The culture of microorganisms has been the basis of microbiology. However, revolutionary tools such as metagenomics have made it possible to describe uncultivated bacteria, but several breakthroughs have occurred in culture leading to a revival of these techniques. In this review we focus on new applications that have successfully cultivated previously uncultivated bacteria. We also review the axenic cultivation of intracellular bacteria such as Tropheryma whipplei and Coxiella burnetii. These successes provide new tools for the design of axenic media for intracellular bacteria, such as Rickettsiae and Chlamydiae, or historically uncultivable pathogens, such as Mycobacterium leprae and Treponema pallidum. The future axenic culture of these microorganisms will facilitate antibiotic susceptibility testing and will provide insight into their microbial ecology and pathogenicity.