2.4.1.2. Single spore isolation

  1. Add 10 ml 0.05 % Triton-X on the culture agar plate
  2. Rub its surface gently with a sterile Drigalski spatula to loosen the conidia
  3. Transfer the suspension conidia into a 15-50 ml sterile tube
  4. Wash the suspension twice (to remove agar and hyphal fragments)
    4.1. Centrifuge 3 min at 7000 g for 3 min
    4.2. Discharge the supernatant and add 10 ml 0.005 % Triton-X
    4.3. Centrifuge 3 min at 7000 g for 3 min
    4.4. Discharge the supernatant and add 10 ml 0.005 % Triton-X
    Prepare a serial dilution (remember to whirl mix before pipetting)
  5. Count the spore concentration in a haemocytometer (described in the BEEBOOK paper on miscellaneous methods (Human et al., 2013))
  6. Transfer 100 µl of a spore solution at 5 X 102 spore per ml to a new plate
  7. Incubate at 25 °C for two-four days
  8. Transfer a single small colony to a new plate

To harvest conidia for experimental purposes the above procedure can be used (minus step 6-8). It is important to use Triton-X or another detergent to avoid spore clumping.