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Growing Mushrooms the Easy Way



[CAVEAT FROM RUSH WAYNE, POSTED SO WE COULD GET HIS PERMISSION TO HOST THIS---
1) the manuals you have posted are out of date

2) I have given away a number of my (up-to-date) manuals for free to people
who have asked for them, both people who have not had the money to buy them
but very much wanted the information, and people working for non-profit
organizations, or academics, or deserving students.

3) Anyone who receives the manuals directly from me, but only from me,
whether for free or by purchase, also can receive free technical support
from me for the manuals by e-mail correspondence. Since it is easy to make
mistakes in the procedures and conclude that the methods are worthless. this
technical support can be an important part of the manuals, and I have never
sold the manuals through bookstores or resellers for this reason. Users who
download the documents posted on your site do not qualify for technical
support.----]

Home Mushroom Cultivation

with Hydrogen Peroxide


Volume I



by R. Rush Wayne, Ph.D.

Nan's Nook : Archives : Misc Tek : Straw Tek : Sterilize Straw with Peroxide : Volume II


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Growing Mushrooms the Easy Way
Home Mushroom Cultivation with Hydrogen Peroxide
Copyright © 1999
R. Rush Wayne. Ph.D.

All rights reserved. No part of this work may be reproduced or used in any form or by any means without permission of the author.

ooops...

First published as Growing Mushrooms with Hydrogen Peroxide, December 1996
Third Edition revised November 1999

Visit the Updates Page of the Growing Mushrooms the Easy Way Web site
(http://members.aol.com/PeroxyMan/Updates.html)
for periodic corrections and updates to this manual, notes on sources of supplies,
and news about the peroxide method.



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CONTENTS



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Introduction

Preliminaries

The Mushrooms

Hypsizygus ulmarius (Elm oyster)
Pleurotus eryngii (King oyster)
Hericium erinaceus (Lion’s Mane)
Agaricus subrufescens (Almond mushroom)
Lentinula edodes (Shiitake)
Pleurotus ostreatus (Oyster mushrooms)
Ganoderma lucidum (Reishi mushroom)
Coprinus comatus (Shaggy Mane)
Hypsizygus tessulatus (Shimeji)
Stropharia rugosa-annulata (King stropharia)

Equipment You Will Need

Specialized Supplies You May Need

The Basics on Peroxide



What peroxide does

Biological effect of peroxide
Advantages of using peroxide in mushroom culture
Contaminant resistance to peroxide


What peroxide does not do
The need for caution when exposing mycelium
Peroxide is not a sterilant at mushroom-growing concentrations

Safety and environmental considerations for hydrogen peroxide

Peroxide and the spirit of organic growing
Lack of effect of peroxide on substrate or mushroom cultures

Stability

In pure solution
At higher temperatures
In the presence of peroxide-decomposing enzymes
Guarding the purity of peroxide stock solution

Variations in peroxide concentration from commercial sources

Measuring peroxide concentration : Calculating how much peroxide solution to use


Growing and Maintaining Agar Cultures

Preparing agar plates

MYA medium

Use the lowest effective concentration of peroxide in agar

No-pressure agar medium

Hazards of agar drips and importance of clean plates
Reusing peroxide agar medium

Acquiring mushroom cultures

Importance of starting with good strains
Cloning Mushrooms

Strain storage.

Distilled water method
Keep storage cultures peroxide-free

Inoculating and handling agar cultures

Cooling hot scalpels
Inoculating from storage cultures or peroxide-free medium
Preventing occult contamination with bottom inoculation: cleaning the mycelium
Incubating inoculated plates and storing uninoculated plates

Making Mushroom Spawn

Economic advantage of making your own spawn
Advantages of sawdust-based spawn

"Ten minute" no-autoclave sawdust spawn

Nitrogen supplements compatible with Ten Minute Spawn
Importance of clean containers in making Ten Minute Spawn

Pressure-sterilized sawdust spawn

Grain spawn

Difficulties of grain spawn and pitfalls in spawn making

Spawn containers

Inoculating spawn

Agar chunk method
Use peroxide-adapted mycelium for spawn inoculation
Incubating and shaking the spawn
Liquid culture


Colonization of bulk substrate

Importance of choosing substrates lacking peroxide-decomposing enzymes
Pasteurizable substrates compatible with peroxide
Recipes for fruiting substrates

Wood chips and substrate density

Preparing supplemented sawdust with peroxide



Nitrogen supplements for bulk substrate

Calculating how much supplement to add

Measuring pH of substrate



Culture containers

Trash bags as culture containers
Excluding fungus gnats
Plastic buckets as an alternative to bags

Inoculating supplemented sawdust

Breaking up spawn for inoculation into fruiting substrates
Adding the spawn to the substrate


Growing mushrooms on straw

Hydrated lime soak method
Inoculating straw

Mushroom formation

General procedures for fruiting oyster-like mushrooms and Hericium
Protecting yourself from spores
Mushrooms needing a casing layer

Seasonal planning

Outdoor vs. indoor growing



Harvesting

Trouble shooting.

Conclusion

About the Author



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Introduction

Back to Contents

When I first took an interest in growing mushrooms, I checked out a well-known book on mushroom cultivation from the library and eagerly read through it. But my interest soon turned to general discouragement as I read about all the equipment and procedures the book insisted were necessary to grow mushrooms without getting the cultures contaminated. I would need a sterile laboratory space with a laminar-flow hood fitted with electrostatic and HEPA filters and an ultraviolet light. This space would need a sterile air-lock entry way with a foot wash, and I would need to have special clothing to enter it, so that I could wash down the floors with chlorine bleach every day. My fruiting mushrooms would have to be grown in a separate building altogether, so as to avoid getting spores into the sterile laboratory. These fruiting cultures would have to be grown in specially designed plastic bags with microporous filter patches attached, so that the mushroom mycelium could get oxygen without letting mold spores or bacteria get in. Of course, I would need an autoclave or at least a specially designed pressure cooker to sterilize the media that went into these bags.

After considering these requirements briefly, I put aside the thought of growing mushrooms. I wasn’t about to get all that equipment, and I figured I probably wasn’t cut out for the job anyway. From what I could gather, my house would be a death trap for mushroom cultures. Neither my wife nor I are careful housekeepers. We have unabashed dust and clutter, and green and white fuzzy things can be found in and outside the refrigerator. Although I was skilled at sterile technique from my years as a graduate student in biochemistry, I didn’t think that would save me from the legions of eager contaminants that would surely dog my every movement should I attempt to grow anything so delectable as mushrooms.

Still, the thought of growing mushrooms didn’t disappear entirely. Instead, a year or so later, it resurfaced again in the form of a new idea. I had read that culture media used for growing orchid seeds could be rendered free of contaminants if hydrogen peroxide was added to the medium. While the peroxide killed bacteria, yeast, and fungal spores, it left the orchid seeds viable because they contained enough peroxide-decomposing enzymes to protect themselves. Then the orchid seeds could be germinated and tended easily by relative beginners without the need for strict sterile technique.

So here was a question: could added peroxide be used to keep mushroom-growing media, like orchid-seed media, free of contaminants? If it could, then perhaps mushroom growing could be made accessible to beginners, just like orchid seed germination. So I resolved to try it with mushroom mycelium.

What followed was a fairly complicated and non-linear process of learning about growing various mushrooms, experimenting with adding hydrogen peroxide, trying different concentrations, learning about different culture media and how they interacted with the mushrooms and the peroxide, trying various degrees and techniques of pasteurization and sterilization, going back over earlier ground with better pH measurements, experimenting with supplements, tracking down sources of contamination, tightening my procedures, and on and on, until I developed some fairly reliable guidelines for what I was doing. It all took far longer than I ever would have guessed. But the upshot of it was that, yes, mushroom growing can be made accessible to beginners without the need for sterile facilities, air filtration, or even a pressure cooker, if one adds hydrogen peroxide to help keep the mushroom culture media free of contaminants. Using the techniques I developed, all the stages of mushroom culture can now be carried out by relative beginners, with a wide variety of mushrooms, and without investing a small fortune in equipment and facilities.

I have written the current book as a guide for the home hobbyist interested in mushroom growing, one that could serve as a basic stand-alone primer on home cultivation of several delectable mushroom species, and one that anyone can use, including the beginner. My previous manual, Growing Mushrooms with Hydrogen Peroxide, was written for the mushroom grower who is already familiar with traditional methods of mushroom cultivation, and its focus is on the use of hydrogen peroxide as an improvement to the traditional methods. While that manual made it possible for the first time to perform all phases of gourmet mushroom cultivation in the home without sterile facilities and air filtration, the current book goes even further and presents procedures that do not require pressure sterilization.

Of course, not all of the procedures described in this book were created by me. In particular, any procedures not specifically required for using the peroxide technique, or not specifically made possible by the peroxide technique, are likely to have originated elsewhere.

For in depth accounts of the traditional methods of mushroom cultivation, as well as the growth requirements for a wide range of mushroom species, please consult Stamets, Growing Gourmet and Medicinal Mushrooms, Chilton and Stamets, The Mushroom Cultivator, or another basic text. These books are valuable reference volumes for anyone who seriously wants to pursue mushroom growing in detail. Also, as you glance through these books, with page after page of discussion of elaborate sterile procedures and sources of contamination, you will enhance your appreciation for the simple techniques contained in this manual.

Note that sterile or aseptic technique, which the procedures in this book require to some extent, is always better demonstrated than described. It is my hope that the reader will seek out direct instruction in this regard. Your local mycological society may be helpful to that end.

Also note that this book is not intended as a guide for commercial cultivation of mushrooms, although the methods described here may well prove valuable to small scale commercial growers as well as to home hobbyists.

Preliminaries

The Mushrooms

Back to Contents

Just about any mushroom that is currently cultivated can be grown in the home. However, some are easier to grow than others, and some, though easy, are not as rewarding as others that are more difficult.

I currently focus on four mushrooms in my own home growing. These are:

Hypsizygus ulmarius, the elm oyster or white elm mushroom: although it is not a member of the oyster mushroom family, it is an oyster-style mushroom in appearance and habit. It grows aggressively on sawdust or straw, it rarely has contamination problems following the techniques described here, and it does well in a variety of conditions and temperatures, fruiting either vertically or horizontally. When well cultivated, the mushrooms are large and attractive, rather like strange white flowers, and they are in my opinion the most delicious of the oyster-style mushrooms (not counting P. eryngii).

