Shroom Culture

terile tissue culture has revolutionized the biological sciences. For the first time in the history of human evolution, select organisms can be isolated from nature propagated under sterile conoii 'ons in the laboratory, and released back into the environment. Since a competitor-free environment does not exist naturally on this planet*, an artificial setting is created—the laboratory—in which select organisms can be grown in mass.

Louis Pasteur (1822-1895) pioneered sterile technique by recognizing that microorganisms are killed by heat, most effectively by steam or boiling water. Tissue culture of one organism—in absence of competitors—became possible for the first time By the early 1900*» growing organisms in pure culture became commonplace. Concurrently, several researchers discovered that mushroom mycelium could be grown under sterile conditions. However, the methods were not always successful. Without benefit of basic equipment, efforts were confounded by high levels of contamination and only after considerable effort was success seen. Nevertheless, methods slowly evolved through trial and error.

Figure 62. Diagrammatic representation of the effectiveness of filtration media. Dirty air is first filtert through a coars prefilter (30% @ 10 p), then an electrostatic filters (99% @ 1 p), and finally through a Hif Efficiency Particulate Air (HEPA) filter (99.99% @ .3 p). Only some free-flying endospores of bacteria and viruses pass through.

The monumental task of creating a sterile environment has been difficult, unt¡I recently. The invention of hign efficiency particulate air filters (called HEPA filters) has made sterile I' ssue culture aciiieveable to all. In vitro ("within glass") propagation of plants, animals, fungi,

* Scientists have recently discovered a group of heat resistant bacteria thriving in the fumaroles of submerged, active volcanoes.These bacteria thrive where no other life forms live.

** Many types of filiation systems are available. Ionizers, for our purposes, are insufficient in their air cleaning capacity. For a comparison of filtration systems, which rates HEFA filtration as the best, please refer to Consumer Report ;, Oct. 1992, pg. 657. A new generation of micron filters, the ULPA filters screen out particulates down to. 1 microns with a 99.9999% efficiency. This means only 1 particle measuring. 1 microns in diameter of every 1,000,000 flows through the filtration medium.

bacteria, protozoa became commercially possible. Today, the HEPA (High Efficiency Particulate Air) filter is by far the best filtration system in use, advancing the field of tissue culture more-so than any other invention. ** When air is forcibly pressed through these filters, all contaminants down to .3 microns (p) are elimi • nated with a 99.99% efficiency. This means only 1 of every 10,000 particulates exceeding. 3 p pass through. For all practical purposes, a sterile wind is generated downstream from the filter. The cultivator works within this air-stream This unique environment calls for unique techniques. A d ifferent set of rules now presides, the violation of which invites disas ter.

Sterile tissue culture technique fails if it solely relies on mechanical means. Sterile tis-

sue culture technique is also a philosophy of behavior, ever-adjusting to ever-changing circumstances. Much like a martial art, the cultivator develops keen senses to constantly evaluate threats to the integrity of the sterile laboratory. These enemies to sterile culture are largely invisible and are embodied within the term "contaminant".

A contaminant is anything you don't want to grow. Classically, Pénicillium molds are contaminants to mushroom culture. However, if you are growing Shiitake mushrooms, and a near-by fruiting of Oyster mushrooms generates spores that come into the laboratory, then the Oyster spores would be considered the "contaminant". So the definition of a contaminant is a functional one— it being any organism you don't want to culture.

The laboratory environment is a sanctuary, a precious space, to be protected from the turmoils of the outside world. Maintaining the cleanliness of a laboratory is less work than having to deal with the aftermath wreaked by contamination. Hence, contaminants, as soon as they appear, should be immediately isolated and carefully removed so neighboring media and cultures are not likewise infected.

The stages for cultivating mushrooms parallel the development of the mushroom life cycle. The mass of mycelium is exponentially expanded millions of times until mushrooms can be harvested. Depending upon the methodology, as few as two petri dishes of mushroom mycelium can result in 500,000-1,000,000 lbs. of mushrooms in as short as 12 weeks! If any contaminants exist in the early stages of the spawn production process they will likewise be expanded in enormous quantities. Hence, the utmost care must be taken, especially at the early stages of spawn production. Several tracks lead to successfully growing mushrooms. For indoor, high-intensity cultivation, three basic steps are required for the cultivation of mushrooms on straw (or similar material) and four for the cultivation of mushrooms on supplemented sawdust. Within each step, several generations of transfers occur, with each resulting in five-to hundred-fold increases in mycelial mass.

