Preparation of Spores for Molecular Analyses
Anytime mycorrhizal fungi are cultured on living plants in open pot culture, then a microbial milieu develops over time regardless of the sterility of a potting medium at the time of startup. The amount and activity of this microbiota is a function of many variables, but the dominant ones are (i) associated microbes in original inoculum, (ii) amount of organic matter in potting medium, (iii) moisture and temperature during active culture growth, and (iv) microbial activity in watering solution (usually tap water).
In INVAM cultures, routine counts of “colony forming units” from 3-month-old pot cultures indicates an average background of 103 actinomycetes, bacteria, and fungi per cm3 contents. Despite this background, multitudes of healthy spores are used to extract DNA that produce reproducible results. Exceptions do occur (D. Redecker, M. Hijiri, H. Dulieu, & I. R. Sanders, 1999. Fungal Genetics and Biology, 28:238-244), and they can be a molecular biologist’s worst nightmare. Selective primers are one way to overcome any chances of product contamination. Another is to be extremely thorough in the screening of spores extracted from a pot culture, especially if universal primers are going to be used. The procedure routinely used by INVAM on spore material sent out to users requiring quality material is illustrated and described in a PDF document. In essence, it is a screening process in which spores are extracted, examined closely over a period of at least 3 days while incubated in water at 4°C.
Once spores have been screened to select only the healthiest ones, they are collected in sterile distilled water and added to sterile sand in a 2-ml screw-top plastic vial for shipment or storage. The purpose of the sand is to minimize direct contact between spores, so if any contaminants are missed, any regrowth is limited to a small part of the total sample (and should be detected by the recipient in extracted spores if that person knows what to look for).
Below are links to pages of selected species showing appearance of spores prior to
and after screening and examples of changes in appearance of spores as a result
of degradation, natural senescence, and parasitism.