Journal article Open Access
**Time required:** Cells are grown 6 hours to overnight; a total of less than 5 minutes bench time for each strain. ### Procedure: **Day 1** 1. Inoculate a 15 ml culture tube containing 5 ml of LBM or LBM+antibiotic selective medium with a freshly grown isolated colony. Incubate at 37 degrees C until culture is in late log or stationary phase (usually 5 hours to overnight). **Day 2** 1. For each strain to be stored at -80 degrees C for the archives prepare a sterile labeled cryovial. Pipet 225 ul sterile 80% glycerol into the cryovial. Add 1.0 ml of the bacterial culture (frozen stock will be 15% glycerol). Mix well (vortex) and place tube at -80 degrees C. - For each strain to be stored at -20 degrees C as a liquid glycerol " working" stock pipet equal volumes 80% glycerol and bacterial culture into a labeled polypropylene tube. Mix the contents well (if not well mixed ice crystals will form decreasing the viability of the cells). Place the tube in a -20 degrees C freezer. If possible check the viability of the cells after 1 week. To recover a strain from the -80 degrees C glycerol stock use a sterile toothpick to scrape some of the ice then streak out the cells on the appropriate medium e.g. LBM + ampicillin. Do not thaw the frozen stocks because each freeze-thaw cycle will result in a 50% loss in cell viability. To use the -20 degrees C working stocks pipet 50 to 100 ul as inoculum for a 5 ml overnight culture.