I. Culturing Mycelium on Nutrified Agar Media: Mushroom mya lum is first grown on sterilized, nutrified agar media in petri dishes and/or in test tubes. Once pure and grown out, cultures are transferred using the standard cut-wedge technique. Each culture incubating in 100 x 15 mm. petri dish can inoculate 10 quarts (liters) of grain spawn. (See Figure 63) If the mycelium is chopped in a high-speed stirrer and diluted one petri dish culture can effectively inoculate 40-100 quarts (liters) of sterilized grain. These techniques are fully described in the ensuing pages.

II. Producing Grain Spawn: The cultures in the petri dishes can be expanded by inoculating sterilized grain housed in bottles, jars, or bags. Once grown out, each jarcan inoculate 10 (range: 5-20) times its original mass for a total of three generations of expansions. Grain spawn can be used to inoculate pasteurized straw (or similar material) or sterilized sawdust. Grain spawn is inoculated into sawdust, straw, etc. at a rate between 3-15% (wet mass of spawn to dry mass of substrate).

III. Producing Sawdust Spawn: Sawdust spawn is inoculated with grain spawn. Sawdust spawn is best used to inoculate a "fruiting substrate", typically logs or supplemented sawdust formulas. One 5 lb. bag of sawdust spawn can effectively inoculate 5-20 times its mass, with a recommended rate of 10:1. Sawdust-to-saw-

Figure 63. Creating and expanding grain spawn. Sterilized grain is first inoculated trom agar-grown futures. Once grown out, each jar can inoculate ten more every two weeks.

dust transfers are common when growing Shiitake, Nameko, Oyster, Maitake, Reishi or King Stropharia. Once grown out, each of these bags can generate 5-10 more sawdust spawn bags (S1 to S2). No more than two generations of expansion are recommended in the production of sawdust spawn.

IV. Formulating the Fruiting Substrate:

The fruiting substrate is the platform from which mushrooms arise. With many species, this is the final stage where mushrooms are produced for market.The formulas are specifically designed for mushroom production and are often nutrified with a variety of supplements. Some growers on bulk substrates expand the mycelium one more time, although I hesitate to recommend this course of action. Oyster cultivators in Europe commonly mix fully colonized, pasteurized straw into ten times more pasteup ?;ed straw, thus attaining a tremendous amount of mycelial mileage. However, success occurs only if the utmost purity is maintained. Otherwise, the cultivator risks losing everything in the gamble for one more expan sion.

This final substrate can be amended with a variety of materials to boost yields. With Shiitake, supplementation with rice bran (20%), rye flour (20%), soybean meal (5%), molasses (3-5%), or sugar (1 % sucrose) significantly boosts yields by 20% or more. (For more information the effects of sugar supplementation on Shiitake yields, see Royse et al., 1990.)

and pouring nutrified agar media into petri dishes. A variety of vessels

Figures 64, 65, 66 and 67. Freparing can be used for media preparation.

and pouring nutrified agar media into petri dishes. A variety of vessels

CHAPTER 12

any formulations have been developed for the cultivation of j mushrooms on a semi-solid agar medium. Agar is a seaweed- I ; derived compound that gelatinizes water. Nutrients are added to the j agar/water base which, after sterilization, promote healthy mushroom j i mycelium. The agar medium most commonly used with the greatest | success is a fortified version of Malt Extract Agar (MEA). Other | nutrified agar media that I recommend are: Potato Dextrose Agar ; | (PDA), Oatmeal Agar (OMA), and Dog Food Agar (DFA).

By supplementing these formulas with yeast and peptone, essential vitamins and amino acids are provided. These supplements not only | | greatly stimulate the rate of growth but the quality of the projected j mycelial mat. Most agar media are simple and quick to prepare. What j : follows are some of my favorite nutrified agar recipes—of the 500 or j more published.

Malt Extract, Yeast Agar 1000 milliliters (1 liter) water 20 grams agar agar 20 grams barley malt sugar 2 gram yeast (nutritional)

1 gram peptone (optional, soybean derived) (The above medium is abbreviated as MYA.

With the peptone, which is not critical for most of the species described in this book, this medium is designated MYPA.)

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