Steps for Creating Second and TMrd Generation Ci sin

After sterilizing grain in 1/2 gallon or gallon jars, standard procedures for inoculation are followed. For every quart Grain Master, five 1/2 gallon jars are recommended essentially a 1:10 expansion.

Step 1. Select a Grain Master showing even, luxuriant growth. Avoid spaw a jars having zones of heavy growth, discoloration, or excess liquid.

Step 2. Using a cleaned nibber tire, carefully slam the jar against it, looening thegrain. If the spawn is overgrown, more forcible shaking is required before the spawn kernels will separate. Do not strike the jar against the palm of your

Figure 110 Jars furthest downstream »re inoculated first and removed.
Figure 109. inoculating G2 gallon jars of stei ilized gr„in from 1/2 gallon (2 liter) G1 masters.

cleanest items should be prioritized nearest to the micron filter. Adherence to sterile inoculation techniques should be strictly observed.

for Creating Second

After sterilizing grain in 1/2 gallon or gallon jars, standard procedures for inoculation are followed. For every quart Grain Master, fi^e 1/2 gallon jars are recommended, essentially a 1:10 expansion.

Step 1. Select a Grain Master showing even, luxuriant growth.Avoid spawn j'.xs having zones of heavy growth, discoloration, or excess liquid Step 2. Using a cleaned rubber tire, carefully slam the jar against it. lot ening thegrair. If the spawn is overgrown, more forcible shaking is required before the spawn kernels will separate, Do not strike the jar against the palm of youi

Figure 110. Jars furthest downstream »re inoculate first and removed.

Figure 111. Grain inoculated with mycelium but contaminated with bacteria. Note greasy appearance of grain kernels. Bacterially contaminated grain emits a distinct, unpleasant odor.

hand! Be careful! (This author, at the t:me of this writing, is recovei :ng from a slic ;d wrist after a br s k visit to the hospital emergency room, caused by a glass jar shattering on his palm during shaking, requiring muf'ple stitches.)

Step 3. Once the Grain Master has been shaken, loosen the lids of the jars which will re ceive the spawn. Remove the lid of the Grain Master and set it aside. With your favored hand, move the grc:n master upstream to the first jar, hovering: nches above it. With your other hand, remove the ] d and hold it in the air. By tilting downwards and rotating the Grain Master, ker nels of spawn fall into the awaiting ar. Replace the lid of the jar just inoculated and continue to the next. By the time the tenth jar is inoculated, the spawn jar should be empty. Repeated transfers eventually lead to an even dispersal of spawn each time. Precise measurement is desirable but not absolutely critical with this suggested rate of expansion. However, as one becomes more experienced, inoculation rates achieve a high degree of regularity.

Step 4. Once inoculated, the lids are tightened securely. Each jar is then shaken to evenly disperse the Grain Master spawn kernels through the sterilized grain. Thorough shaking encourages fast grow-out. As the jars are shaken, note the rotation of the myceliated grain kernels throughout the jar.

Step 5. Set the Second Generation Spawn jars upon a shelf or rack in a room maintained at 75° F. (24° C.). The jars should be spaced at least 1/2 inch apart. Closely packed jars self-heat and encourage contamination. I prefer that jars incubate at an incite, allowing for more trans ,:;ration.

Step 6. After 3-4 days, each jar is shaken again. As before, the grain can be loosened by si riking the jars against a rubber tire or similar surface. Grasping each jar firmly, accelerate each jar downwards in a spir J, pulling back at the end of each movement 1'his technique sends the top grain kernels deep into the bottom recesses of the jar, in effect rotating and mix ing the grain mass.

Step 7. In 7-10 days, re-inspect each jar to determine even dispersal of growth sites. Should some jars show regions of growth and no-growth, another shaking is in order. Those showing good dispersion need not be disturbed. Here the discretion of the cul ":vator plays an important role. If any unusual pungent odors are noticed, or if the grain appears greasy, contamination may be present although not yet clearly visible.

Step 8.By day 14, all the jars should be thoroughly colonized by myce';um. With Oyster, Shiitake, Enokitake, Reishi, King Stropharia, the mycelium has a grayish-white appearance

Figure 111. Grain inoculated with mycelium but :ontaminated with bacteria. Note greasy appearance of grain kernels. Bacterially contaminated *rain emits a distinct, unpleasant odor.

">ar:d! Be careful! (This author, at the time of his writing, is recovering from a sliced wjist af-¿r a b ' sk visit to the hos r tal emergency room, :aused by a glass jar shattering on his palmdur-ng shaking, requiring mult'-le st;i:ches.)

Step 3. Once the Gra:,i Master has been haken, loosen the lids of the jars which will re ;eive the spawn. Remove the lid of the Grain Vlaster and set it aside. With your favored hand, nove the grain master upstream to the first jar, lovering inches above it. With your other hand, "emove the lid. and hold it in the air. By til ng lownwards and rotating the Grain Master, ker lels of spawn fall into the awaiting jar Replace he lid of the jar jusr inoculated and continue to he next. By the time the tenth jar is inoculated, he spawn jar should be empty. Repeated transfers eventually lead to an even dispersal of

48 hours before flushing out with bright white mycelium.

