EXTRACTION OF SPORES

The procedure detailed here focuses on extraction of spores from greenhouse-grown pot cultures (cone-tainers, deepots, pots). In all of the various pot types, a pie-shaped slice is removed from the side that extends almost to the center and from top to close to the bottom. In all cases (including field soils), it is critical that a representative sampling of roots is included in the sample because some species in all genera except Gigaspora produce intraradical spores.		click on image for larger photo
The sample is placed in water in a Waring Blendor and blended at high speed for approximately five seconds. The purpose of this step is to break up root fragments and release spores and also to release spores from hyphal aggregates attached to roots or in the soil (especially those of species with robust thick-walled hyphae). Longer blending times do not affect spores, but can break up roots to the extent that more organic detritus is associated with spores in the extracted spore prep (see below).
The blended material is immediately poured through two sieves. Most sand remains in the blender. The bottom sieve has openings of 38, 45, or 53 µm (any of these three work well for most species, although there are some small hyaline Glomus species that require the 38 µm sieve). It captures the majority of spores. The top sieve generally has 500 µm openings. It captures roots, debris, and large spores. Despite the amount of organic material, spores are large enough to be easily detected and collected. click on image for larger photo		click on image for larger photo
Generally, we do not process material in the top sieve through a centrifugation step because of the amount of organic detritus. It is washed, transferred to a large petri dish, and viewed through a stereomicroscope. The material on the bottom sieve is collected in a 50-ml beaker and then transferred into 50-ml tubes containing a 20/60% gradient. These tubes are centrifuged (approx. 960 x g) for 2-3 minutes. Soil and other particulates sediment. Spores and fine organic detritus is suspended in sucrose (now somewhat blended).		click on image for larger photo
click on image for larger photo	click on image for larger photo	click on image for larger photo
The supernatant in each tube is quickly decanted into smaller sieves -- either commercial stainless steel ones made by Tyler or homemade units of two types: (i) made from plastic vials with base removed and nylon mesh held in place by the plastic cap (with center removed using a heated cork borer) and (ii) nylon mesh glued to the rim of thick-walled PVC pipe.	click on image for larger photo	click on image for larger photo
The material collected on these smaller sieves is washed for 1-2 minutes under tapwater and transferred to a glass petri dish. Spores are collected manually with an extruded glass 9-inch pipette to separate from organic material. Once this is done (see example below), they can be stored at 4C for up to 30 days (checking weekly for parasitized spores which then are immediately removed).
click for larger photo	spores separated from organic detritus in extract	click on image for larger photo
The final (and most laborious) step is to separate spores of each morphotype (to mount and preserve, inoculate plants, extract DNA, perform germination assays, etc.). Some taxonomic expertise is needed here, but if the spores are in good condition, then anyone with observation skills can make the separations and initiate monospecific cultures. Identification to species can be another matter. Click here to see a composite photo of the different species in the extract pictured above (from a fresh-water wetland trap culture).