6516172Broad Institute2157-02_Inhibitor_Dose_DryPowder_Activity201397Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_ActivityKeywords:Mycobacterium tuberculosis, drug resistance, non-replicating, carbon starvationAssay Overview:This assay determines the inhibitory effect of small molecules against M. tuberculosis during logarithmic growthExpected Outcome:Compounds are designated as active if 90% inhibition of growth was observed relative to DMSO control wellsMedia: 7H9 Complete (4.7 gm Middlebrook 7H9 powder, 900 mL water, 100 mL OADC (BD/Difco), 4 mL 50% glycerol, 2.5 mL 20% Tween-80)Strain: M. tuberculosis H37RvAssay steps: A master plate containing a 2-fold dilution series of a small molecule in DMSO was created in 96 well format (top concentration 50 mM, bottom concentration DMSO only).  4uL of the master plate dilution series was transferred to the first row of a clear 96 well assay plate (Costar, 3790).  196 uL of media was added to this row.  After pipetting up and down to mix, 50 uL was transferred to the next three rows, so that the top four rows of the plate contain 50 uL of diluted small molecule in media.  This process was repeated with a new small molecule in the bottom four rows of the assay plate.  The assay plate was then brought into the BSL3.In the BSL3 a logarithmic-phase M. tuberculosis culture (OD600 between 0.4 and 0.8) was diluted to an OD600 of .05.  50uL of the diluted TB culture was added to each well of the 96 well plate.  The plate was incubated at 37 C in a sealed Tupperware container for 14 days containing a wet kimtech shop towel.  After 14 days the wells were mixed by pipetting to disrupt clumps.  The OD600of the plate was read using M5 Spectramax (Molecular Devices) plate reader.  The active concentration is defined as the concentration that results in 90% inhibition of growth relative to the DMSO controls. All inactive compounds received an activity score of 1.  The active compounds were ranked on a scale of 10-100, with 100 representing the most active compound tested in this assay.48892983332Mycobacterium tuberculosis1ActiveConcentration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