This protocol describes a method for storing bacterial cultures containing glycerol which are growing in liquid media.

Reagents

• Bacterial culture growing in appropriate liquid medium
• Glycerol, 60%, sterile
• Nutrient (LB, TSB, etc.) agar plate containing the appropriate antibiotic

Equipment

• Incubator, preset to correct temperature for growth
• Inoculating loop, sterile/sterile toothpicks/sterile glass capillary tube
• Tube, storage, with screw cap and air-tight gasket

Method

• Sterilize glycerol by autoclaving for 20 minutes at 15 pounds per square inch (psi) (1.05 kg/cm2) on liquid cycle.
• To 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol in a labeled storage tube (final glycerol concentration of 15%). Label the top with a strain ID and the side with more detail (strain, date, initials).
• Vortex the culture to ensure that the glycerol is evenly dispersed.
• Transfer the tube to the -80°C freezer, and record its location and contents in your notebook.
• To recover the bacteria, scrape the frozen surface of the culture with a sterile inoculating loop, and then immediately streak the bacteria that adhere to the needle onto the surface of an LB agar plate containing the appropriate antibiotic. Return the frozen culture to storage at -80°C. Incubate the plate overnight to get individual colonies.

Adapted from OpenWetWare