80 ml of

80 ml of sterile molten Sabouraud Dextrose Agar (, , ) or Mueller Hinton Agar (MHA) () were maintained in a water bath at 45°C to prevent solidification of the medium, and were aseptically inoculated with 200 µl of bacteria or yeast suspension. Microbial inoculum and medium were well mixed and dispensed into sterile diameter Petri dishes and allowed to solidify at room temperature in a sterile cupboard. After solidification, discs of in diameter previously impregnated with 10 µl of test samples were placed aseptically on the solid plates. This gave a charge of 1 mg or

100 µg of test substances per disc. The Petri dishes were Inhibitors,research,lifescience,medical left at +4°C for 2 h to allow extracts and compounds to diffuse from the discs into the medium. The test media were then incubated at 35°C for 24 h (for bacteria) and 48 h (for yeasts). Antimicrobial activity was evaluated by selleck chem FTY720 measuring the clear zone of growth inhibition on agar surface

around the discs. The assay was done in triplicates. Gentamicin (Sigma-Aldrich, Inhibitors,research,lifescience,medical , ) and nystatin (Merck, ) were used as positive controls for bacteria and yeasts respectively Inhibitors,research,lifescience,medical at 10 µg per disc. Dimethylsulfoxide solution (10% v/v) was used as a negative control. Determination of minimum inhibitory concentrations (MICs) and minimum customer reviews microbicidal concentrations (MMCs) Minimum inhibitory concentration values were determined by broth micro dilution method as reported by Nyaa and co-workers.16 The test samples were Inhibitors,research,lifescience,medical first of all dissolved in DMSO. The solution obtained was then added to Mueller Hinton Broth (MHB) for bacteria or Sabouraud Dextrose Broth (SDB) for yeasts to give a final concentration of 2000

µg/ml. This was serially diluted two folds to obtain a concentration range of 0.48 to 2000 µg/ml. One hundred microliters Inhibitors,research,lifescience,medical of each concentration was added into each well (96- wells microplate) containing 95 µl of MHB or SDB and 5 µl of inoculum (106 CFU/ml for bacteria and 5×105 spores/ml for yeasts) to obtain final concentrations varying from 0.12 to 1000µg/ml. The final concentration of DMSO in the well was less than 1% (v/v). Preliminary analysis with 1% (v/v) DMSO did not inhibit the growth of the test organisms. The negative control wells consisted of 195 µl of MHB or SDB and 5 µl of the inoculum. The plates were covered with sterile lids, then agitated to mix the contents of wells Dacomitinib using a plate shaker and incubated at 35°C for 24 hours for bacteria, 48 hours for Candida sp, or 72 hours for Cryptococcus neoformans. The assays were repeated thrice. The MICs of samples were detected following the addition of 50 µl of a 0.20 mg/ml p-iodonitrotetrazolium violet (INT) solution followed by incubation at 35°C for 30 min. The colorless tetrazolium salt acts as an electron acceptor and is reduced to a red-colored formazan product by biologically active microorganisms. Where microbial growth was inhibited, the solution in the well remained clear after incubation with INT.

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