"

From 2013.igem.org

The optical density of yesterday culture was measured, however it was not sufficient enough to put it into sporulation media. so the cultures we left for further incubation for 3 hours. At this time new sporulation media was prepared and autoclaved. When the OD600 measurements were 2.5-3 the cultures were manipulated according the following procedure: - Transfer 1 ml culture to a sterile, disposable 15-ml tube and centrifuge 5min at 1200xg (3000rpm). - Pour off supernatant and resuspend cells in 5ml sterile water. Vortex to resuspend cells and repeat spin. - Pour off supernatant and resuspend cells in 1ml liquid sporulation medium supplemented with nutritional requirements of the particular diploid. - Shake at 30C for 3-6 days. - Look for sporulation microscopically.