One liter of malt extract agar medium wili pour 20-40 lOOx 15 mm. petri dishes, depending upon the depth of the pour. Before pouring an agar medium, the table top is thoroughly wiped clean with an 80% concentration of isopropanol (isopropyl alcohol). Plastic petri dishes usually come pre-steiilized and ready to use. Glass petri dishes should be first washed and sterilized in a petri dish-holding rack simultaneous to the steril ization of the agar medium in an autoclavable flask. Pre-pouring media into glass dishes and then sterilizing is awkward. Media separation occurs, and any movement during the steriliza tion cycle (or while transferring the pressure cooker to the clean room) causes the liquified media to spill out of the petri-dishes. A huge mess results. However, this problem can be avoided if the pressure cooker cools overnight, for instance, and is then opened the next day. Be; forewarned that if you choose this alternative, your pressure cooker must either form a vacuum, safely protecting the media before opening, or be placed into a HEPA filtered airstream to prevent contamination entry during cool-down
Figures 68-69. Pouring malt agar into sterile petri dishes in front of laminar flow hood.
Figure 70. Glove boxes are considered "old tech". To retrofit a glove box into a laminar flow hood, simply cut out the back panel, replace with a similarly sized HEPA filter and build a6"-deep plenum behind the filter. A squirrel cage blower is mounted on top, forcing air into the plenum. Air is forced tnrough the filter. Downstream from the filter, a sterile wind flows in which inoculations can be conducted.
With the micron filters mounted horizontally, and facing the cultivator, every movement is prioritized by degree of cleanliness. The cleanest articles remain upstream, the next cleanest downstream in second position, etc. The cultivator's hands are usually furthest downwind from the media and cultures.
The surest method of starting a mushroom strain is by cloning. Cloning means that a piece of pure, living flesh is excised from the mush
The surest method of starting a mushroom strain is by cloning. Cloning means that a piece of pure, living flesh is excised from the mush room and placed into a sterilized, nutrient enriched medium. If the transfer technique is successful, the cultivator succeeds in capturing a unique strain, one exhibiting the particular characteristics of the contributing mushroom. These features, the expression thereof, are called thephenotype. By cloning, you capture the phenotype. Later, under the proper cultural conditions, and barring mutation, these same features are expressed in the subsequently grown mushrooms.
Several sites on the mushroom are best for taking clones. First, a young mushroom, preferably in "button" form, is a better candidate than an aged specimen. Young mushrooms are in a state of frenzied cell div!"' m. The clones from young mushrooms tend to be more vigorous. Older mushrooms can be cloned but have a higher contamination risk, and are slower to recover from the shock of transfer. Two locations resulting in a high number of successful clones are: the area directly above the gills, and the interior tissue located at the base of the stem. The stem base, being in direct contact with the ground, is often the entry point through which larvae tunnel, carrying with them other microorgar. sms For this reason, I prefer the genetically rich area giving rise to the gills and their associated spore-producing cells, the basidia.
The procedure for cloning a mushroom is quite simple. Choose ihe best specimen possible, and cut away any attached debris. Using a damp paper towel, wipe the mushroom clean. Lay the specimen on a new sheet of paper towel. Flame-sterilize a sharp scalpel until it is red hot. Cool the scalpel tip by touching the nutrient agar medium in a petri dish. This petri dish will be the same dish into which you transfer the mushroom tissue. Carefully tear the mushroom apart from the base, up the stem, and through
Figure 71. Ideal sites for cloning a mushroom:«irectly mushrooms are ,-est candidates for cloning.
the cap. With one half of this split mushroom, cut a small section ("square") of flesh about the size of a kernel of grain. Quickly transfer the excised tissue to the nutrient-filled petri dish, and submerge the tissue into the same location where the scalpel tip had been cooled. By inserting the tissue part way into the agar medium, in contrast to resting it on the surface, the mushroom tissue has maximum contact with the life-stimulating nutrients. Each time a clone is taken, the scalpel is re-ster'"zed, cooled and then the tissue is transferred into a separate petri dish following the aforementioned steps.
One carefully keeps the hot scalpel tip and the freshly poured media plates upstream of the mushroom being cloned or the mycelium being transferred. Next downstream is the cultivator's hands. No matter how many times one has disinfected his hands, one should presume they are replete with con-
above the gills or at the base of the stein. Young, firm taminants. (To test this, wash your hands, disinfect your fingertips with alcohol and fingerprint newly poured media plates. In most cases, the plates will contaminate with a plethora of microorganisms.)