Each Second Generation spawn jar can be used for inoculating another set of grain jars for instance, five-gallon jars containing twice the amount of grain as the 1/2 gallon containers, i-effect another 1:10 expansion. (2112 gallon (10 liter) jars or bags can be used at a similar rate. (See Figure 112.)) These would be denoted as G3. Third Generation grain spawn is inoculated in exactly the same fashion as Second Generation grain spawn However, contamination is likely to go unobserved. Some large spawn laboratories successfully generate Fourth Generation spawn. However, contamination outbreaks discourage most from pushing this expansion any further. As the mass of sterilized grain is increased within each larger container, anaerobic conditions can more easily prevail, en couraging bacteria. These larger containers require more aeration, a feat that is accomplished with frequent shaking (every 48-72 hours), greater filter surface area, and near-horizontal incubation. When the large containers are laid horizontally, the surface area of the gr?in-to-au is maximized, providing better respiration for the mushroom mycelium.

Throughout every stage in the grain expansion process, any hint of contamination, especially smell, the texture of the grain or unusual colora tions, should be considered warning signs. The spawn maker soon develops a sixth sense in choosing which spawn jars should be expanded and which should be avoided. Most spawn pro ducers only select a portion of the spawn inventory for further propagation. The remainder are designated as "terminal," and are not used for further expansion onto sterilized grain. Un-

Figure 112.11 lbs. (5 kg.) of grain spawn in gallon (10 :1 liter) gla., jar and polyprcpy'- : ag^ Glass jars are resuable whereas spawn bags are currently not recycled. However, since bag spawi s easier to handle, this i* ■ form most commonly sold to mushroom growers by commercial spawn laboratories.

Figure 112.11 lbs. (5 kg.) of grain spawn in gallon (10 :1 liter) gla., jar and polyprcpy'- : ag^ Glass jars are resuable whereas spawn bags are currently not recycled. However, since bag spawi s easier to handle, this i* ■ form most commonly sold to mushroom growers by commercial spawn laboratories.

Figure 113. Gallon jars of 3rd generation grain spawn incubating.

less contaminated these terminal spawn jars usually are of sufficient qua,;ty for inoculating bulk substrates. Of course the spawn manager can always exercise the opt;on of using First, Second or Third Generation grain spawn for inoculating sawdust or straw.

In effect, the spawn maker has taken a single petri dish and in three generations of transfers created 250 gallons of spawn.Therefore, a stack of twenty petri dishes can give rise to 5000 gallons of spawn! This places a whole new perspective on the sheer biological powei Cerent within a single test tube slant, which can ®sily inoculate a sleeve of 20 petri Jishes. Most laboratories do not fully reaiize the potential of every culture. In many cases, spawn expansion is terminated at G2. Many spawn managers choose not to "chase" the optimum. Few laboratories are large enough to accommodate the end result of the methods described here.

An alternative method for generating spawn is vie. Liquid Culture. This method saves time, money, and is less s usceptible to contamination. These techniques are described further cn.

The next step is for each of theseThirrt Generation spawn units to inoculate ten to twenty its mass in sawdust or straw. See Chapters 16 and 17.

Autoclavable bags have been used by the mushroom industry for nearly 40 years. Primary uses for autoclavable bags are for the incubation of grain and sawdust. Preferences vary Widely between cultivators. Flat, non-gussetted bags are popular for incubating grain spawn. The more grain filled into a bag, the greater the danger of poor gas exchange, a ma jor factor leading to contamination Three dimensional gusseted bags are used pri-

Figure 114. Grain spawn ready for use.

140 GENERATING GRAIN SPAWN

Fig.i

Fig.i

Sept 16, 1938 —P-jy^ä -MOTU«« 2,831,821 such as imssow spmn

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- "Vi

~ i V'

Sept 16, 1938 —P-jy^ä -MOTU«« 2,831,821 such as imssow spmn

I'l-HUF. GUIOCSiON

Fi lire 115. The use of heat tolerant plastic ba&s greatly advanced the practicality o he bulk processing of gl and sawdust One of the first patents for this innovation was awarded to Guiochon. (ÜÄ Patent #2,851,821) in 1958.

marily for holding non-supplemented and supplemented sawdust.The proper handling of these bags is critical to their successful use. Bags contacting hot surfaces become elastic, deform, and fail. Currently the industry uses polypropylene or poly methylpentene bags with and without microporous filters.

Over the years, a number of patents have been awarded, some long since expired. The use of plastic bags has had a drastic impact on the way many cultivators generate spawn. Numerous patents have been awarded for bags specifically designed for mushroom culture. The earliest patent I can find is from 1958, awarded to a Frenchman by the name of Guiochon (U. S. #2,851,821). His cylindrical bag resembles those still widely in use by Asian cultivators. (See Figure 115). In 1963 several similar patents were awarded in London (#985,763; 1,366,777 and 1,512,050). R. Kitamura and H. Masubagashi recieved a patent (#4,311,477) for a specialized mushroom culture bag in 1982.*

About a dozen bags are currently available to mushroom cultivators, some borrowed from the hospital supply industry. Cellophane deserves re-examination since it is made from wood cellulose and is completely biodegradable. If problems with seam integrity, tensile strength, and heat tolerance could be impioved, spawn bags made of this environmentally friendly material could eliminate the widespread use of throw-away plast ;s. An advantage of cellophane-like materials is that the mushroom mycelium eventually consumes the very bag in which it has been incubated.

