3.5.2. Preservation via ultra-low freezing

  1. Inoculate MYPGP broth (see section 3.1. for recipe) using a fresh culture of P. larvae.
  2. Grow overnight at 37oC with moderate aeration.
  3. Growth in the morning should show light to moderate turbidity.
    Note: Turbidity should not reach the point where the culture has become opaque. A light turbidity will place the culture in early to mid-exponential growth. Also, growth can be washed from the surface of a solid medium using fresh liquid medium.
  4. Examine the culture microscopically to gauge contamination while chilling the culture on ice (optional).
  5. Add an appropriate volume of a sterile solution of 100 % glycerol to the culture to produce a bacterial suspension containing 20 % glycerol (i.e. 0.25 ml of glycerol per 1.0 ml of culture).
  6. Aliquot 0.5 ml of the bacterial / glycerol suspension into cryovials.
    Snap-top microcentrifuge tubes also work, but may result in faster loss of viability during storage.
  7. Label and date the vials.
  8. (Optional) Quickly freeze the aliquots in an ethanol/dry ice bath.
  9. Place the bacterial suspensions in a pre-chilled storage box and store at -80oC.