1.4.4.1. Production and harvest of in vitro spores

  1. Cut agar plugs from the border of well-growing isolates with different mating types
  2. Place the plugs of the + and – strain a distance of approximately 4 cm on MY20 agar (see paragraph 1.4.1.1.)
  3. Incubate the plate in the dark at 30-34°C at least 3 weeks to ensure production of mature spores
  4. Use a scalpel to scrape off the spore cysts from the black mating stripes
  5. Transfer them to a glass mortar with a ground-glass pestle (glass tissue grinder; Fig. 6a and b)
  6. Add 5-10 µl sterile water and grind the suspension 1 min to release spores from the spore cysts and balls
  7. Add 50 µl sterile water and grind again 1 min
  8. Add 200 µl sterile water to the mortar
  9. Pipette spores for the mortar into an 1.5 ml Eppendorf tube
  10. Add additional 750 µl water to get a medium density of spores
  11. Let large particles unbroken sporocysts, spore clumps etc. in the suspension settle for 30 min
  12. Pipette, from just below the surface, approximately 500 µl into a new tube, which should contain mostly released individual spores
  13. Prepare a dilution series to 10-2 or 10-3 for counting and calculating the concentration of the undiluted suspension with a haemocytometer (refer to the BEEBOOK paper on miscellaneous methods (Human et al.,


Fig. 6. Spore germination test: A. & B. First grind the sporocysts to release spores from cysts and balls; C. & D. incubate the spores in liquid medium and flush with CO2 incubate the spores 32 hours at 34 °C; E. & F. check for spore enlargement. Photos: A B Jensen.

Figure 6