Cord formation in Mycobacterium tuberculosis has been traditionally related to virulence since avirulent M. tuberculosis strains do not form cords. These structures consist of bacilli aligned in parallel, end to end, and side to side, along the long axis of the cord. However, similar structures were recently found in non-pathogenic and in opportunistic Mycobacterium species such as M. abscessus, M. chubuense, M. gilvum, M. haemophilum, M. marinum, M. obuense, M. parafortuitum, and M. vaccae. Although the visualization of cords has been traditionally achieved using Ziehl-Neelsen staining, the resolution of light microscopy does not allow to distinguish among cords, pseudocords, clumps or loose aggregates. Nevertheless, the observation of these formations at high resolution is crucial for two essential applications. Firstly, to identify the unknown mycobacterial components responsible for cording formation, an important step to understand virulence. Secondly, to demonstrate that cording is not specific of M. tuberculosis, since many clinical mycobacteriology laboratories base the tuberculosis diagnostic on the observation of cording morphology, and may mistakenly identify non-tuberculous mycobacteria as M. tuberculosis. Thus, patients would be exposed to an ineffective drug regimen with many adverse side effects. Here, we focused on sample treatment in order to certainly observe cords by using scanning electron microscopy (SEM).