The oil spreading assay was developed by Morikawa . For this assay, 10 μl of crude oil is added to the surface of 30 ml of distilled water in a petri dish to form a thin oil layer. Then, 10 μl of culture or culture supernatant are gently placed on the centre of the oil layer. If biosurfactant is present in the supernatant, the oil is displaced and a clearing zone is formed. The diameter of this clearing zone on the oil surface correlates to surfactant activity, also called oil displacement activity.