Pleurotus eryngii or King oyster: a member of the oyster mushroom family, but it does not have the usual oyster mushroom appearance or habit. A native of Europe, it grows up from the ground in a regal stance, rather than on trees and logs. It is large and thick-fleshed. Its substrate requirements are more narrow than other oysters, as are its temperature requirements. I have read that it prefers sawdust to straw, although I have not experimented enough with it on straw to confirm or deny this. It fruits best in fall or spring temperatures, growing at a glacial pace in the cold of winter, and dying back in the hotter parts of the summer. It is one of the most delicious of cultivated mushrooms if cooked properly, sauteed rapidly in a wide pan, without being allowed to stew in its own juice, then lightly salted.

Hericium erinaceus or Lion’s Mane, also called Pom Pom mushroom: a fungus that lacks the stalk and cap of a traditional grocery store mushroom, instead appearing as a kind of snowball covered with white icicles. It grows rapidly on sawdust substrates, and fruits easily over a range of temperatures. I have heard that it can be grown on straw as well, although I have never tried it. Chefs love this mushroom, and indeed it has a delicious gourmet flavor sometimes tasting like lobster.

Agaricus subrufescens, or almond mushroom: a member of the family that includes the domestic button mushroom and the Portabellas. It is distinguished by its unmistakable flavor of almond extract. Like the domestic mushroom, it prefers to grow on compost, but it can also be grown on straw, wood chips, or supplemented sawdust. It is a warm weather mushroom, but it will also fruit indoors in the winter in a heated room, making it a good candidate for year-round cultivation. This mushroom generally needs a casing layer--a layer of friable, soil-like mixture usually containing peat--applied to the surface of the culture in order to form fruiting bodies.

Some other mushrooms to consider include:

Lentinula edodes or Shiitake: ever popular, grown by many people and written about extensively elsewhere. I am not a shiitake grower, but the methods of culture handling, spawn and substrate preparation described here will all work for shiitake as well as for the mushrooms that I normally grow. Be sure to get a shiitake strain selected for growth on sawdust if you decide to grow this species using pellet fuel as your bulk substrate. Warm weather and cool weather strains are available.

Pleurotus ostreatus and other oyster mushroom species: like H. ulmarius, these are among the easiest mushrooms to grow, racing through sawdust or straw or any of a variety of other substrates. They were the first mushrooms I fruited using the peroxide method. Strains exist for most temperature ranges. The spores of P. ostreatus, which are released in great quantity from mature fruiting bodies, can cause health problems.

Ganoderma lucidum or Reishi: a top flight medicinal mushroom with immune stimulating properties. This mushroom grows on hardwood sawdust in warm temperatures. A related species from the Pacific Northwest, Ganoderma oregonense, prefers cooler temperatures. The woody mushrooms are broken up and made into tea.

Coprinus comatus or Shaggy Mane: a mild tasting, short-lived mushroom that grows best on compost. I never got it to fruit indoors, but after I discarded the cultures in my yard, it appeared in my garden for a couple of seasons.

Hypsizygus tessulatus, or Shimeji: a cute, small round mushroom with a crunchy texture, grows on straw or sawdust-based substrates. The strain I bought required near freezing temperatures to initiate fruiting, so I didn’t experiment much with it, but there are presumably others that will fruit without that.

Stropharia rugosa-annulata, or King Stropharia: a large mushroom that grows on beds of wood chips or straw and requires a casing and warm weather to fruit. The mycelium grows slowly, and only one variety is currently known to fruit indoors without regards to season of the year.

Equipment You Will Need

Back to Contents

The methods described in this manual require very little in the way of equipment for growing your own mushrooms at home. Handling and measuring hydrogen peroxide requires only a measuring pipette (10 ml volume) and a graduated cylinder (probably 100 or 250 ml volume). These can be purchased from scientific supply stores. To measure the peroxide concentration in the bottles you get from the drug store, you will also need a small test tube with a lip, and a balloon. Preparing mushrooms spawn requires jars with lids (pint, 26 oz, or quart jars), a covered pot for steaming big enough to hold the jars, a small scale or balance for weighing, and some clear plastic food storage bags. Preparing agar cultures requires in addition a set of petri dishes. I recommend reusable plastic petri dishes if you can find them. I purchased mine at my local scientific supply store. A pressure cooker, although not absolutely necessary, will be useful. These can often be found used at second hand stores that carry kitchen implements, or new in the kitchenware section of hardware stores and department stores. Make sure the cooker you get is tall enough to accommodate your jars. You will NOT need a glove box, HEPA filters, ultraviolet lights, a sterile laboratory, laminar flow hoods, air locks, foot washes, etc. etc.

Preparing and handling bulk pellet fuel substrate requires a covered pot for boiling and cooling water, a second pot such as a teapot to boil water for pasteurizing containers, and a larger container such as a five gallon plastic bucket with a tight-fitting lid. Five gallon buckets can often be scavenged or purchased cheaply from ice cream factories and various other kinds of food preparation establishments (avoid buckets that have been used for paint, solvents, or other toxic substances).

For growing out the bulk substrate, you’ll also need some small boxes (usually no bigger than about 500 cubic inches, or 8"x8"x8") and some fresh 0.5 mil or less high density tall kitchen bags (avoid the thicker soft plastic bags), or a set of 2 to 3 gallon plastic buckets with lids. For helping the mushrooms along, you’ll need a hand mister, and a cool space. Later, if you are growing a lot of mushrooms, you may want a fan and an automatic misting system.

Specialized Supplies You May Need

Back to Contents

For making agar medium, you will need agar, light malt powder, and (if you plan to pressure cook your medium) yeast extract flakes, among other things. Agar is available at some health food stores, or through scientific supply houses, or commercial mushroom supply dealers. Note that although agar by itself is more expensive than ready-mixed MYA medium, the latter is only half or less agar by weight, so it is not necessarily a better deal. Malt powder is available from home brew supply stores or scientific supply houses. Yeast extract flakes are available from health food stores.

For spawn making and bulk substrate, you may need paper fiber pellets and wood pellet fuel. The paper fiber pellets are sold in my area as Crown™ Animal Bedding or Good Mews™ Cat Litter. (Check grocery stores, pet supply stores, farm and garden supply stores, etc.) In rural areas of the U.S. and Canada, wood fuel pellets can be found in grocery stores, hardware stores, farm supply stores, stores that sell pellet stoves, etc. In urban areas, check your phone book for distributors of wood pellet stoves, or check with your rural friends. It may take a drive out of town to get some. Try to find out what kind of wood they are made from, and look for hardwood for most mushrooms (fir pellets, however, will work well for P. eryngii and A. subrufescens).

The Basics on Peroxide

What peroxide does

Back to Contents

The peroxide radical is a reactive form of oxygen which attacks various organic compounds. In living cells, it damages the genetic material, cell membranes, and whatever else it finds to react with. By doing so, peroxide in sufficient concentration can kill bacteria, bacterial endospores, yeast, and spores of fungi, including mushroom spores. It apparently can also kill small airborne particles of fungi, and the contaminants associated with human skin cells, which are said to be continually flaking off of the mushroom cultivator. Hydrogen peroxide thus acts to some extent against all commonly-encountered airborne contaminants of mushroom culture, including mushroom spores themselves. By contrast, antibiotics generally act only against bacterial contamination, and fungicides act only against yeasts and fungi.

The beauty of peroxide is that it does not kill established mushroom mycelium or interfere with its growth and fruiting. Despite peroxide’s wide range of action against the common contaminants of mushroom culture, there is a relatively wide range of concentrations at which peroxide will allow the growth and fruiting of mushroom mycelium. The established mycelium, because of its ability to produce high levels of peroxide-decomposing enzymes, is evidently able to defend itself against much higher concentrations of peroxide radical than can isolated spores, cells or tiny fragments of multicellular organisms. So we can add hydrogen peroxide to mushroom cultures, and the mycelium will grow but the small contaminants will die.

This arrangement has a number of advantages. Most obvious is that it reduces the need for costly and elaborate facilities and equipment for environmental contaminant control. By adding hydrogen peroxide to mushroom culture media, it becomes possible to perform all phases of traditional mushroom cultivation, from isolation to fruiting, successfully in non-sterile environments with unfiltered air. Gone is the need for special clean rooms, HEPA filters, pre-filters, laminar-flow hoods, UV lights, air locks, glove boxes, or any other equipment associated with environmental control of microbial contamination--even microporous filters on bags and jar lids become superfluous. Using peroxide, the equipment minimally required for contamination control comes down to some measuring implements, a source of boiling water, and a large pot for steaming (or a pressure cooker for added security) --little more elaborate than is found in many kitchens. And whereas the traditional methods of mushroom culture required skillful sterile technique and immaculate personal cleanliness for success with agar cultures and spawn, use of peroxide allows success with only modest sterile technique and only minimal attention to personal hygiene. What’s more, it becomes possible to fruit mushrooms--even those that have the highest spore load--in the same building used to maintain agar cultures and grow spawn, without the fear that spores released by the fruiting bodies will diffuse into the agar cultures and ruin them. Hydrogen peroxide uniquely will kill the spores of the very same mushrooms whose mycelium it protects.

Do contaminants develop resistance to peroxide, the way they do to ordinary antibiotics? Yes and no. Many of the contaminants are already resistant to peroxide, and once they have established a colony, they will grow vigorously. Live Aspergillus (blue green) mold is very resistant to peroxide. But evidently peroxide at sufficient concentration overwhelms the resistance mechanisms of the single-celled organisms and isolated spores, and those of very small, isolated multicellular organisms as well.