Autoclavable bags are inoculated with Grain Masters and are Second or Third Generation. Agar-to-graLi inoculation from petri dish cultures to bags is awkward and impractical unless liquid inoculation techniques are employed. (These techniques are fully desc,' bed later on.) Bags are filled with pre-moistened grain, with the lips folded closed. Some spawn producers use sp "ng-activated clothespins, paper clips plastic tape, to hold the folds closed. I prefer to simply press the bags together with flaps folded. As the bags are sterilized, the contents exceed the boiling point of water, and gases are released. If the bags are sealed before loading explosions or "blow-outs"—holes where live steam has vented—are likely.

In the standard 18x8x5 inch gussetted autoclavable spawn bag featuring a 1 inch filter patch, no more than 3500 grams of dry grain should be used. * OneAll-American ™ 941 pressure cooker can process 50 lbs. of dry rye grain in one run. However, the pressure cooker—with its tightly packed contents—should be kept at 15+ psi for 4-5 hours to insure even and full sterilization.

If the grain is first boiled or simmered in hot water before filling, even moisture absorption is assured. Exces s water collecf ng at the bottom of the bags often leads to d: 1 aster If this water is reabsorbed back into the media by frequent shaking or by turrng the bags so that the excessively moist grain is on top, the cultural environment is soon re-balanced in favor of mycelial growth. Standing water, at any stage in the musliroomcult; vat'on process, encourages compeftors. Many spawn producers add 20-30 grams of calcium sulfate to the grain, when dry, to help keep the kernels separated after autoclaving

After sterilizaf jn, 2 hours at 15 -18 psi if the bags are separated or 4-5 hours if the bags are tightly packed, the bags are removed and allowed to cool in the pure wh dstream coir ing from the laminar flow bench. An alternative is to allow the grain bags to cool v ithin the pressure vessel, provided it is of the type that holds a vacuum. The vacuum is then "broken" by allowing clean-room air to be sucked in. If the pressure cooker does not hold a vacuum, then it should cool within the sterile laboratory to preserve sterility. In either case, I place a pre-sterilized cotton towel, soaked in alcohol, over the vent cock to act as a filter. For equalizing the pressure in a larger autoclave, air passes directly through a microporous filter into the vessel's

* Other patents, too numerous to list here were also awarded. Many re-designed the seam, the filtration media, and/or sometimes the wording to qualify for a new variation.

1 Please see »ormulas on page 130. Spawn incubation bags are available from suppliers listed in Appendix IV: Resource. Director}'

interior. (See Figure 137.)

Once the bags are cooled, they are unfolded by hand, being careful to only touch the outer surfaces of the plastic. A jar of spawn is selected, shaken and opened. Using a roll-of-the wrist motion, spawn free-falls into each bag at a recommended rate of 1:10 to 1:40.The bag is then laid down so as to open into the airstream. The top 2 inches of the bag is positioned over the element of a clean heat sealer and expanded open, again by only touching the outer surfaces of the plastic. The clean air coming from the laminar flow filter inflates the bag. I gently press on the sides which further inflates them before sealing. The top arm of the sealer is brought forcibly down, often times two or three times in rapid succession, pausing briefly to allow the plastic seam to re-solidify. Each bag is squeezed to determine whether the seam is complete and to detect leaks. (Often, pin-hole leaks can be detected at this stage. Having a roll of plastic packing tape, 3-4 inches wide, handily solves this problem by simply taping over the puncture site.)

If the bags hold their seal with no leaks, the spawn should be mixed through by shaking each bag. This cultivator strives to capture enough air within each bag so that when they are sealed, each bags appears inflated. Inflated bags are much easier to shake and support better mycelial growth than those without a substantial air plenum. (See Figures 125-129.)

Spawn bags should be set on a shelf, spaced 1/2 inch or more from each other to counter-act heat generation. After four days, each bag should be carefully inspected, laid on a table surface, and rotated to disperse the colonies of mycelium. In another week, a second shake may be necessary to ensure full and even colonization.

The advantages of using bags for processing grain spawn are:

1. In the limited space of a sterilizer, more grain can be treated using bags than jars.

2. Bags, if they break, are not dangerous. Being cut by glass jars is one of the occupational hazards of spawn producers.

3. Since the bags are pliable, spawn can be more easily broken up into individual kernels and distributed into the next substrate. The process of spawning is simply easier.

A rain storm is a form of liquid inoculation. The earliest fungophile, unwittingly or not, used liquid inoculation techniques. Every time mushrooms are eaten, cooked or washed, spores are disseminated in liquid form. Nature's model can be modified for use within the laboratory. Currently several strategies incorporate liquid inoculation methods.The advantages of liquid inoculation ar ethe speed of colonization, the purity of spawn, and the ease of handling.

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