Spore Selection and Cleaning

Prepare to Select Suitable Spores

These steps are especially important for spores obtained from open pot cultures. However, the same steps are followed for spores from root organ culture to ensure no hyphal fragment or root particulates remain attached (and invisible) on spore surfaces. A discussion of how spores from a bulk extract are prepared to select suitable spores can be viewed on the Cleaning Spores page. On this page are links to pages of selected species with images of the range in phenotypes from healthy to degraded or parasitized spore.

Briefly, spore extraction by sucrose-density gradient centrifugation (from pot cultures), wash them thoroughly in tap water and transfer them to a petri dish. Refrigerate for a minimum of one day, so that degraded, dead or parasitized spores become more visible and can be removed. Collect enough healthy spores to photograph and make a voucher slide if there is any doubt about species identity or origin.

Collect the spores and place in a small petri dish or a 1.5 mL centrifuge tube containing distilled water.

Place the petri dish or 1.5 mL centrifuge tube in a jewelers sonicator for about 30 seconds to remove debris from the spore surface and break up any spores are structurally unsound.

Transfer spores to a clean, sterilized watch glass. Under a dissecting microscope, remove all debris and replenish the distilled water several times until only healthy spores remain. Below are two images: the left one shows spores of Acaulospora scrobiculata before sonication; the right photo shows spores after sanitation.

Before sonication : red-starred spores would not have been picked because they show some signs of degradation.

After sonication : white-starred spores do not have typical contents and therefore are removed.

• Transfer each healthy spore selected for DNA extraction to its own sterile watch glass containing fresh sterile-distilled water.

• For each spore, flush and remove distilled water with a sterile pipette several times.
• Inspect the spore one more time to verify absence of any visible surface debris or particulates.
• Store each spore at 4oC in sterile-distilled water until it is to be used to extract DNA.