What peroxide does not do

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One thing peroxide does NOT do is eliminate all need for concern about sterile technique. To repeat, although added peroxide will kill isolated spores, yeast, and bacteria that find their way into your cultures, and these are a big part of routine contamination problems, peroxide does NOT kill established live multicellular organisms (such as green mold) beyond a certain size. It also will not do very well against high local concentrations of mold spores. Evidently multicellular organisms and high concentrations of germinated spores are able to produce enough peroxide-decomposing enzymes to defend themselves against high concentrations of external peroxide. And since both multicellular organisms and concentrations of spores can be microscopic and reside on your hands or on particles of dirt or dust, you still have to take sensible precautions to keep your hands and all non-sterile particulate matter out of your early-stage cultures, even with peroxide added. Although you don’t have to be afraid to leave cultures open to the air for brief times, to perform manipulations or otherwise check on them, you’ll still want to use common sense in avoiding contamination. You wouldn’t want to use the lid to a petri dish after you dropped it on the floor, for instance. Neither would you want to allow visible, non-sterile debris of any sort to fall into your cultures, or insects of any kind to enter them. It is a good idea to periodically wipe the dust off shelves used to incubate cultures. You will still need to flame or otherwise sterilize whatever instrument you use to transfer chunks of mycelium from one culture to another. And I make it a regular practice to wipe my fingers with rubbing alcohol before performing inoculations of spawn or agar cultures. I do the same with any counter surfaces I use to perform manipulations with my petri dish cultures. This reduces the chances of larger particles making it into the cultures and helps protect the exposed mycelium.

It is also especially important to know and remember that peroxide does NOT protect the mushroom mycelium itself from aerobic contaminants. The mycelium decomposes peroxide that comes in contact with it, so any aerobic contaminants associated with the mycelium will be shielded from the deleterious effects of peroxide. Thus, as a general rule, peroxide only protects the culture medium or substrate from aerobic contamination. So your most careful procedure should be reserved for transferring mycelium, or performing any operation which exposes mycelium to unfiltered air. And once your mycelium is contaminated, you will need to start over with a fresh, uncontaminated culture, or purify your mushroom tissue in some way to free it of contaminants. I’ll discuss this more later.

Finally, peroxide is not a sterilant except at concentrations too high to use for mushroom growth. That is, you generally cannot use hydrogen peroxide by itself to sterilize culture media or mushroom substrates. At the concentrations that are compatible with mushroom growth, hydrogen peroxide will not kill live mold contaminants resident in the medium, and the peroxide itself will be rapidly destroyed by the peroxide-decomposing enzymes in non-sterile organic materials. Although some spores and bacteria may be killed by adding peroxide to non-sterile medium, there will be far more contaminants that will easily survive and grow within a short time. Therefore the general rule is: all culture materials and containers must be pasteurized before adding peroxide or peroxide-containing medium to them; culture materials that contain raw, unprocessed organic matter must be pressure-sterilized to destroy the peroxide-decomposing enzymes. And a corollary: any water used without pressure-sterilization in peroxide-treated medium should be clear and free of obvious particles, since any bits of organic or even inorganic material introduced with the water could harbor live contamination and/or peroxide-decomposing enzymes that would not be destroyed by pasteurization.

Safety and environmental considerations for hydrogen peroxide

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There are no special safety precautions required for handling 3% hydrogen peroxide. Its toxicity is very low, and it decomposes completely to water and oxygen when it is spilled or ingested. It is odorless and does not stain or burn. It is generally not even active as a bleach until it reaches 60oC, the temperature of very hot tap water. Every evidence suggests that it is environmentally benign.

Since commercial peroxide is prepared chemically, rather than extracted from natural sources, it probably would not be considered compatible with organic certification standards following the criteria currently in vogue. However, I consider the use of peroxide to be in the spirit of organic cultivation. Since the peroxide added to mushroom cultures decomposes entirely to water and oxygen as the mushroom mycelium occupies the substrate, there can be no trace of the added peroxide left in the mushroom crop, beyond what is naturally there due to metabolic processes. Moreover, hydrogen peroxide itself is found naturally in all aerobic living organisms and in a variety of natural environments. From time immemorial, honeybees have secreted enzymes which add peroxide to their nectar, protecting it from bacteria, yeasts, and mold, and imparting antibacterial properties to the resulting honey. The mycelia of at least certain mushrooms produce their own peroxide to help break down the woody substrates the organisms encounter. And peroxide is even a part of the healing defenses of the human organism. Indeed, around the world, thousands of proponents of a system of healing called oxygen therapy actually ingest food-grade peroxide solution on a daily basis to cure various ills and promote vitality, and some people have done so for many years (I do not necessarily recommend this, however). Finally, the use of peroxide circumvents the need for resource-intensive equipment, facilities and supplies, simplifying every stage of the mushroom cultivation process.

There is some question as to the effect peroxide oxidation may have on the mushroom substrate itself. Chlorine, when it reacts with organic materials like paper pulp, produces small amounts of dioxin, a very dangerous, cancer-causing chemical. Hydrogen peroxide does not produce dioxin, and as a result, environmentalists are campaigning to get paper companies to bleach their paper fiber with peroxide rather than chlorine. Still, it is conceivable that peroxide could produce some other harmful substance when it reacts with the organic materials in mushroom substrates. I have not ruled out this possibility, but I consider it unlikely. For one thing, aerobic living organisms have evolved for millions of years with hydrogen peroxide both in and around them. Peroxide is generated by normal aerobic metabolism, and it is also naturally formed by the reaction of water with oxygen in response to the ultraviolet rays in sunlight. This means that aerobic organisms most likely have developed metabolic machinery to deal safely with the variety of oxidation products that result from the reaction of peroxide with biological materials. In addition, hydrogen peroxide is chemically quite stable in sterilized mushroom substrates, and the concentration of peroxide we’re using is so low that the amount of substrate oxidation going on has to be very low indeed. Finally, I have seen absolutely no evidence of any mutagenic or toxic effect of peroxide-treated mushroom substrate on the mycelium or fruiting bodies. Agar cultures containing hydrogen peroxide give fine, healthy halos of mycelium, and the final fruiting cultures produce mushrooms as beautiful as any grown by traditional methods.

Stability

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The 3% hydrogen peroxide solution available at supermarkets and drug stores, with phosphoric acid stabilizer added, is quite stable on the shelf when kept relatively cool. When added to heat-sterilized and cooled mushroom culture media, hydrogen peroxide evidently decomposes only slowly. Precisely how long it will last is presumably a complex function of media composition, peroxide concentration, and temperature. However, my experience so far is that peroxide continues to exclude contaminants for long enough to allow the mycelium of a variety of mushroom species to safely colonize their substrates.

On the other hand, hydrogen peroxide should generally not be added to hot culture media, unless you are going to add extra to compensate for losses from decomposition. Since peroxide becomes active as a bleach above 60oC, it will decompose readily when in contact with complex organic materials at this temperature and above. So wait until your medium has cooled -- if not to room temperature, then at least to a temperature that is comfortable to the touch--before adding peroxide.

In contrast to its behavior in pure solution or sterilized media, peroxide breaks down rapidly in the presence of peroxide-decomposing enzymes, as happens when you put peroxide on a wound. The broken skin cells and blood vessels of a wound contain peroxide-decomposing enzymes in abundance, and they rapidly break down peroxide solution and release oxygen bubbles. Similar enzymes, known as catalases and peroxidases, are found in all kinds of living or once-living material, unless it has been heat treated or extensively processed. So, uncooked grain, flour, sawdust, wood, etc. all will destroy peroxide in short order. This means that you will need to keep all such materials out of your stock peroxide solution. It also means that if you want to incorporate such materials into a culture medium, you have to be sure everything in that medium gets thoroughly heat-treated or cooked clear through to destroy peroxide-decomposing enzymes before you add peroxide.

I take several measures to guard the purity of my stock peroxide solution. When I am about to withdraw peroxide, I first wipe down the lid and upper part of the bottle with rubbing alcohol, to keep out particles that might contain live contaminants. Then I either free-pour to a pasteurized measuring vessel or I use a clean, pasteurized pipette with the mouth-end plugged with cotton to draw up the solution. Pipettes do not need to be autoclaved, but they should be at least steeped in boiling water (filled somewhat beyond the top graduation, but below the cotton plug) for a few minutes, then cooled, before using them to withdraw peroxide. A one hundred milliliter graduated cylinder makes a convenient vessel for steeping a 10 ml pipette in boiling water. The heat will kill any live organisms in the pipette, while the peroxide itself will kill remaining heat-resistant spores. I also take care never to set the peroxide bottle cap down unless I am certain it will not contact contamination.

Variations in peroxide concentration from commercial sources

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One annoying fact of life when using peroxide is that the solution you get from the drugstore or supermarket, labeled as containing 3% hydrogen peroxide solution, U.S.P., may or may not actually contain a 3% solution. The concentration can vary considerably, both above and below 3%. You can protect yourself somewhat from buying "worn out" peroxide by looking for the expiration date on the bottle, and getting one with the latest date, if there is a date at all. (The bottles of peroxide I get list only the month of expiration, not the year). However, even the expiration date gives no absolute assurance that the concentration is really 3%. It is important, therefore, to have a way to measure the peroxide concentration in the solution. This can be readily done by decomposing a sample of the peroxide and measuring the released oxygen, which I do with a simple balloon technique.

Here is my method for getting a rough measurement of peroxide:

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Get a clean test tube (preferably one with a lip or screw cap), a small birthday-party type balloon, and a slice, small enough to fit into your test tube, of the stalk of any mushroom you have handy (for best results, use a young, rapidly growing mushroom and take a piece of stalk, trimming off the natural skin to expose plenty of broken cells). If you don’t have any mushrooms, a piece of banana or other skinned vegetable should do just as well). You will also need your peroxide solution, a rubber band, a pasteurized measuring pipette, a 100 ml graduated cylinder, and a pot of water.

1) With the pasteurized measuring pipette, withdraw 5 ml of the peroxide solution from the bottle and transfer it into the test tube.
2) Place the slice of mushroom in the upper part of the tube (don't let it slip into the peroxide yet).
3) Make sure the balloon is empty of air and stretch the mouth of the balloon over the mouth of the tube (tilt the tube to keep the slice of mushroom from slipping into the solution until the balloon is in place.
4) Put a rubber band around the mouth of the balloon on the tube, to keep gas from escaping as the pressure builds (I have found it most effective to use a broken rubber band that can be wound tightly around the threads of the tube, over the mouth of the balloon).
5) Once the balloon is sealed in place, let the mushroom slice slip down into the peroxide solution. The solution should begin bubbling oxygen immediately.
6) Agitate the tube. The peroxide solution should be largely decomposed in five to ten minutes, depending on the amount of catalase/peroxidase in your mushroom slice.
7) When decomposition is almost complete, youÕll see that the bubbling will have slowed and the bubbles will have become quite small. Meanwhile, the balloon should have become taut as it began to fill with released oxygen.

