3.4.2. Sporulation in liquid growth medium

  1. Inoculate TMYGP broth (6 ml in a 20 x 150 mm loosely capped screw-cap glass culture tube, see section 3.1. for recipe) with P. larvae NRRL B-3650.
    Note: Other bacterial strains must be tested separately because they may sporulate poorly in this medium and under these growth conditions.
  2. Incubate the culture at 37oC in a rotary incubator shaker adjusted to 195 rpm.
    The culture tube is held at a 45o angle in a wire test tube rack during incubation and aeration.
  3. Incubate for 3 to 4 days while microscopically monitoring cellular growth and sporulation.
    Other strains of P. larvae may require a longer incubation time for sporulation to occur.
             Alternatively, see Genersch et al. (2005) for sporulation of P. larvae in the liquid part of Columbia sheep-blood agar slants.
  4. Collect and concentrate spores via centrifugation.
  5. Wash the spores four times with 30 ml cold sterile H2O (as described in section 3.4.1.).
  6. Suspend the washed spores in a final volume of 5 ml cold H2O.
  7. Store at 4oC.
  8. Obtain spore counts (i.e. heat resistant counts) by serial-dilution plating of the spore suspension onto MYPGP plates following heating of the suspension at 65oC for 15 min or determine counts by direct microscopic counting.
    Note: Heat resistant spore counts are usually about 6 % of direct microscopic spore counts (Dingman and Stahly, 1983).