Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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Glomaceae Acaulosporaceae Gigasporaceae
Glomus Entrophospora Acaulospora Gigaspora Scutellospora
Archaeosporaceae
Archaeospora
Paraglomaceae
● vesicles
auxiliary cells ●
● hyphae ●
● arbuscules ●
Al-Agely and Ogram © 2004
METHOD
1. SPORE ISOLATION - procedure will be demonstrated at takedown of experiment
described below. For 1st exercise, spores will be provided in Petri dishes and prepared
slides for observation at higher magnification.
Spores of VA mycorrhizal fungi are separated from soil by decanting and wet-sieving,
followed by sucrose centrifugation. Place 100 g of soil in a one-liter beaker and add tap
water. Stir vigorously, allowing the sand to settle for 10-15 sec, and pour supernatant
through a No. 40 (425 µm) and No. 325 (45 µm) sieve. Roots will be on the coarse sieve.
Spores will be on the fine sieve. Wash spores into 50 ml centrifuge tubes and fill to
within 2 to 3 cm of the top with tap water. The sieving should not be deeper than 1/2
inch thick at the bottom of the tube. If so, use two tubes for the sample. Remember to
balance the centrifuge. Spin at top speed for 3 min and allow stopping spinning by itself.
Gently remove tube and, in one smooth motion, pour off the water, taking care not to
pour off any sieving. You may need to remove floating organic matter that will adhere to
the upper walls of the tube with your finger.
Next, fill the tube up to within 2 to 3 cm of the top with cold 40% sucrose solution and
centrifuge at top speed for 1-1.5 minutes. Manually stop the centrifuge, or use the brake
to minimize the time the spores are in the sucrose solution due to potential damage by
osmosis. Pour the supernatant through the No. 325 sieve to collect the spores, rinse