Step 1. Visually ascertain the purity of a mushroom culture, selecting a petri dish culture showing greatest vigor. Ideally, this culture should be no more than two weeks old, and there should be a margin of uncolonized media along the inside peripheral edge. This uncolonized zone, approximately 1/2 inch (1. 30 cm.) in diameter, can tell the cultivator whether or not any viable contaminant spores have recently landed on the media. Once the mycelium has reached the edge of the petri dish, any contaminant spores, should they be present, lie dormant and invisible upon the mushroom mycelium only to wreak havoc later.
Step 2. Although the contents w ithiJI may be sterilized, the outer surface of the pressure cooker is likely to be covered with contami nants which can be transferred via hand contact. Therefore, the outside of the pressure cooker should be thoroughly wiped clean prior to the sterilization cycle. Open the pressure cooker in the laboratory clean room. Ideally, the pressure cooker has formed a vacuum in cooling. If the pressure cooker in use does not form a vacuum, outside air will be sucked in, potentially contaminating the recently sterilized jars. The pressure cooker should be placed in the clean room directly after the sterilization cycle and allowed to cool therein. (I usually place a paper towel saturated with isopropanol over the vent valve as an extra precaution, to filter the air entering the pressure cooker.) Another option is to open the pressure cooker in front of a laminar flow bench at the moment atmospheric pressure is re-achieved. Remove the sterilized grain jars from the pressure cooker. Place the grain-filled jars upstream nearest to the laminar flow filter. Then sterilize the scalpel by flaming until red hot.
Step 3. Directly cut into the petri dish culture (The blade cools instantly on contact.) Drag the blade across the mycelium-covered agar, Bat-
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Figure 108. Transferring several squares of mycelium from the nutrified agar medium into sterilized grain.
ing 8 or more wedges. Replace the petri dish lid
Step 4. Loosen the lids of the jars to be -n oculated so you can lift them off later with one hand. Re-flame the scalpel. Remove the petri dish cover. Spear two or more wedges simultaneously. Replace the petri dish cover. While moving the wedges of mycelium upstream to the jars, remove the lid of the jar to be inoculated, and thrust the wedges into the sterilized gram Replace and screw the lid tight. Repeat and shake each jar so the wedges move throughout the interior mass of the grab, with the intention that strands of mycelium will tear off onto the contacted grain kernels.
Step 5. Set the inoculated jars of grain onto the shelf and to incubate them undisturbed for several days.
Step 6. Three days from inoculation, inspect each jar to determine two preconditions: first recovery of the mycelium, "leaping off' onto contacted grain kernels and secondly, the absence of any competitor molds, yeasts, mites or bacte ria. If these preconditions are satisfied, to the best of your knowledge, continue to the next step.
Step 7. Seven days after inoculation, shake each jar again. Ten to fourteen days after inoculation, incubated at 75° F (24° C), each jar should be fully colorized w:th mushroom mycelium. If colonization is not complete three to four weeks after inoculation, something has probably gone awry v 'th the process Some of the more common causes of slow colonization include unbalanced moisture content, contaminants, weak strain, residual fungicides in the grain, poor quality grain, etc
SpawSZ|t the peak of cell development is the hefet^|i|p correlat" g to about two weeks af-tema^iSion. The key concept here: to keep tblffycMam runr. ingetitsmaxitttum potential throughout the spawn generation process. With over-incubation, the grain kernels become difficult to break apart. It is important for the myceliated grain kernels to separate so they can be evenly dispersed throughout the next generation of substrates. Over-incubation results in clumping and disease
Grain masters can be kept at room temperature for a maximum of four to eight weeks from the time of original inoculation. Best used within a week of full colonisation, some farms refrigerate grain masters until needed. I strongly discourage this practice. The rule here: use it or lose it.
The next generation of spawn jars is denoted as G2. Each Grain Master cai, inoculate 5 to 20 times ;ts mass. Many start with narrow mouth quart mason jars for Grain Masters, and use 1/2 gallon or 2 liter j rs for Second Generation spawn. (For use of bags as spawn containers, see ])g. 138.).Third Generation spawn is typically in bag form and is sold to consumer-growers.
A standard inoculation rate would be 1 quart (liter) Grain Master to five 1/2 gallons, in other words a 1:10 expansion A diluted inoculation, on the verge of being unsuccessful would be 1 quart Grain Master to twenty 1/2 gallons, in other words a 1:40 expansion. Exceeding a 1:40 expansion of mycelium is likely to be associated with a >20% failure rate, a percentage unacceptable to any spawn laboratory. Not only can the loss be measured in terms of failure to mature, but each failed spawn jar is likely to be center stage for releasing thousands of contami nants back into the laboratory. Liquid inoculation techniques allow a much greater exponent of expansion than the traditional method described he~e. (See Liquid Inoculation Techniques described on Page 146.)
Step-by-step instructions follow for a clas sic grain-to-grain inoculation. As before, the cleanest items should be prioritized nearest to the micron filter. Adherence to sterile inoculation techniques should be strictly observed.
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