3.4.2. Sporulation in liquid growth medium
- 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.
- 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.
- 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.
- Collect and concentrate spores via centrifugation.
- Wash the spores four times with 30 ml cold sterile H2O (as described in section 3.4.1.).
- Suspend the washed spores in a final volume of 5 ml cold H2O.
- Store at 4oC.
- 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).