Now, my college chemistry training tells me that 5 mls of a 3% solution of hydrogen peroxide should generate about 49 mls of oxygen when the peroxide decomposes completely at room temperature and one atmosphere pressure. To measure the oxygen released from your peroxide solution:

1) Fill a graduated cylinder with water and turn it upside down in a pot of water, making sure all bubbles are out.
2) Twist the balloon on your test tube to trap the released oxygen, remove the balloon from the tube holding the twist tightly, and put the balloon under the water in your pot.
3) Carefully release the gas from the balloon up into the inverted graduated cylinder, displacing the water inside it.
4) Keeping the open end of the cylinder under water, read the volume of oxygen off the graduated cylinder.

The first time I did this, I got 52 mls of gas inside my graduated cylinder from 5 mls of peroxide solution. Given that there may well have been about 3 mls of air in the flat balloon before I started, the peroxide solution probably generated pretty close to the theoretical amount of oxygen for 5 mls of 3% solution.

Here’s how to calculate the amount of peroxide solution you will need, if you solution tests higher or lower than 3%:

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1) Divide the volume of oxygen expected for 5 mls of 3% solution (49 mls if the balloon is completely empty to begin with, or 52 mls in the above example, counting the few milliliters of air initially trapped in the balloon) by the volume of oxygen you actually got.
2) Multiply the previous number by the volume of peroxide solution you would add to your medium or substrate if it were really a 3% solution (this volume is given in appropriate section of this manual, for instance, in the section on agar culture, you will find that you would need to add 6 mls of 3% peroxide for 1 liter of pressure-cooked agar medium).

Knowing the precise concentration of peroxide is most important when you are making agar plates (see below), since you will be working at concentrations close to the lower limit of effectiveness. When you are making spawn, you will be working at a considerably higher concentration, so there will be much more leeway for variation. I use less peroxide for bulk substrate than for spawn, but there is still some room for variation there, as well. I recommend you do the balloon test to check each new bottle of peroxide solution you use for making agar plates, and check the peroxide you use for making spawn and bulk substrate at least until you know how reliable your local product is. That way, you will know for sure that you are giving your cultures the protection you expect. Also, you may want to experiment with the peroxide sources in your local area to see who sells the most reliable product. Paradoxically, cheapest may be best, because there will be regular turnover of the stock where the price is lowest. If peroxide is not readily available at local stores where you live, you will probably want to order it from a chemical supply house. They will often carry 30% or 35% solution, which can be diluted. Swimming pool supply stores also may carry similar solutions. Note, however, that these concentrated solutions are considerably more hazardous than the standard 3% solution. Read all the precautions and warnings on the container and act accordingly.

Growing and Maintaining Agar Cultures

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The first stage of mushroom growing is the propagation and maintenance of mushroom tissue (the mycelium) on agar as petri dish cultures. These first-stage cultures are used to store, propagate, and maintain the mushroom strains in a healthy state by serial transfer, and to inoculate the second stage cultures, the spawn.

Preparing agar plates

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There are many recipes for agar medium that can be used to grow mushroom mycelium on petri dishes. I have tried several of these, but I currently use only one: malt yeast agar medium, also known as MYA. This medium has worked respectably for every mushroom species I have attempted to grow. It is not so rich that it contaminates instantly, yet most strains grow across a petri dish of MYA in two or three weeks. In my opinion, if you are using peroxide in your medium, there is not much point to growing the mycelium any faster than that, since it will just force you to make up more agar plates sooner, to keep the mycelium fresh. Also, after repeatedly transferring the mycelium from plate to plate, some growers recommend that you start anew with mycelium from a storage culture, to avoid problems of senescence (aging) of the mycelium. The faster the mycelium grows, according to this view, the sooner one has to go back to storage. If this is true, I would just as soon have the mycelium grow relatively slowly.

I maintain all my petri dish cultures on peroxide-containing medium. Contamination on peroxide plates is rare, as long as a few precautions are followed, and you won’t need to buy a laminar flow hood or build a glove box to keep contaminants out. You can pour your plates in the open air in your kitchen, and you can store and incubate your plates almost wherever you like, as long as the spot is relatively clean and the environment is compatible with mycelial growth. However, see my recommendations at the end of this section.

MYA Medium

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Here is the recipe I use for one liter of MYA medium:

12 gms (0.35 oz) agar
12 gms (0.42 oz) light malt powder
1 gm ((0.035 oz) nutritional yeast powder
0.5 gm (0.017 oz) grain flour (I rotate between wheat, rye, corn, rice, oats, and millet)
0.5 gm (0.017 oz) rabbit feed (or other animal feed pellets)
5-7 wood fuel pellets (the number of wood pellets can be increased for those wood-decomposing species that do poorly on agar)
1 liter tap water

If you purchase ready-mixed MYA medium from a mushroom supply house, it will probably only contain the first three ingredients: agar, malt, and yeast. (You can add the others). Check the instructions to see how much of the powder the manufacturer recommends you use per liter of water. Usually it will be something like 40 to 50 gms. Depending on the proportion of agar to malt powder, you should be able to cut the recommended usage in half and get a medium that is actually better for the long term health of your mushroom cultures.

I prepare the agar medium for plates as follows:


1) I add all the ingredients to a jar with the desired amount of water.
2) I adjust the pH with a little baking soda (my water is acidic, but you could use vinegar if yours is alkaline. Also, see my "Note on Measuring pH of Substrate" below in the section on preparing bulk substrate).
3) I then use my ordinary kitchen pressure cooker to melt and sterilize the medium. (I use tap water and have not had any problems with it. In fact, when I grew mushroom mycelium on medium prepared with distilled water, growth was noticeably slower). I put lids loosely in place and pressure cook at 15 psi for no more than 10 minutes, allowing an initial ten minutes of steaming to melt the agar before putting on the pressure regulator. (If you are using ready-mixed MYA medium, the instructions may say to pressure cook for much longer times, for example, 45 minutes. DonÕt do it! 20 minutes is the most youÕll need, and any longer will produce carmelization products in the medium that are harmful to the mycelium). I also sterilize a set of petri dishes along with my medium, placing the dishes in a large tomato can covered with aluminum foil (I use plastic reusable petri dishes, and a liter of medium fills up about 30 plates).
4) When the cooking is finished, I allow the pressure to drop on the cooker, remove the jar containing the medium and let it cool in the open air on my counter top. There is no need to avoid entry of unsterilized air, assuming there is not a great deal of heavy dust, since the peroxide will kill the airborne contaminants when it is added.
5) When I can handle the jar quite comfortably, I usually put the jar of agar in a pot of warm water for the last part of the cooling process, since the agar is close to solidifying at this temperature.
6) Then I add my peroxide solution with a pasteurized pipette and quickly mix the peroxide into the medium by moving the jar with a circular motion, reversing directions a couple of times (but doing my best to avoid making a lot of bubbles, which will end up on the surface of the agar).
7) Once IÕve added the peroxide, I go straight to my petri dishes, which I have set out on a clean counter, and I free-pour the medium into the dishes, closing the lids as I finish.
8) When the agar has solidified, I set aside the plates to dry for a few days in a lightly covered tray.

To be on the safe side with my plate cultures, I use the lowest concentration of peroxide that I have found effective in agar medium, which is about 0.018%, or 6 mls per liter of medium. (You can add twice this much without visible harm to the mycelium of the species I have grown, but note that very slow-growing species such as Stropharia may be more sensitive to peroxide exposure. The production of protective peroxide-decomposing enzymes seems to be roughly parallel to the rate of growth of the organism). When the plate is inoculated, the concentration presumably begins to drop slowly below the initial level as the peroxide is decomposed by the spreading mycelium. Eventually, the peroxide should disappear completely when the agar is overgrown, if not earlier. Once this stage is reached, colonies of mold may begin to appear at the edge of the agar plate.



No pressure agar medium

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If you do not own a pressure cooker, or do not want to use one, you can still make serviceable agar plates by boiling/steaming the agar medium, provided you alter the above recipe somewhat. You will need to replace the ingredients that contain peroxide-decomposing enzymes with other ingredients that are free of those enzymes. In the above recipe, agar, malt powder, and pellet fuel do not contain peroxide-decomposing enzymes, but yeast powder, flour, and rabbit chow all do. In order to use peroxide in our agar medium, we ordinarily have to pressure cook the medium to destroy the peroxide-decomposing enzymes in these ingredients. However, other ingredients can be used in their place. The yeast powder provides vitamins, so this ingredient can be replaced by a bit of a fresh synthetic B-complex vitamin pill. Because it is synthetic, it will not contain enzymes. Grain flour and rabbit chow provide protein/nitrogen, so these ingredients should be replaced by other peroxide-compatible protein sources. Typically, only highly processed substances are free of peroxide decomposing enzymes, substances like gelatin, soy milk powder, non-fat milk powder, low sodium soy sauce, etc. To test for the presence of the enzymes, mix a little of the substance in question with some 3% peroxide solution and watch for evolution of bubbles. No bubbles means you are in the clear.

Here then is a recipe for one liter of "No pressure" agar medium:

12 gms agar
12 gms light malt powder
0.5 gm processed nitrogen source (rotate between gelatin, soy milk powder, milk powder, low sodium soy sauce, etc.)
5-7 wood fuel pellets
small chip (0.1 gm? enough to turn the solution slightly yellow) from synthetic B vitamin complex pill
1 liter clear water, free of particulates

1) Let your jar containing this medium (and your petri dishes, if reusable) sit in boiling water in a covered pot with a heavy lid for about forty-five minutes, which allows time to melt the agar and kill any live organisms in the medium (it may be advisable to steam reusable petri dishes even longer).

2) Then remove the jar and let it cool, adding the peroxide as in the first recipe. The peroxide will kill any spores remaining in the medium. I add slightly more peroxide to non-autoclaved plates, about 8 mls per liter of medium. In general I find that non-autoclaved peroxide plates contaminate more often than autoclaved peroxide plates, but they still do considerably better than plates made without peroxide.

Watch out for drips of agar medium that land on the outside of your petri dishes. If these are not cleaned off, they will grow mold within a few days, and the spores will diffuse into the plates and start germinating at the outer edge of the agar.

If you are working with reusable petri dishes as I am, clean them carefully after you take out the old agar. Even the smallest amount of old medium left in a plate, if it is not in contact with the peroxide in the new agar the next time you use the plate, can grow mold and become a jumping-off point for contamination.

