title: Optimisation of the microplate resazurin assay for screening and bioassay-guided fractionation of phytochemical extracts against Mycobacterium tuberculosis.
abstract: BACKGROUND
  Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate resazurin assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the assay can be problematic and unreliable when screening methanolic phytochemical extracts.
  
  
  OBJECTIVE
  To optimise the MRA for the screening and bioassay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra.
  
  
  METHODS
  The effects of varying assay duration, resazurin solution composition, solvent (dimethyl sulphoxide - DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioassay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity.
  
  
  RESULTS
  The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL resazurin in 5% aqueous Tween 80 over 96 h incubation.
  
  
  CONCLUSIONS
  The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The assay procedure has been used successfully for the screening and bioassay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants.
authors.affiliation: Department of Biology, University of New Brunswick, Saint John, New Brunswick, Canada, E2L 4L5.
authors.forename: Taryn E
authors.initials: TE
authors.lastname: O'Neill
authors.forename: Haoxin
authors.initials: H
authors.lastname: Li
authors.forename: Caitlyn D
authors.initials: CD
authors.lastname: Colquhoun
authors.forename: John A
authors.initials: JA
authors.lastname: Johnson
authors.forename: Duncan
authors.initials: D
authors.lastname: Webster
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Gray
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001681:Biological Assay; D005591:Chemical Fractionation; D019684:Magnoliopsida / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D010078:Oxazines / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D012293:Rifampin / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry*
chemical_list: D000995:Antitubercular Agents; D010078:Oxazines; D010936:Plant Extracts; D014966:Xanthenes; C005843:resazurin; D012293:Rifampin
keywords: Mycobacterium tuberculosis H37Ra; anti-mycobacterial activity; bioassay screening; bioassay-guided fractionation; medicinal plants; microplate resazurin assay