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The cultivation of mycobacteria often requires the use of several antibiotics to limit the growth of other rapidly growing micro-flora present in the growth medium. This antibiotic cocktail is one of the most expensive reagents required for mycobacterium culture. Here we present a customized antibiotics mix that is easy to prepare at a fraction of the cost of the commercially available antibiotic mixture that protects against transient flora, which are normally present in lungs, without affecting mycobacterial colony number.