3.4.1. Sporulation on solid growth medium

 

  1. Create a 2-fold dilution series of the P. larvae bacterial culture being studied using MYPGP broth and spread each dilution onto several MYPGP agar plates (see section 3.1. for recipe).
  2. Incubate plates at 37oC for 6-7 days and select plates exhibiting 50 to 5,000 colonies per plate.
  3. Note: When high numbers of colonies are present on a plate, sporulation efficiency can decline. Also, maximum sporulation obtained in relation to the plate colony number will vary between bacterial strains.
  4. During the 6-7 days of incubation, microscopically monitor cellular growth and sporulation via single colony analysis.
  5. After incubation, remove spores from the surface of the agar medium by washing three times (5 ml sterile H2O per wash).
  6. Combine the three washes.
  7. Once H2O is added to a plate, use gentle rubbing of the agar surface with the sterile glass pipette to loosen spores from the surface.
  8. To produce a spore stock following removal of spores from a plate surface, concentrate the spore suspension via centrifugation (12,000 x g, 15 min, 4oC).
  9. Discard the supernatant.
  10. Suspend the resulting spore pellet in 30 ml cold sterile H2O.
  11. Perform alternate centrifugation and pellet suspension four times.
  12. Suspend the spore pellet in a final volume of 5 ml cold sterile H2O.
  13. Store at 4oC.
  14. Note: Spores must be removed from the solid growth medium for preservation. When left on the agar plates for extended time, heat-resistant counts decline rapidly (Dingman, 1983).  Also, long-term survival of washed spores that have been desiccated is not known.
  15. Obtain spore concentration by heating a portion of the spore stock at 65oC for 15 min.
  16. Perform serial-dilution plating onto MYPGP or T-HCl-YGP (Steinkraus and Morse, 1996) agar plates.
  17. Incubate inoculated plates 6-7 days at 37oC to determine colony counts (i.e., spore counts). Alternatively, determine spore number by direct microscopic counting.
  18. Note: The latter will give an overestimation. Heat resistant spore counts are usually about 6 % of direct microscopic spore counts (Dingman and Stahly, 1983).