220.127.116.11. Spore viability and germination test (Fig. 6)
- Place a sterile Teflon coated slide in a sterile petri dish lined with wet filter paper
- Prepare a concentration of 2.0 x 107 spores per ml
- Mix 100 µl spore suspension with 500 µl GLEN (see recipe below)
- Place 10 µl of the GLEN / spore mix onto the spot of the Teflon coated slides
- Place the petri dish in an airtight container and flush with CO2 30 sec to ensure a minimum of 10 % CO2 concentration
- Incubate 32 hours at 34°C
- Add a cover slip and count the spores directly on the Teflon slide under a microscope at 400 magnification
- Evaluate 100 spores for enlargement or germ tube
formation in three different fields of view on the slide
The AnaeroGen system (Oxoid; Basingstoke, UK) has been used with success in cases where the laboratory is not equipped with CO2. A packet is placed together with the petri dish in a sealed jar and will generate 9 to 13 % CO2.
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.