Bacteria Microbiology

scientificprotocols authored about 3 years ago

Time required:

Cells are grown 6 hours to overnight; a total of less than 5 minutes bench time for each strain.


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.


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