A benefit of pouring plates when the agar is so cool, is that there is considerably less condensation on the inside of the lids, than if you pour hot. This obviously means that you won’t have to take special measures to get rid of condensation, such as shaking out the lids, or warming the plates to evaporate the droplets. And you’ll be better able to see what is happening in your plates. However, the agar surface still needs some drying, so I let the plates sit at room temperature for a day, lightly covered with a sheet of wax paper to keep dust off, before I use them. Plates with agar medium that has been steamed will be wetter than plates with medium that has been pressure cooked, because of the lower cooking temperatures and shorter cooking time, so they will need to be dried for a longer time.

If you have extra agar medium after your plates are poured, the excess will remain sterile stored in the refrigerator. When you get around to using it, you can melt the agar again, but note that you will need to add peroxide again, because the heat of melting will have destroyed what you added the first time.

Acquiring mushroom cultures

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There are several ways to acquire a culture of mushroom mycelium to grow out on your agar plates. Spores from a mushroom can be germinated in nutrient medium. Tissue can be cut aseptically out of a fresh mushroom and coaxed to sprout mycelium on nutrient agar. Or a mushroom tissue culture can be purchased from a commercial supplier, usually in the form of an agar tube culture or a petri dish culture.

Since peroxide-containing nutrient medium kills mushroom spores, I have not worked with germinating spores to acquire mushroom cultures. Instead, I prefer to purchase tissue cultures from a reputable supplier. The way I see it, the supplier has already gone to the trouble of isolating a mushroom strain with desirable characteristics, and by purchasing a tissue culture, I am relatively assured of obtaining a mycelial isolate carrying the same desirable characteristics. By contrast, if you try to grow a mushroom strain you’ve isolated from spores or cloning and it doesn’t fruit, you won’t know whether it is the conditions you are providing, or the strain that is causing the problem. (Spores are like seeds: they may or may not have the same genetic characteristics as the parent). And you can waste enormous amounts of time trying to fruit a worthless strain. Moreover, once you work out the conditions for growing an isolate in your situation, if you ever lose the culture, unless you purchased from a commercial supplier, you can’t go back to the same supplier and get another "copy." You’ll have to start all over and work out the conditions again for a new strain.

When you purchase a tissue culture from a commercial supplier, it is generally understood that you will use that culture to grow--and sell if you choose to--spawn, fruiting cultures, and fruiting bodies of that mushroom strain. It is also presumed that you will not take that culture and use it to establish your own commercial strain bank, selling agar cultures to others. If you want to sell agar cultures, the ethical route is to isolate your own strains by cloning from the wild or germinating spores.

Cloning mushrooms

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It can be fun, regardless, to clone your own mushroom culture from a specimen you collect in the wild. Perhaps it will provide you with a first-rate fruiting strain. If you’d like to try it, you will need some nutrient agar plates containing peroxide (see below), a scalpel, an ethanol-fueled alcohol lamp, and a fresh mushroom.

To clone a mushroom:

1) Clean off the external surface so that there is no loose debris.
2) Break open the mushroom cap (or base of the stalk) as cleanly as you can.
3) Light your alcohol lamp and flame your scalpel blade. Then cut out a small piece of clean tissue within the mushroom that does not contact any external surface. This will obviously be easier with thick, fleshy mushrooms than with thin ones.
4) When you have piece of mushroom on your scalpel, transfer it to the middle of one of your nutrient agar plates. Since the chances of failure are high, take a few more pieces if you can and transfer each one to its own agar plate.
5) Finally, stack the plates, wrap them in a clear plastic bag, and set them in a convenient place to incubate at room temperature.

6) Mycelial growth, if it occurs, should become visible in a number of days, spreading out from the chunk of mushroom tissue. Mold or bacteria may grow as well, in which case you may have to cut out a bit of clean mycelium and transfer it to a fresh plate. If you decide to subclone the mycelium away from mold contaminants in this way, be sure you do it before the mold has matured enough to darken in color and form spores. Otherwise you will simply be transferring mold with the mycelium.

If you are trying to clone mushroom mycelium from the wild, remember that hydrogen peroxide in your medium will not by itself clean your mycelium of resident contaminants. If your material is dirty, and you can’t get a piece of clean tissue by breaking open the stalk or cap of the mushroom you want to clone, peroxide in the agar will not improve matters. It is not a sterilant. However, if your material is basically clean, peroxide in your agar will at least reduce the incidence of background contamination on your cloning plates.

Strain storage

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Once you have acquired a mushroom culture, you will need a way to safely preserve samples of it for long periods, so that you can go back to these preserved samples if anything happens to the active culture you use every day. The storage method I use simply requires scraping a bit of mycelium off an agar plate and transferring it to a screw cap tube of sterile distilled water (thanks to Joe Kish for bringing this technique to my attention). Once in the distilled water, the mushroom mycelium goes dormant and will last indefinitely for some strains (oyster-type mushrooms last only about a year in my experience). Refrigeration is not even required.

Although agar slants are the "traditional" way to store cultures for those who don’t have liquid nitrogen, slants do not preserve strains very long--six months at best.

Now, when you make storage preparations of strains you want to preserve for long term storage, I recommend that you prepare these without resort to hydrogen peroxide. The reason for this is that I don’t really know what long term effects peroxide exposure may have on mushroom storage cultures. Could it accelerate senescence? Does it weaken the strains gradually? Are there gradual genetic changes? I am simply not in a position to rule out all the problems that could occur with all the different species you may want to store. In addition, actively growing cultures are better able to defend themselves against added peroxide than dormant storage cultures, which may be more subject to damage. So, although slants and distilled water storage tubes can easily be prepared with peroxide, peroxide-free storage of strains is the safest course. Besides, it is so easy to prepare good clean distilled water storage tubes by dispensing the distilled water into the tubes, lightly screwing on the caps, then pressure cooking for half an hour (if you don't have a pressure cooker, I would try boiling for an hour with a few drops of 3% peroxide; the peroxide will kill the heat-resistant spores, and then the lengthy boiling will destroy the peroxide). And unlike petri dishes, screw cap tubes can be flamed on opening and closing, making it easier to keep them sterile without air filtration while inserting mycelium. I wrap the final storage tubes, containing mycelium, in clear plastic "food storage" bags before putting them in a safe spot in my basement.

Inoculating and handling agar cultures

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I inoculate agar plates and slants by sterilizing a scalpel with the flame of my alcohol lamp, then transferring to each fresh plate or slant a small chunk of mycelium-impregnated agar cut from a plate that has a healthy halo of mycelium growing across it. When using a flamed scalpel to cut out agar chunks, I first cool the scalpel by plunging it into the agar of the plate containing the culture I want to transfer. (Traditionally, you would cool the scalpel by plunging it into the agar of the new, unused plate. But the hot scalpel may decompose some peroxide in the plate at the site of the cut. In unfiltered air, this spot then might become a locus for contaminant growth, since it is less protected. This is not a problem with the plate I inoculate from, since it will be discarded. But it may be a concern with the new plate. So I cool the scalpel in the old plate).

If you are inoculating a plate from a storage culture devoid of peroxide, don’t use an inoculating loop except to fish out a large clump of mycelium. In my experience, the minor mycelial fragments picked up by an inoculating loop are not enough to establish colonies readily in the presence of the concentrations of peroxide that are effective against contaminants, especially when the culture has not been growing on peroxide previously. The mycelium has a much better chance of taking hold if you can transfer a clump of mycelium from a distilled water storage tube, or a chunk of mycelium-containing agar excised from a slant using a scalpel or other sharp sterile implement. (Admittedly, it is somewhat awkward and sometimes frustrating to dig pieces of agar out of a slant with a scalpel).

Cultures that have not been exposed to peroxide medium previously will often lag at first, as the mycelium adjusts to this new feature of its environment. Sometimes the mycelium will appear to be trying to grow away from the peroxide agar at first. (You may observe similar behavior when transferring from a plate that originally contained peroxide but that has been overgrown with mycelium for a few days so that all added peroxide has decomposed). Sooner or later, however, the mycelium will settle down and grow normally over the surface of the new medium.

I have never observed any problem with my strains which I could attribute to continuous peroxide exposure. Typically, I transfer my strains about ten times on peroxide-containing medium before returning to peroxide-free storage cultures, although the choice of ten transfers is an arbitrary one, and returning to storage cultures may not be necessary at all.

Note that peroxide protects only the portion of an agar plate that does not have mycelium growing on it. The mycelium itself is unprotected, since it decomposes the peroxide as it grows. Therefore, older plate cultures that have been overgrown with mycelium for more than a few days have an increased likelihood of harboring contaminants.

Preventing occult contamination with bottom inoculation

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It is also possible for contaminants to accumulate on mycelium if you transfer it repeatedly in unfiltered air, even if there is always peroxide in the agar and the mycelium never covers the entire plate. Although you may never see contaminants growing on the mushroom mycelium in your plates, invisible contaminants will slowly build up. This "occult contamination" can be a problem whether or not you are using peroxide in your spawn and in your fruiting substrate as well. However, if your spawn or fruiting substrate will be peroxide free, there is an even greater chance that the occult contaminants could bloom when they enter the unprotected medium.

To guard against the possibility of such occult contamination, I use a simple trick: I regularly inoculate the bottom of the agar when I do my transfers (How often you do this depends on how you store your plates, and for how long. The safest course is to perform this operation at every transfer, at least with those plates used for transferring mycelium to subsequent plates. But you may be able to get away with putting it off for two or three transfers before it starts affecting your success rate). I perform the bottom inoculation as follows:

1) Turn the plate upside down.
2) Lift one side of the plate bottom as if it were hinged to the top, and gently pry the agar disk out into the lid of the plate with a flamed scalpel. (If your agar regularly tears or breaks at this step, you will need to increase the amount of agar you add for making your medium.)
3) Close the plate back up until you're ready, then transfer a chunk of mycelium to the exposed underside of the agar with a flamed, cooled scalpel.
4) Finally, after inoculating the underside, close the plate, turn it right side up again, and gently pry (again with a flamed scalpel) the agar disk back to its usual position in the bottom of the plate, now sitting on top of the chunk of mycelium.

