There is a real and urgent need for new antibiotics which are able to kill Mycobacteria. This group of bacteria includes Mycobacterium tuberculosis, a deadly human pathogen that latently infects a third of the world\u2019s population. Multi-drug resistant (MDR) and totally-drug resistant (TDR) isolates have also now evolved. Due to the difficulties and dangers involved in culturing M. tuberculosis, an airborne Biosafety Level 3 pathogen, faster-growing, and less pathogenic mycobacterial species are routinely exploited for TB research and anti-mycobacterial drug discovery. M. marinum shares conserved virulence determinants with M. tuberculosis and is a pathogen of ectotherms (fish, amphibians, and reptiles), as well as causing granulomatous skin infections in humans.Because of the slow growth of many mycobacterial species, we routinely use luciferase-tagged strains (1) for our assays. As bacteria only produce light when alive, bioluminescence is an excellent non-destructive real-time reporter to assay for anti-mycobacterial activity in microtitre plate formats using a luminometer (2-4) or in vivo using sensitive imaging equipment (5). This protocol is modified from the method described by Dalton et al (4).References:Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible UE, Robertson BD, Wiles S (2010). Optimisation of bioluminescent reporters for use with Mycobacteria. PLOS One. 5(5): e10777 (doi:10.1371\/journal.pone.0010777).Andreu N, Fletcher T, Krishnan N, Wiles S, Robertson BD (2012). Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence. Journal of Antimicrobial Chemotherapy. 67(2): 404-14 (doi: 10.1093\/jac\/dkr472). Dalton JP, Uy B, Phummarin N, Copp BR, Denny WA, Crosier PS, Swift S, Wiles S (2016). Effect of common and experimental anti-tuberculosis treatments on Mycobacterium tuberculosis growing as biofilms. PeerJ. 4:e2717 (doi: 10.7717\/peerj.2717).Dalton JP, Uy B, Okuda K, Hall CJ, Denny WA, Crosier PS, Swift S, Wiles S (2017). Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model. Journal of Antimicrobial Chemotherapy 72(2):421-427 (doi: 10.1093\/jac\/dkw421).Andreu N, Zelmer A, Sampson SL, Ikeh M, Bancroft GJ, Schaible UE, Wiles S, Robertson BD (2013). Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy. 68(9):2118-27 (doi: 10.1093\/jac\/dkt155).