In this experiment, milk agar is used to demonstrate the hydrolytic activity of these extracellular enzymes. The medium is composed of nutrient agar supplemented with milk that contain the protein substrate casein. Following inoculation and incubation of the agar p cultures, microorganisms secreting protease will a zone of proteolysis, which is demonstrated by a clear area surrounding bacteria growth. In absence of protease activity, the medium surrounding the growth of microorganism remain opaque, which is a negative reaction.