This arrangement forces the mycelium to grow up from the bottom of the agar through the medium to the surface of the plate, leaving any accumulated contaminants behind in the process. Certain strains may not respond well to this arrangement, but so far I have not had any problem as long as the strain I was using was able to grow vigorously on the medium. However, because of the amount of manipulation involved, this procedure does carry an increased risk of contamination compared to simple transfers. Whereas I rarely see contamination on peroxide plates inoculated in the usual way until they are old, I lose perhaps one in five plates inoculated on the underside of the agar. Wiping down your counter surfaces and your fingers with rubbing alcohol before you begin may help cut down on such failures.

A tricky but important point in the bottom-inoculation procedure is to avoid scraping bits of agar onto the rim of the petri dish bottom when you close the plate after inoculating the bottom of the agar. Bits of agar that get on the rim, or outside of it, tend to sprout contaminants because of their proximity to ambient air. It is also advisable to use plates that have been dried sufficiently to eliminate obvious surface drips. If the agar is still very wet when pried into the lid, it may leave enough medium behind in the lid to cause troubles at the edges of the plate later on.

One final point: Be sure not to cut all the way through the agar when you remove wedges for inoculation from a bottom-inoculated plate. Doing so will defeat the whole purpose of the procedure by bringing along the occult contaminants we’re trying to confine to the bottom of the plate. To leave these contaminants behind, excise wedges only from the top of the agar.

Once my agar plates are inoculated (I keep four at a time for each strain), I place them inside fresh clear-plastic food-storage bags, which I close with twist ties. The closed bag provides a still-air environment and helps keep out marauding fungus gnats and mites. I put three or four petri dishes in a single bag. They then can be incubated anywhere that is convenient--on a bookshelf, in a closet, on a counter top, etc. However, I do not recommend storing plates in the refrigerator, because of the condensation that is produced, and I also don't recommend incubating plates on a shelf above a heater, because the on-off cycles of heating and cooling will cause contaminants to be drawn into your plates.

Being able to store fresh (uninoculated) plates easily is one of the benefits of peroxide. I keep a set of fresh plates stored in a cool spot--again, not the refrigerator. Like the inoculated plates, I keep these wrapped in plastic food storage bags. Each time I use one of my growing cultures to inoculate spawn, I also take out one of these fresh plates and inoculate it to replace the culture I’ve used. At the same time, I replace any cultures that may have developed mold colonies at the edge. This way, the number of plates I have growing remains constant, and I rarely find myself short.

Making Mushroom Spawn

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Production of spawn is the second stage in mushroom growing. Spawn is the "starter" used to inoculate bulk fruiting substrates, or to make more spawn. Traditionally, spawn making was best left to commercial spawn suppliers, who had the sterile facilities to keep spawn free of contaminants. With the development of the peroxide method, however, spawn making is just another step in the mushroom growing process, and an easy one at that.

Being able to make your own spawn without a sterile facility has a significant economic benefit for the small grower or hobbyist. At $20 to $25 for a few pounds of spawn, purchasing spawn represents a significant expense. If you make the same few pounds yourself with the help of peroxide, grain will cost you about a dollar or two, and the peroxide -- ten cents. (Sawdust, if not free, will cost quite a bit less than grain). And you won’t have to spend a small fortune building an air filtration set-up or special clean rooms for incubating the spawn.

For my own mushroom growing, I have switched almost entirely to using sawdust-based spawn. With my current methods, sawdust-based spawn medium can be prepared more quickly and easily than grain spawn, without soaking of grain, and even without autoclaving or pressure sterilizing (see below). Also, mature sawdust spawn colonizes sawdust-based substrate more quickly, and in my experience, with a lower incidence of mold contamination, than grain spawn. And contamination of the spawn itself is rare, perhaps one jar in one hundred, as might be expected just from occasional, inevitable mistakes in technique. True, sawdust spawn does not contribute as much nutrition to the bulk substrate as grain, but it is not particularly difficult to add the missing nutrition directly to the substrate from other sources that do not require pressure cooking.

For most mushroom species, grain spawn is recommended for straw, since the grain adds to the nutritional base of the substrate. And that grain has to be pressure sterilized. Still, there are two good species that will grow well on straw using sawdust-based spawn instead of grain: Hypsizygus ulmarius (the elm oyster) and Hypsizygus tessulatus (Shimeji). Since H. ulmarius grows every bit as easily and tastes considerably better than traditional oyster mushrooms of the Pleurotus family, I can see no reason to incur the added difficulty of grain-spawn making simply to grow oyster mushrooms on straw.

Ten Minute Spawn

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My own procedure for preparing sawdust-based spawn originally required sterilizing separately enough water to add diluted peroxide to the spawn after it had been pressure-cooked and cooled. This is an awkward procedure at best, so I sought alternatives. My search lead to the development of "10 minute spawn," a form of sawdust-paper pellet spawn that only requires a ten minute steaming and no pressure cooking at all. This is probably the fastest method yet in existence for preparing sawdust-type spawn. In this "one step" procedure, all the solid ingredients are placed in a jar, then peroxide is added with all of the water, and the spawn medium is briefly steamed and cooled. Enough peroxide evidently survives the brief steaming to keep the spawn contamination-free.

Here's the recipe for making "ten minute spawn:"

Wood fuel pellets - 1.5 oz (42.5 gm, or about 4 tablespoons)
Paper fiber pellets - 3 oz (85 gm, or about 1/2 cup plus 1 tbs)
Ground limestone - 0.015 oz (0.4 gm, or about 1/4 teaspoon)
Gypsum (optional) - 0.015 oz (0.4 gm, or about 1/4 teaspoon)
Nitrogen supplement - 2% by weight (see below) - (usually about 3/4 tablespoon total)
Hot water - 150 mls, mixed with 3% hydrogen peroxide - 20 mls
Jar with lid containing fitted cardboard disk (see below under "Spawn Containers")

1) Place into a 26 oz or similar size jar the wood fuel pellets (ordinary sawdust will NOT work), paper pellets (Crown™ Animal Bedding or Good Mews™ Cat litter, etc.), lime, gypsum (optional) and nitrogen supplement (see below). The wood fuel pellets must be made of a relatively light wood, such as cottonwood or fir, so that they disintegrate easily as well as heat up and cool down quickly. Fresh ground oyster shell lime will substitute for limestone.
2) Add the hot water with peroxide to the jar, and mix slightly.
3) After allowing a few minutes for the liquid to be absorbed and the wood fuel pellets to disintegrate, shake the jar with a temporary lid in place to mix the ingredients, then knock the jar on a padded surface to dislodge substrate from the upper part of the jar back down into the rest of the substrate.
4) Slightly moisten the cardboard disk in the final lid and put the lid loosely in place on the jar.
5) Place the jar in a steamer pot containing about a half inch to an inch of water, cover the pot with a fitted lid, bring the pot to steaming temperature, and let it cook for just ten minutes over a rolling boil. (I want the pot to reach steaming temperature quickly, so I start with hot tap water. The jars sit on a rack that elevates them slightly from the bottom).
6) When the ten minutes are up, withdraw the jar and let it cool rapidly to room temperature.
7) Wet the cardboard disk inside the jar lid with some 3% peroxide by free-pouring a little peroxide into the lid and wiggling the lid to spread the liquid. Pour off any excess.
8) The spawn is then ready to inoculate.

In the above procedure, I mix one part sawdust with about two parts pelletized paper. The pellets allow the spawn to break up on shaking in jars after the mycelium has grown through the substrate. Of course, you can also prepare your sawdust spawn in heat-resistant plastic bags, and then you won't need paper pellets, since you can break up the spawn by manipulating the bag.

Agar colonizes sawdust by itself with difficulty, which is why I have added an additional nitrogen source to my sawdust spawn in the procedure. The standard recipe calls for one part bran for every four parts sawdust, but if you use any such "raw" supplement as bran, you will have to pressure sterilize your spawn after all, to eliminate peroxide-decomposing enzymes. Therefore, I have identified several nitrogen supplements that do not require pressure sterilization.

Two readily available choices are powdered soy milk and powdered cows milk. I have used each of these substances successfully in the above recipe for 10 minute spawn by adding 0.3 oz (or a little less than a tablespoon) to the specified amounts of paper fiber pellets and wood pellet fuel.

Sylvan Corporation sells two processed supplements, one based on denatured soy protein (Millichamp 3000), and the other based on corn gluten (CG60), and these serve the purpose quite well (I add 0.30 oz in the above recipe for "10 minute spawn"). Neither of these commercial supplements decomposes peroxide when the supplement is fresh, although older Millichamp 3000 and a third supplement sold by Sylvan, CS36, does.


Artificial fertilizer can also provide a workable nitrogen source (for example, about 0.1 oz of "Schultz Instant" brand 20-30-20 fertilizer works well in the above recipe). I have used this successfully with both P. eryngii and H. ulmarius. However, be forewarned that mushroom mycelium takes some time to adapt to chemicals such as these, so the growth will start off quite slowly.

Perhaps you don't like the idea of using artificial fertilizer. Well, since human urine contains nitrogen primarily in the form of urea, it can be used as an organic supplement in place of the fertilizer. In that case, you could replace roughly half of the water required with fresh urine.

To use other supplements, the idea is to add enough to bring the percent nitrogen in the spawn medium to about 0.4, or the percent protein to about 2.5. See the section on supplements under bulk substrate preparation for details of making this kind of calculation.

Two final notes on this ten-minute spawn procedure: first, be careful to use clean containers and implements, use only clear water, free of particulate matter, and if you are working in a kitchen, make sure you don't get flour, crumbs, or other organic matter into the jars or the containers you use for weighing out the medium. Also, make sure none of your ingredients (or your cardboard disks for the lids) is so old it has had a chance to get moist and start to decompose. This will introduce live contaminants containing active peroxide-decomposing enzymes. The procedure works because there are no peroxide-decomposing enzymes in any of the ingredients, so you need to ensure that this remains the case.

Second, the procedure also works because the small amount of material I am using for a 26 oz. jar can be heated and cooled quickly, so that some intact peroxide remains after the steam treatment. Larger quantities of spawn will both take longer to heat through and longer to cool, so they will likely require the addition of greater amounts of peroxide to assure that any survives. You will have to perform your own experiments to determine the amount of peroxide to add.

