To collect spores, sever the cap from the stem of a fresh, well cleaned mushroom and place it gills down on a piece of clean white paper or a clean glass surface such as a microscope slide. If a specimen is partially dried, add a drop or two of water to the cap surface to aid in the release of spores. To lessen evaporation and disturbance from air currents, place a cup or glass over the mushroom cap. After a few hours, the spores will have fallen according to the radiating symmetry of the gills. If the spore print has been taken on paper, cut it out, fold it in half, seal in an airtight container and label the print with the date, species and collection number. When using microscope slides, the spores can be sandwiched between two pieces of glass and taped along the edges to prevent the entry of contaminant spores. A spore print carelessly taken or stored can easily become contaminated, decreasing the chance of acquiring a pure culture.
Psilocybe cubensis, Agaricus brunnescens and many other mushroom species have a partial veila thin layer of tissue extending from the cap margin to the stem. This veil can be an aid in the procurement of nearly contaminant-free spores. The veil seals the gill from the outside, creating a semi-sterile chamber from which spores can be removed with little danger of contamination. By choosing a healthy, young specimen with the veil intact, and then by carefully removing the veil tissue under aseptic conditions, a nearly pure spore print is obtained. This is the ideal way to start a multispore culture.
Techniques for Spore Germination
Once a spore print is obtained, mushroom culture can begin. Sterilize an inoculating loop or scalpel by holding it over the flame of an alcohol lamp or butane torch for five or ten seconds until it is red hot. (If a butane torch is used, turn it down to the lowest possible setting to minimize air disturbance). Cool the tip by inserting it into the sterile media in a petri dish and scrape some spores off the print. Transfer the spores by streaking the tip of the transfer tool across the agar surface. A similar method calls for scraping the spore print above an opened petri dish and allowing them to freefall onto the medium. When starting a new culture from spores, it is best to inoculate at least three media dishes to improve the chances of getting a successful germination. Mycelium started in this manner is called a multispore culture.
When first produced, spores are moist, inflated cells with a relatively high rate of germination. As time passes, they dry, collapse at their centers and can not easily germinate. The probability of germinating dehydrated spores increases by soaking them in sterilized water. For 30 minutes at 1 5 psi, sterilize an eye dropper or similar device (syringe or pipette) and a water filled test tube or some spores onto a scalpel and insert into sterile water. Tightly seal and let stand for 6-12 hours. After this period draw up several milliliters of this spore solution with the eye dropper, syringe or pipette and inoculate several plates with one or two drops. Keep in mind that if the original spore print was taken under unsanitary conditions, this technique just as likely favors contaminant spores as the spores of mushrooms.
Characteristics of the Mushroom Mycelium
With either method of inoculation, spore germination and any initial stages of contamination should be evident in three to seven days. Germinating spores are thread-like strands of cells emanating from a central point of origin. These mycelial strands appear grayish and diffuse at first and soon become whitish as more hyphae divide, grow and spread through the medium. Once the mushroom mycelium has been identified, sites of germinating spores should be transferred to new media dishes. In this way the cultivator is selectively isolating mushroom mycelia and will soon establish a pure culture free of contamination. If contamination appears at the same time, cut out segments of the emerging mushroom mycelia away from the contaminant colonies.
Since many of the common contaminants are sporulating molds, be careful not to jolt the culture or to do anything that might spread their spores. And be sure the scalpel is cool before cutting into the agar media. A hot scalpel causes an explosive burst of vapor which in the microcosm of the petri dish easily liberates spores of neighboring molds.
Ramifications of Multispore Culture
Multispore culture is the least difficult method of obtaining a viable if not absolutely pure strain. In the germination of such a multitude of spores, one in fact creates many strains, some incompatible with others and each potentially different in the manner and degree to which they fruit under artificial conditions. This mixture of strains can have a limiting effect on total yields, with the less productive strains inhibiting the activity of more productive ones. In general, strains created from spores have a high probability of resembling their parents. if those parents have been domesticated and fruit well under laboratory conditions, their progeny can be expected to behave similarly. In contrast, cultures from wild specimens may fruit very poorly in an artificial environment. Just as with wild plants, strains of wild mushrooms must be selectively developed.
Of the many newly created strains intrinsic to multispore germination, some may be only capable of vegetative growth. Such mycelia can assimilate nutrients but can not form a mushroom fruitbody (the product of generative growth). A network of cells coming from a single spore is called a monokaryon. As a rule, monokaryons are not capable of producing fertile spore-bearing mushrooms. When two compatible monokaryons encounter one another and mate, cytoplasmic and genetic material is exchanged. The resultant mycelium is a dikaryon that can produce fertile offspring in the form of mushrooms. Branching or networking between different dikaryotic strains is known as anastomosis. This process of recombination can occur at any stage of the cultivation process: on agar; on grain; or on bulk substrates. The crossing of different mushroom strains is analogous to the creation of hybrids in horticulture.