Initiation of single-spored genetically pure cultures: Without exception, every molecular phylogenetic study in mycology must begin with a pure or axenic culture of the fungus. Failure to initiate a culture that is faithful to the original material will result in a number of "down-stream" failures, not the least of which are Genbank DNA & protein accessions that do not correspond phylogenetically with the specific epithet of the taxon in question. This has unfortunately also occurred in the literature, where single fungal isolates, used as representative of the genus, family and even order in question, have proved in the end to be a "mis-identified" culture. Of course, if the fungal isolate can fruit in culture or produce a recognizable anamorph in culture, then it is a simple task to verify the culture prior to further analysis. Unfortunately, this is the exception rather than the rule for many fungi. For example, although many anamorphs have been described for the Hysteriaceae & Mytilinidiaceae, many isolates do not produce them in culture, even after prolonged incubation lasting many months. Also, in this group coelomycetous anamorphs tend to possess few morphological characters useful for species confirmation.

Thus, a certain amount of effort must be put into establishing a single-spored culture suitable for DNA/RNA isolation and eventual deposit in a culture collection. And this requires a certain degree of mycological expertise. Routinely, upon receipt of material from the field or from collaborators overseas, under the stereomicroscope, using a sterile razor blade, I remove a single ascoma. I then affix the fruitbody to the underside of a Petri plate lid, using double edged sticky tape, such that the area of dehiscence (ostiole, apothecia, sulcus etc.) is facing downwards towards the agar surface. I use water agar plates, so that whatever is eventually deposited on the surface of the agar will not grow very fast or far (e.g., weed molds, yeast & bacteria). I never use antibiotics on any mycological media because I want to "see" all that is there, contaminants and all. On the contrary I want to encourage contaminants so that I may be able to avoid or remove them. I then incubate the plate in the dark for up to 96 hrs. which, for this group of fungi, seems to be enough time to rehydrate & begin spore discharge. Most importantly, every 12 hrs., I rotate the Petri plate lid 45°, such that an even series of spore deposits result. There is an interesting difference between unitunicate hymenoascomyetes and bitunicate loculoascomycetes with regard to spore deposits. Unitunicates tend to discharge all of the spores, all at once in a sudden burst, so that the deposits appear densely clumped, hindering the retrieval of individual spores. Bitunicates, on the other hand, tend to shoot their spores one or a few at a time, due to the nature of the fissitunicate dehiscence mechanism of the bitunicate ascus, thus greatly facilitating recovery of individual spores for this group of fungi. Viable ascospores of Hysterium and Hysterographium have been isolated from herbarium material over three years old.

Some fungi are recalcitrant to spore release using the methods described above, even after prolonged hydration / incubation, and must be "forced" to liberate their ascospores. What I do is place a single fruitbody in a 1.5mL microfuge tube & add 300uL of sterile water, vortex, aspirate off the water, & repeat several times to clean the surface of the fruitbody of adhering contaminant bacterial, fungal and yeast spores. Sometimes I add 5uL of Tween (5% stock) to the water. Very dilute bleach works OK as well. But then I must wash with water several more times to remove the Tween or bleach. Then with a sterile glass rod I macerate the fruitbody thoroughly to release ascospores and create a dense spore suspension. Then I dilute aliquots 1:10 to as much as 1:100,000,000 in 200uL of sterile water volumes and plate this material out. Use a sterile bent glass rod & Petri plate rotator table to insure an even spread on the agar plate. Use a water agar plate, not a nutrient medium (e.g., PDA), or else everything will germinate & grow out (bacteria and yeast that were not removed initially with water rinses). I usually only plate out the final set of dilution series. Then under the 10X lens I find single isolated spores of the fungus, circle them with a marker, and, under the stereomicroscope, transfer individual spores onto a PDA plate, whereupon they germinate after a day or two. This technique works well for ascomycetes that "refuse" to shoot out their spores. Also, if it's the "wrong season" (e.g., winter) and the spores are still not mature in the ascus (e.g., as judged by the lack of full septation and pigmentation), they can sometimes be induced to mature by several repeated cycles of freeze-thawing of the material. Hysteriaceous fungi respond well to this, and material collected in the winter months, that do not release their spores, can be induced to do so using this technique (Lohman 1933a).

Whatever method one is forced to use, it is very important to actually check the morphology of the individual ascospores prior to their removal onto nutrient media. This is done by pouring very thin water agar plates (< 1mm deep) which allows one to invert the plate and use a compound microscope with the 10X lens to ascertain their morphology. This should not be done under a stereomicroscope, except for those fungi with very large spores (e.g., Hysterographium fraxini or H. flexuosum). Confirmation of spore morphology, requires the pouring of thin water agar plates, that can be inverted and examined under a compound microscope using the 10X lens. At this magnification, you can clearly make out spore morphology. You need to have a long working distance 10X lens for this to really work. Alternatively, you can use a 4.5X lens and still make out the morphology. Don't assume a spore deposit represents the fungus at hand until the spores have been morphologically identified under the compound scope. This is very important! Once an isolated spore has been found that corresponds to the morphology of the fungus in question, I circle scribe the bottom of the Petri plate with a thin Sharpie marker. The plate is then transferred back to the stereo microscope and using a sterile needle, under maximum power, a small water agar block is cut out containing the spore and transferred to another Petri plate containing nutrient media (e.g., PDA). I usually plate out half a dozen spores on one plate. After colonies have formed, I check to confirm that they are all identical and then arbitrarily take one to transfer to a PDA slant and designate it the culture for that particular specimen.