Pressure-sterilized sawdust spawn

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If you are not going to use wood pellet fuel as a source of sawdust, or if you want to use an unprocessed nitrogen supplement like bran, you will have to pressure sterilize your sawdust spawn, and add diluted peroxide to the medium after it has cooled. You'll want to sterilize enough water separately to dilute the peroxide in about one-third to one-half the total water added to the substrate. After measuring out the diluted peroxide you need, pour it into the spawn medium and then shake well to distribute the liquid.

Pressure-sterilized sawdust spawn

If you are not going to use wood pellet fuel as a source of sawdust, or if you want to use an unprocessed nitrogen supplement like bran, you will have to pressure sterilize your sawdust spawn, and add diluted peroxide to the medium after it has cooled. You'll want to sterilize enough water separately to dilute the peroxide in about one-third to one-half the total water added to the substrate. After measuring out the diluted peroxide you need, pour it into the spawn medium and then shake well to distribute the liquid.

Here's the procedure as I used to do it:

1) Add roughly half the total water you'll need to the spawn medium in as many containers as you want to prepare.
2) Measure out and sterilize enough water to add the other half of the total water, with peroxide, to each of the containers later.
3) Dilute your peroxide into the sterile water when it is cool, to make a 1 to 10 dilution (that is, add a volume of 3% peroxide that is roughly a tenth of the total volume of the water). 4) Measure out the individual amounts of water for each spawn container in a graduated cylinder pasteurized with boiling water.
5) Free-pour the measured water into each spawn container (resulting in an additional 1 to 2 dilution, since the containers already contained half of the water) making sure to wipe off drips running down the outside of the cylinder so they won't fall into the spawn during pouring, and mix immediately. The total dilution comes to about 1 to 20, or a peroxide concentration of roughly 0.15%, same as for grain spawn.

Grain Spawn

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Now, if you have decided that you need grain spawn, I have to caution you--especially if you have never made grain spawn before--that making grain spawn can prove difficult even with peroxide addition. This is because the grain available to you locally may carry a high load of endogenous contaminants that cannot effectively be eliminated by pressure cooking.

So, although I have employed lengthy sterilization periods and plenty of peroxide, I have not been able to consistently make contamination-free rye spawn with the rye grain I get locally. Fortunately, I have been able to substitute a grain called soft white wheat. It has a much higher initial moisture content than rye berries (30% vs 8%), but for whatever reason it is much cleaner than the rye I can get. Soft white wheat has worked well for me when I have added a measured amount of hot water and let the grain stand overnight before pressure cooking, or when I have steeped the grain with excess hot water. I get contamination-free grain spawn virtually every time with this grain. Unfortunately, soft white wheat is sometimes unavailable, and store personnel are prone to mix it up with hard red wheat, a low moisture grain which gives me the same problems as rye.

Whatever grain you choose, you'll want to be sure that 1) your substrate is completely sterilized before you add peroxide, and 2) you have removed all traces of medium on the outside of your containers.

Of course, the problem of thorough sterilization also exists in preparing spawn in filtered-air environments. If there are mold spores or bacteria inside the grain kernels or other substrate particles, and these are not killed by autoclaving/pressure cooking, they can germinate and spoil the spawn despite filtered air or added peroxide. With peroxide as well, however, incomplete sterilization means that some peroxide-decomposing enzymes are left in the grain, creating pockets of medium that are unprotected by peroxide.

The second problem also exists in conventional cultivation practice. If traces of culture medium get on the outside of culture containers, these bits of medium can become loci for contaminant growth and spore dispersal. If this happens with peroxide-protected substrate, the culture will often remain clear until it is shaken to distribute the mycelium. But a few days later, the contaminants will bloom, taking advantage of the lack of peroxide protection in the newly multiplied zones of mycelial growth. This problem can be prevented by cleaning reusable containers carefully, both inside and out, before use, and wiping down the outsides of the containers with rubbing alcohol after the spawn has been inoculated.

Here's how I make soft white wheat spawn:

1) I weigh out 7 oz of grain into a 26 oz jar.
2) I then add an excess of hot tap water and a tiny amount of baking soda to offset the acidity of my tap water.
3) Next, I steep the grain at near-boiling temperatures for an hour or two to swell the kernels, draining off the excess water when the grain has about doubled in volume.
4) Finally, I sterilize the jar of grain in a pressure cooker for an hour. The exact length of time you use will depend on your grain and pressure cooker.
5) When the jar has cooled, I add 10 mls 3% hydrogen peroxide (or 20 mls peroxide for every 16 oz grain initially added), then shake well to coat the grain.

One grower adds food color to his peroxide, so that he can see whether he's done a thorough job of distributing the peroxide over the grain. (If your grain clumps significantly, it will be difficult to coat the kernels completely, so take care to adjust your water content, and don’t soak or cook your grain too long). The final peroxide concentration is high, about 0.15%, but mushroom mycelium still grows well, if somewhat more slowly than without added peroxide. (If you make your spawn by adding a measured amount of water, remember to subtract the volume of peroxide from the water you add, to achieve the correct moisture content). You may well be able to get away with adding less peroxide, but if you add less than 20 mls 3% solution for each 16 oz of grain, you will most likely need to dilute your peroxide into a larger volume of sterile water before addition, to assure thorough coating of the grain by the solution. On the other side, you can add up to 40 mls peroxide without seriously affecting mycelial growth in most cases.

Spawn containers

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I grow my spawn in 26 oz pasta sauce jars, since I can get these readily. They have one-piece lids. Quart canning jars will work just as well, especially if you have one-piece lids, but a two-piece lid can be serviceable if you put a slightly oversize cardboard disk inside the lid, so that it holds the top of the lid within the band. Be sure to keep the inside of the lids clean for each use, as well as the lid threads on the jars. Traces of old medium around the mouth of the jar or in the lid can cause major problems. Rusty spots on the insides of the lids can also catch such traces of medium and provide a place for microbial growth.

Note that peroxide addition makes unnecessary the use of lids fitted with microporous filters as were traditionally required. However, the jar lids are a vulnerable area, even with peroxide added to the medium, since you will be shaking the jars to distribute mycelium, and the shaking can bring airborne mold spores that have diffused inside the lid (or bits of mold that have grown in the crevices of a poorly cleaned lid) into contact with the mycelium (which itself is unprotected). To compensate for lid vulnerability, I do the following:

1) I prepare a set of thin cardboard disks cut to fit inside my jar lids (cereal box cardboard works well; just trace a circle around the lid onto the cardboard with a pen, then cut slightly inside the traced circle with a scissors).
2) For "10 minute spawn," I mix the ingredients with a separate lid, then I put the lids with cardboard disks in place just before steaming.
3) As the spawn cools, I open the lids and wet the cardboard disks with 3% peroxide by free-pouring a couple milliliters into each lid. The peroxide-moistened disks then form a barrier to airborne contaminant entry.

For grain spawn or other spawn that requires pressure sterilization, I wrap the disk-containing lids individually in aluminum foil, sterilizing them separately from the jars of spawn, which I sterilize with temporary lids in place. Then, after adding peroxide to the sterilized spawn substrate and shaking it in, I remove the temporary lid and put in place one of the sterile lids with a cardboard disk. I then wet the disk with 3% peroxide solution.

Inoculating spawn

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Sterile containers of spawn medium can be inoculated in a couple of ways. You can cut chunks of mycelium out of agar cultures with a sterile scalpel and drop the chunks into the container. (If you do this, first tip the jar or bag to make the substrate slope to one side, so you can get the chunks of agar a ways down into the substrate, but still at the side of the container where you can check for growth). Or you can shake the container after adding the chunks. I prefer not to shake the container, because the chunks often end up sticking above the medium, unprotected by peroxide, and they are difficult to dislodge by further shaking. With peroxide addition as well, there is no clear benefit from shaking the chunks with the substrate. The small fragments of mycelium that are broken off this way seem to be too small to effectively recover and grow in the presence of peroxide at the high concentration used in spawn medium. Therefore, I drop the agar chunks (three pieces has been adequate for slow growing strains) down into the substrate and close the container. With sawdust spawn of H. erinaceus, I then tap the jar on my counter to pack down the spawn medium around the agar chunks, since the organism seems to prefer a dense, closely-packed substrate.

Note that peroxide-treated spawn medium should only be inoculated with peroxide-adapted mycelium, that is, mycelium that has grown out on peroxide-containing agar. Otherwise, the unadapted mycelium may die off or take a very long time to initiate new growth when confronted with the relatively high concentration of peroxide I have suggested for spawn making. (Peroxide-treated bulk substrate, however, contains a much lower concentration of peroxide, so it can safely be inoculated with spawn that has not been adapted to peroxide.)

My original procedure was to put my inoculated jars inside fresh plastic food storage bags tied closed. (I would do this immediately after wiping down the jars with rubbing alcohol). I used the plastic bags to provide a still-air environment, and to keep out stray fungus gnats. (The bags can be reused, provided they are still clean). Lately, however, I have been incubating the spawn jars without enclosing them in food storage bags, and this appears to work almost as well.

Finally, I make sure the jars are sealed completely and I let the mycelium grow out from the agar for several days. Decomposition of the added peroxide provides oxygen to support mycelial growth up to this stage, and carbon dioxide levels are not yet very high. When I have a halo of growth about a centimeter wide, I then shake the spawn, and this now results in new growth appearing at many new places in the medium within a few days. (Don’t wait too long to shake your spawn, as the amount of peroxide left to protect the mycelium steadily declines as the halo of mycelial growth from the agar chunks grows wider). I used to loosen the lid to the jar slightly after shaking, to allow gas exchange, but I now find this to be unnecessary. The cardboard disk evidently allows enough gas exchange even with the lid tightened down.

The spawn is ready to use when the mycelium has grown through the spawn medium lightly but completely. I usually wait until the mycelium has started to extend about a half a centimeter or more above the top surface of the medium before I use a spawn jar for inoculation.

If you are using spawn bags, rather than jars, the procedure is essentially the same. You won’t have to worry about contaminants entering the bags as they cool--any that do enter will be killed by the added peroxide.

What about using peroxide to make liquid cultures? I have not pursued this possibility, for two reasons. The first is that any method of inoculating a liquid culture is likely to require blenderizing the inoculum (or in some other way breaking up the mycelium), which releases significant quantities of peroxide-decomposing enzymes into the medium upon inoculation. The second reason is that, even assuming the first problem could be overcome, I would still expect the peroxide concentration to decline rapidly in a liquid culture as the intact fungal material with its internal peroxide-decomposing enzymes circulates throughout the liquid. (With solid substrates, the mycelium is confined to one area, and the peroxide concentration in the remaining substrate stays at a desirable level). The decline in peroxide could be compensated for by regular addition of fresh peroxide, but this might require a method of measuring peroxide concentration in very dilute solutions.

