Retrieving the bags rom the autoclave

Bags of sawdust, havingbeen removed directly from the autoclave, cool to room temperature by being placed in the windstream of a laminar flow hood. Once the bags are below 100° F. (38° C.) inoculations can proceed.

3. Opening the bags. The bags are opened by pulling the outside plastic panels outwards from the outside. The inoculator's hands never touch the interior surfaces. If they do, contamination is likely. Once ten bags have been fully opened, the inoculator wipes his hands with isopropanol and brings a gallon of grain spawn to the table directly downstream from the newly opened bags. The jar lid is loosened to a point where it can be lifted off easily with one hand.

4. Inoculating the bags in a specific sequence. If using jar spawn, remove the liu and place it upside down, upstream and away from the bags to be inoculated. Since you may wish to return the lid to the spawn jar should all its contents not be used, pay attention to the manner in which it is handled. Grasping the spawn jar with one hand, palm facing up, position the jar opening above the first bag to be inoculated. If you are right handed, inoculate each sawdust bag in sequence going from left to right. (Left handers would logically do this in the reverse.) With a roll of the wrist, angle the jar so that grain spawn free-falls into the opened bag. The spawn at most 4 weeks. Carefully scrutinize the filter disc zone, inside and outside, to discern the presence of any molds or unusual signs of growth. Only cottony spawn, void of wet spots or areas of no-colonization, should be chosen. Since the spawn generally chosen is Second or Third Generation, bag spawn is preferred for this stage. The grain kernels of each spawn jar are loosened by shaking the bag or slamming the jar against a cleaned rubber tire or similai substance.

2. Retrieving the bags from the autoclave

Bags of sawdust,havingbeen removed directb from the autoclave, cool to room temperatun by being placed in the windstream of a lamina flow hood. Once the bags are below 100° F. (38 C.) inoculations can proceed.

3. Opening the bags. The bags are openei by pulling the outside plastic panels outward from the outside. The inoculator's hands neve touch the interior surfaces. If they do, contami nation is likely. Once ten bags have been full opened, the inoculator wipes his hands wit isopropanol and brings a gallon of grain spaw to the table directly downstream from the newl opened bags. The jar lid is loosened to a poii where it can be lifted off easily with one ham

4. Inoculating the bags in a specific si quence. If using jar spawn, remove the lid ar place it upside down, upstream and away fro the bags to be inoculated. Since you may w; to return the lid to the spawn jar should all i contents not be used, pay attention to the ma ner in which it is handled. Grasping the spav jar with one hand, palm facing up, position t! jar opening above the first bag to be inoculate If you are right handed, inoculate each sawdi bag in sequence going from left to right. (L handers would logically do this in the revers With a roll of the wrist, angle the jar so that gn spawn free-falls into the opened bag. The spa'

Figure 125. Sawdust spawn of Reishi (Ganoderw.a lucidum). Note inflated atmosphere within bag.

must be well separated for this technique to result in a consistent rate of inoculation for each bag. Through trial-and-error and experience, a h iDhly rhythmic and exact amount of spawn is approportioned amongst the ten sawdust bags.

If there are, for instance, 4 rows of 10 bags 11 front of the laminar flow bench, then a right-handed person would inoculate bags starting from the far left, rear bag. Each bag to the right would then be inoculated until the back row is finished. In turn, the third row would then be inoculated with the next gallon of gr ^n spawn, aga'n from left to nght. In this fashion, the nands of the :noculator can not jeopardize the sanctity of the upstream bags. To inoculate the first row nearest to the face of the micron filter would endanger downstream bags from the debris coir'ng from the inoculator's hands and/ or undetected contaminants from a spawn jar.

5. Sealing the sawdust spawn bags. Few steps are as critical as tl 's one. The simple mechanical act of sealing sterile airflow bags can have extraordinarily disparate results for the success of the spawn incubation process, All other steps in this process can be perfectly ex ecuted, and yet failure to achève a continuous seal can be disastrous

I attempt to create a positively inflated bubble at the time of se^-ng. (See Figure 129). Although the filter patch allows the transpiration of gases, it is not at a rate that causes the bag to noticeably deflate, even wnh gentle squeezing. When this bubble environment is created at the ;me of sealing, two advantages are clearly gained. First, the grain spawn mixes and rotates easily through the sawdust, making shaking easy. Secondly, each bag now has a voluminous plenum, a iri"i-biosphere with an

Figure 126. Universal hand position used for opening bags., lifting petri dish lids, and reiuoving spawn jar caps-

Figure 126. Universal hand position used for opening bags., lifting petri dish lids, and reiuoving spawn jar caps-

Figure 127. Inoculating sawdust witn grain spawu

if the problem is at the seal or not. Roll the sealed region several times into a tight :old and ushdov , The bag inflates and if there is a leak not at the seal, a distinct hissing sound emanates from the defective site. Should the bag remain tightly inflated with no apparent loss of pressure , then the seal at the top is at fault. Simply re-seal and test again for leaks.

6. Shaking the sawdust spawn bags Once the bags have been properly sealed, they are thoroughly shaken to evenly distribute the spawn kernels. If partially inflated, this process takes only a few seconds. Proper shaking is critical for successful spawn incubation (See

Figure 129).

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