Some use a "cooling dish" into which the hot scalpel tip is inserted before touching the living flesh of a mushroom. Repeatedly cooling the scalpel tip into the same medium-filled petri. dish before each inoculation is not recommended. A mistake with any inoculation could cause contamination to be re-transmitted with each transfer. If. for instance, a part of the mushroom was being invaded by Mycogone, a mushroom-eating fungus, one bad transfer would jeopardize all the subsequent inoculations. Only one cooling dish should be used for each transfer; the same dish that receives the cloned tissue. In this fashion, at least one potential cross-contamination vector is eliminated.
When cloning a mushroom for the first time, I recommend a minimum of 5 repetitions. If the mushroom spec nen is rare, cloning into sev eral dozen dishes is recommended. As the specimen dries out, viable clones become increasingly less likely. With the window of opportunity for cloning being so narrow, ihe cuhivator should clone mushrooms within hours of harvesting. If the mushrooms must be stored Lhen the specimen should refrigerated ai 35-40° F. (1-5° C.).After three to four days from harvest, finding viable and clean tissue forclon-ing is di ficult.
A few days to two weeks after cloning the mushroom, the .issue fragment springs to life, becoming fuzzy in appearance. Contaminants usually become visible at this stage. As a rule, the cultivator always transfers viable mycelium away from contamination, not the other way around. The essential concept here,:s that the cultivator "runs" with ths mycelium, subcultur-ing away from contamination as many times as is necessary until a pure culture is established.
Each transfer from an older petri dish culture to a newer petri dish moves upstream. The scalpel is brought into contact wifh heat. The tip is cooled into the dish destined to receive the mycelium. The lid of this dish is lifted, the seal pel is cooled, and then the liH is replaced. Next, the ,:d of the dish hosting the mature mycelium is opened. The mycelium is cut. The wedge is transferred to the newly poured media plate. With the lid replaced, the culture is labelled and moved aride. The process is repeated until a number of plates are inoculated.
As each 'id lifted, care is taken not to extend the fingers beyond the lip of each top.The-overhangi ng of fingers results in off-flaking of contaminants into the petri di h. Furthermore, the lids are lifted wilh their undersides catch-
ing the sterile airstream. If the lids must be laid down, they are positioned undersides up, upstream of the operations area, so that contaminants are not picked up off the table. Always presume the air coming off the face of the micron filter is cleaner than the work surface in front of i:.
Culture transfers that are fast, evenly repeated, and in quick succession usually Q.re the most successful.The simplest acts dramatically impact sterile technique. Merely breathing over exposed petri dishes significantly affects contamination levels. Singing, for instance, is associated with a high rate of bacterial contamination. One bewildered professor discovered that her soliloquies in the laboratory—she sang as the radio blared—were a direct cause of high contamination rates. An alert student discovered her digression from sterile technique upon passing the door to her lab. This illustrates that the cultivator's unconscious activities profoundly influence the outcome of tissue culture transfers. Every action in the laboratory has significance.
Cloning Wild Specimens vs. Cloning Cultivated Mushrooms
Many people ask "What is wrong with just cloning a nice looking specimen from each crop of cultivated mushrooms to get a new strain?" Although morphological traits can be partially selected for, senescence factors are soon encountered. Generating mycelium in this fashion is a fast-track to genetic demise, quickly lead ing to loss of vigor and yield. By not returning to stock cultures, to young cell lines, one has gone furthest downstream one ^e^chain of cells. Mushrooms, like everi OTjjjfy reproducing organism on this plank^i^Jenerate a limited number of cell division?SeMre vitality falters. Sectoring, slow growth, anemic mushroom formation, malformation, or no mushroom formation at all are all classic symptoms of senescence. Although senescence is a frequently encountered phenomenon with cultivators, the mechanism is poorly understood. (See Kuck et al., 1985.)
In the competitive field of mycology, strains are all-important. With the aforesaid precautions and our present day technologies, strains can be preserved for decades, probably centuries, all the-while kept within a few thousand cell divisions from the original culture. Since we still live in an era of relatively rich fungal diversity, the time is now to preserve as many cell lines from the wild as possible. As bio-diversity declines, the gene pool contracts. I strongly believe that the future health of the planet may well depend upon the strains we preserve this centuiy
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