Spore Germination and Bias Assays

Obvious Reminder: DO NOT contaminate or mix samples at any step

Germination Assay

  1. Follow protocol for “Preparing spore samples”
  2. Prepare petri dishes with water agar (See “Agar Plates”) and write the tube number on the bottom
  3. You can use one petri dish for multiple samples by drawing quadrats on the bottom, but be sure to avoid any cross contamination while plating spores
  4. Vortex the tubes for a few seconds to suspend spores in solution
  5. Shake each tube and dip in the glass rod. Then smear rod tip onto the plate in corresponding quadrat.
  6. Repeat for all samples and sterilize rod each time with ethanol and fire.
  7. Parafilm plates and put in fridge or growth chamber at 16º C for 24 hours
  8. The following day, take your plates and carefully cut out a ½ inch square of agar for each sample. Place it onto a microscope slide and cover it with a cover slip.
  9. You can fit two pieces of agar onto one slide
  10. Look at it under a microscope to get a rough count of germination. Scan the entire agar piece and pick about 5 random spots to count (1) the number of spores germinating with sporidia and (2) the total number of spores. If your total number of spores isn’t over 100, count a few more spots until it is.

Bias Assay (for mating type bias)

  1. Follow protocol for “Preparing spore samples”
  2. Prepare petri dishes with water agar (See “Agar Plates”) and write the tube number on the bottom
  3. You can use one petri dish for multiple samples by drawing quadrats on the bottom, but be sure to avoid any cross contamination while plating spores
  4. Vortex the tubes for a few seconds to suspend spores in solution
  5. Shake each tube and dip in the glass rod. Then smear rod tip onto the plate in corresponding quadrat.
  6. Repeat for all samples and sterilize rod each time with ethanol and fire.
  7. Parafilm plates and place in fridge or growth chamber at 16º C for 6–7 days
  8. After about a week, take a look at the entire plate under 4x or 10x magnification.
  9. Look for a spot that is not very dense and try to determine the bias status by examining the colonies