The culturing of mycobacteria is a standard procedure that is consistent world-wide, with little variation in the growth media constituents, particularly those found in liquid and solid media. Before the 1940s however, the aggregating nature of mycobacteria as well as the characteristic slow growth-rate saw mycobacterial research delay considerably. Dubos and colleagues addressed both these issues and observed that a very small volume of Tween detergent was sufficient to greatly improve the culturing of mycobacteria. Over the years however, evidence of the unfavourable effects of this detergent on a number of morphological, biochemical, pathogenic and host-interacting properties of mycobacteria surfaced. For the first time we bring together literature, past and present to comprehensively review the mycobacterial properties which are, and are not affected by the use of this detergent. We also address other detergents and methods which may circumvent the need to include Tween compounds in mycobacterial culture media.