Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. This protocol describes sample and media preparation and contains instructions for assembling the ODELAM sample chamber. The ODELAM sample chamber is designed to provide a controlled environment to safely observe the growth of Mtb by time-lapse microscopy on an inverted wide-field microscope. A brief description of the ODELAM software is also provided here. ODELAM tracks up to 1500 colony forming units per region of interest and can observe up to 96 regions for up to seven days in a single experiment. This technique allows the quantification of population heterogeneity. ODELAM enables rapid quantitative measurements of growth kinetics in as few as 30 h under a wide variety of environmental conditions. Graphic abstract: Schematic representation of the ODELAM platform.
Keywords: Drug resistance; Growth phenotypes; Live imaging; Microbiology; Microscopy; Mycobacterium tuberculosis.
Copyright © The Authors; exclusive licensee Bio-protocol LLC.