Rapid, semiquantitative assay to discriminate among compounds with activity against replicating or nonreplicating Mycobacterium tuberculosis Academic Article uri icon


MeSH Major

  • Antitubercular Agents
  • Biological Assay
  • Colony Count, Microbial
  • Mycobacterium tuberculosis


  • The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days-about 2 weeks sooner than required to count CFU-fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic.

publication date

  • October 2015



  • Academic Article



  • eng

PubMed Central ID

  • PMC4576094

Digital Object Identifier (DOI)

  • 10.1128/AAC.00803-15

PubMed ID

  • 26239979

Additional Document Info

start page

  • 6521

end page

  • 38


  • 59


  • 10