In natural environments, microbes occur almost always in a mixed population composed of a multitude of different strains and species. For analyzing the properties of a defined organism out of such a mixed population, a pure culture is required. Apart from direct isolation of strains by diluting and plating, enrichment cultures with hydrophobic substrates are very promising for the isolation of biosurfactant producing microbes. Additionally, hydrophobic interaction chromatography and the replica plate technique are also rewarding methods.