Optimization of the microplate resazurin assay as a screening tool for natural products with anti-mycobacterial activity

The microplate resazurin assay (MRA) is commonly used for the isolation of anti-mycobacterial compounds. However, we found the MRA to be problematic in that it provided inconsistent results when used with phytochemical extracts. In this study we optimized the MRA for both screening and bioassay-guided fractionation of phytochemical extracts to facilitate the discovery of potential anti-mycobacterial therapeutics. MRA aspects that were evaluated include: assay duration; optimization of resazurin indicator concentration; solvent (DMSO) effect on mycobacterial growth; and microtitre plate type used. The optimized MRA was validated through bioassay guided isolation of falcarindiol, the major anti-mycobacterial natural product in extracts of Heracleum maximum, a medicinal plant used by the Eastern Canadian First Nations to treat TB.