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BioAssay: AID 651617

Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_Activity

This assay determines the inhibitory effect of small molecules against M. tuberculosis during logarithmic growth ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
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AID: 651617
Data Source: Broad Institute (2157-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-10-04
Hold-until Date: 2013-09-07
Modify Date: 2013-09-10

Data Table ( Complete ):           Active    All
BioActive Compounds: 25
Depositor Specified Assays
488929Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosis - Summarysummary
687034Broad Institute Elucidation of the physiology of non-replicating, drug tolerant Mycobacterium tuberculosis: Probe to inhibit the Transition from Non-Replication to Replication Inhibitor Probe Projectsummary
Mycobacterium tuberculosis, drug resistance, non-replicating, carbon starvation

Assay Overview:
This assay determines the inhibitory effect of small molecules against M. tuberculosis during logarithmic growth
Expected Outcome:Compounds are designated as active if 90% inhibition of growth was observed relative to DMSO control wells
Media: 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 H37Rv

Assay 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.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger

* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH087444-01

Data Table (Concise)