other. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. This method used alamar blue, a resazurinbased oxidationreduction based indicator. With a pure culture, this method can determine antibiotic susceptibility in 6 to 7 days. The use of the microplate alamar blue assay maba to assess the. Microplate alamar blue assay maba and low oxygen recovery assay lora for. Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplate based susceptibility assays. Antituberculosis activity of brotowali tinospora crispa. Microplate alamar blue assay for susceptibility testing of.