### METHOD 1. Place 2 mL of the appropriate sterile medium in a 13 mm yellow-capped culture tube. If more culture is needed, place up to 5 mL in a 16 mm green-capped culture tube. Large amount of culture should be made in growth flasks, using up to 25% flask volume in medium (e.g., no more than 30 mL in a 125 mL flask, or 250 mL in a 1000 mL flask). - Touch a single colony with the end of an applicator stick and hold it in your primary hand (right hand if right-handed). - Holding the test tube in you other hand with palm and lower fingers, remove the cap of the test tube with your thumb and index finger. Dip the inoculated end of the applicator into the sterile medium and, if necessary, wipe the end onto the side of the tube. - Briefly flame the end of the test tube and replace the cap. - Always inoculate a dummy tube with no cells to verify that the medium, tubes, and sticks were sterile. - If inoculating large cultures, dilute an existing culture 1:100. - Grow culture for 12 to 18 hours. After 24-30 hours, cells have entered stationary phase and should not be used for critical procedures. - "Quicky" overnights can be made by rescuing a 2 or 3 day culture. Dilute culture 1:10 in sterile medium and grow for 3 hours. [](http://dx.doi.org/10.5281/zenodo.13543)