The reaction forms water as a by-product and at the same time breaks up the structure of the egg contents as it releases the bacteria into the aqueous phase. The reaction is near completion when the mixture becomes viscous, generally between 1 and 2 min at room temperature. The tube is then centrifuged for 5 min at 10,000g in a microcentrifuge. Centrifugation forms an oily layer with yellow carotenes and oil solubles on the top, a soapy hard layer of esters in the middle, and a clear aqueous layer with soluble bacterial antigen and a pellet of bacterial debris on the bottom (Fig. 2).