Tissue culture is an assured method of preserving the exact genetic character of a living mushroom. In tissue culture a living specimen is cloned whereas in multi-spore culture new strains are created. Tissue cultures must be taken from mushrooms within twenty-four to forty-eight hours of being picked. If the specimens are several days old, too dry or too mature, a pure culture will be difficult to isolate. Spores, on the other hand, can be saved over long periods of time.
Since the entire mushroom is composed of compressed mycelia, a viable culture can be obtained from any part of the mushroom fruit-body. The cap, the upper region of the stem and/or the area where the gill plate joins the underside of the cap are the best locations for excising clean tissue. Some mushrooms have a thick cuticle overlaying the cap. This skin can be peeled back and a tissue culture can be taken from the flesh underlying it. Wipe the surface of the mushroom with a cotton swab soaked in alcohol and remove any dirt or damaged external tissue. Break the mushroom cap or stem, exposing the interior hyphae. Immediately flame a scalpel until red-hot and cool in a media filled petri dish. Now cut into the flesh removing a small fragment of tissue. Transfer the tissue fragment to the center of the nutrient filled petri dish as quickly as possible, exposing the tissue and agar to the open air for a minimal time.
Repeat this technique into at least three, preferably five more dishes. Label each dish with the species, date, type of culture (tissue) and kind of agar medium. If successful, mycelial growth will be evident in three to seven days. An overall contamination rate of a 10% is one most cultivators can tolerate. In primary cultures however, especially those isolated from wild specimens, it is not unusual to have a 25% contamination rate. Diverse and colorful contaminants often appear near to the point of transfer. Their numbers depend on the cleanliness of the tissue or spores transferred and the hygienic state of the laboratory where the transfers were conducted. In tissue culture, the most commonly encountered contaminants are bacteria. Contamination is a fact of life for every cultivator. Contaminants become a problem when their populations spiral above tolerable levels, an indication of impending disaster in the laboratory. If a five, ten or fifteen percent contamination rate is normal for a cultivator and suddenly the contamination level escalates without an alteration of regimen, then new measures of control should be introduced immediately. Once the tissue shows signs of growth, if should be transferred to yet another media dish. If no signs of contamination are evident, early transfer is not critical. If sporulating colonies of mold develop adjacent to the growing mycelium, the culture should be promptly isolated. Continue transferring the mycelium away from the contaminants until a pure strain is established.
One’s attitude towards contamination and cleanliness is perhaps more important than the
installation of any piece of equipment. The authors have seen laboratories with high contamination rates and closets that have had very little. Here are two general guidelines that should help many first-time cultivators.
1. Give the first attempt at sterile culture the best effort. Everything should be clean: the lab; clothes; tools; and especially the cultivator.
2. Once a pure culture has been established, make every attempt to preserve its purity. Save only the cultures that show no signs of mold and bacteria. Throw away all contaminated dishes, even though they may only be partially infected.
If failure greets one’s first attempts at mushroom culture, do not despair. Only through practice and experience will sterile culture techniques become fluent. Agar culture is but one in a series of steps in the cultivation of mushrooms. By itself, agar media is impractical for the production of mushrooms. The advantage of its use in mushroom culture is that mycelial mass can be rapidly multiplied using the smallest fragments of tissue. Since contaminants can be readily observed on the flat two dimensional surface of a media filled petri dish, it is fairly easy to recognize and maintain pure cultures.