Purpose:

Bacterial strains may be stored indefinitely at low temperatures (- 20 degrees C and -80 degrees C) in 15 to 40 glycerol. It is lab policy to prepare a frozen stock of newly acquired or created strains for the archives as soon as possible.

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.
2. 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.