Colonization of bulk substrate

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Colonization of bulk fruiting substrates is the third stage of mushroom growing, leading directly to the production of edible mushrooms.

Because hydrogen peroxide solution is so cheap, it is economically feasible to add enough peroxide solution to fruiting substrates to help keep them free of contaminants. And from a technical standpoint, doing so can make it possible to grow a number of wood decomposing mushrooms without having to autoclave or pressure-cook the substrate. However, the substrate chosen has to be one that is devoid of peroxide-decomposing enzymes, and peroxide will provide little or no benefit with substrates that still have a great deal of biological activity, such as compost, or pasteurized straw, or fresh wood chips that have been treated with boiling water.

I have found one fruiting substrate that is excellent for use with peroxide and reasonably availablein the US and many other parts of the world. It is pellet fuel for wood pellet stoves. This substrate comes previously heat-treated, so it will not cause peroxide to decompose, even without autoclaving. As a result, pellet fuel can be conveniently pasteurized for use as a fruiting substrate by adding boiling water, which becomes part of the process of bringing the substrate to the proper moisture content. (When you add boiling water to fuel pellets, they turn back into the sawdust they were originally made from). Hardwood fuel pellets are generally the best bet for most wood-decomposing mushrooms, but pellets made primarily from Douglas fir may work for your strains too. (I suspect that the heat and pressure used in creating the pellet fuel may break down some of the mushroom-inhibiting resins in the fir). Look for a brand of pellets that does not have any additives--that is, plastic binders. Most do not.

Another substrate that I have used with peroxide is recycled pelletized paper fiber. In my area, this is sold as Crown Animal Bedding™, and as Good Mews™ Cat Litter. These products have been sanitized by a double heat treatment (according to the promotional material). The pellets are still about 30% moisture before adding any water. As with the pellet fuel, the material has no residual peroxide-decomposing activity. The drawback here is cost, as the animal bedding is usually priced about three times higher than pellet fuel on a dry weight basis.

If you have another substrate you’d like to use with peroxide, say paper waste or cardboard, but you plan to pasteurize it rather than autoclaving, you will need to be sure it is free of peroxide-decomposing enzymes after pasteurization. You can test it simply by putting a small amount of the substrate in a cup and adding some fresh 3% peroxide solution. If nothing happens right away, let it sit for a while. When peroxide-decomposing enzymes are present in the substrate, the mixture will bubble and froth. If the enzymes are all gone, the mixture will look no different from substrate mixed with water.

Recipes for fruiting substrates vary from one mushroom species to the next. For wood-decomposing mushrooms, most recipes include sawdust (which we will now derive from pellet fuel), at least 1% powdered lime, water sufficient to give a final moisture content of about 60 to 65%, and from 5-20% of the dry weight as some kind of nitrogen rich supplement like rice bran (which provides about 0.1% to 0.4% nitrogen overall).

Higher nitrogen levels in supplemented sawdust generally translate to higher yields of mushrooms, but traditionally, high nitrogen has also translated to greater risk of contamination. With the peroxide method, the danger of contamination may not increase appreciably with higher nitrogen levels. However, to be on the safe side, I seldom raise the nitrogen level above 0.4%.

Wood chips and substrate density

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Traditional recipes often call for wood chips, but I have never included them in my substrate since it would require the inconvenience of pressure-cooking the chips separately and adding them to the pasteurized bulk substrate later. Some growers believe wood chips are crucial for good growth of shiitake. I have not found them necessary for H. ulmarius, P. eryngii, or H. erinaceus. However, for H. erinaceus, I have found it beneficial to gently but firmly compress the sawdust in my cultures after inoculation by pressing with my hands on the outside of the bag, removing the air space (but not the absorbed water) in the substrate. This may serve something of the same purpose as adding wood chips, by creating a substrate of greater density. I can well imagine that an organism like H. erinaceus, which can grow happily through woods as dense as walnut and cherry, might prefer a dense substrate and thus do better on compressed rather than fluffy sawdust. In traditional methods without peroxide in the sawdust, it would not have been advisable to compress the substrate, because of the danger of creating anaerobic conditions favorable to deleterious organisms. With peroxide in the substrate, however, decomposition of the peroxide provides a beneficial level of oxygen even in compressed substrate, thus making it possible to provide the density some of the organisms prefer without inducing anaerobiosis.

Preparing supplemented sawdust with peroxide

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So here’s what to do with the pellet fuel:

1) First, find a container such as a five gallon plastic bucket with a tight-fitting lid, and clean it thoroughly. (For my routine cleaning, I wipe down the inside of the bucket with a scrub sponge and biodegradable dish detergent, then rinse this out.)
2) Next rinse the container and its lid with boiling water -- a teakettle-full should do. From here on out , you will need to avoid touching the inside of the bucket or the rim.
3) Place the loosely-closed bucket on a scale and scoop in about 8.0 pounds dry weight of pellets if you have oak, or 6.0 to 6.5 pounds if you have a lighter wood like fir (six pounds is roughly a gallon of pellets, if you prefer to measure by volume. I use a one quart glass pot that IÕve boiled some water in on the stove to do my scooping, but it doesn't really need to be pasteurized). You will probably have to make your own adjustments for your local pellet fuel, setting the weight you use according to the amount of sawdust you can fit in your bucket along with your supplements and spawn, while still leaving enough room to mix the contents of the bucket efficiently.
4) If you are using a solid, denatured nitrogen supplement like Sylvan's CG60 or Millichamp 3000, it can be added to the pellet fuel at this stage.
5) Add your lime to the pellet fuel. I use Masons' lime, but crushed limestone is easier and safer to handle if you can get it. Don't use oyster shell lime, since it can "spoil," developing microbial growth with the associated peroxide-decomposing enzymes. Also, do not use dolomite lime, which contains magnesium that can inhibit mushroom development. For mushrooms grown on oak pellet fuel, I use 1 oz of Mason's lime (2 oz of limestone) and half that much for lighter pellet fuel such as cottonwood.
6) Boil in a covered pot half the amount of water you want to add to the pellets. (Your water should be clear and free of any obvious particulates. In some cases this may necessitate filtering). I boil about 3.5 quarts (or about 3.5 liters) for 8.0 lbs of the oak fuel pellets. (If you are using a soluble nitrogen supplement such as artificial fertilizer, it can be added to the water before boiling). Fir pellet fuel sawdust is less dense, so I use only about 6.5 lbs of it, boiling 3 quarts of water. You may want to experiment with different moisture contents for the species you are growing. One advantage of adding peroxide to your cultures is that you can add more water than you could otherwise, without developing anaerobic areas in your substrate that might lead to contamination. However, the pellet fuel sawdust tends to clump as more water is incorporated, which makes it harder to pour into the bags later on without spilling).
7) When the water has boiled for a minute, set the lid of the bucket carefully to one side and pour the boiling water over the substrate. Seal the lid and mix the substrate by turning the bucket for a couple of minutes to distribute the water.
8) Boil in a separate covered pot the other half of the water you want to add to the pellets. When the water has boiled for a minute, turn off the heat and set this pot of water aside to cool with its cover in place. You'll use this water to add peroxide later.
9) Set the bucket of substrate aside to cool, with the lid in place. Cooling usually takes several hours. The bottom of the bucket can still be somewhat warm to the touch at the time of peroxide addition.
10) With a boiling-water-rinsed measuring cup, add about 1/2 cup of 3% peroxide solution to the pot of cooled, boiled water you've set aside.
11) Pour the peroxide mixture into the cooled bucket of substrate and mix thoroughly by turning the bucket. This gives a final peroxide concentration of about 0.03%, or a one to one hundred dilution.
12) Let the substrate finish cooling to room temperature. It is now ready to use.

Perhaps you are wondering at this point whether this procedure can be simplified along the lines of the Ten Minute Spawn procedure. If the peroxide concentration were raised to compensate for decomposition in the hot substrate, maybe peroxide could be added at the beginning of the procedure with all of the water. This may indeed prove possible with a high enough initial peroxide concentration. However, the substrate takes more than twice as long to cool when all of the moisture is added initially as boiling water. Under these conditions, I suspect the peroxide will have a difficult time surviving the exposure to heat, even if the initial concentration is raised several fold.

Nitrogen supplements for bulk substrate

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If you are using traditional nitrogen supplements like millet or rice bran, you will have to pressure cook them. While still hot, the sterilized supplement gets poured into the cooling pasteurized substrate. Be careful to wipe drips off the outsides of the jars before pouring.

Most of the traditional nitrogen supplements for mushroom culture require pressure cooking to eliminate the endogenous peroxide-decomposing enzymes before pasteurization. (These enzymes are remarkably stable, and standard pasteurization procedures are generally not enough to inactivate them, even with delicate supplements like bran). However, as I discussed in the section on making sawdust spawn, I have now found a few supplements that are free of enzymes and so can be added without pressure sterilization, in this case mixed in with the wood pellets. Two of these are commercially manufactured nitrogen supplements already in use in the Agaricus mushroom industry (Sylvan’s Millichamp 3000 and CG 60). They contain denatured soy protein and corn gluten, respectively, and evidently the denaturing process destroys the peroxide-decomposing enzymes. These supplements are an excellent value, but home hobbyists may have difficulty obtaining them. Also, care must be taken to keep them from spoiling in storage, especially with Millichamp 3000.

Some more expensive forms of processed protein are more readily available, such as Texturized Vegetable Protein, powdered soy milk, or powdered cows milk.

Another type of supplement that can be used without pressure sterilization is simply chemical fertilizer, such as a standard 20-20-20 fertilizer. Since these fertilizers do not come from living organisms, they contain no peroxide-decomposing enzymes. Nevertheless, for the most part the nutrients they contain can be utilized by mushroom mycelium after a period of adaptation. If you are going to try this method of supplementation, I recommend that you prepare sawdust spawn using the same supplement, so that the adaptation period will have already taken place by the time you inoculate your bulk substrate. Also, you will get a chance to see how the particular fertilizer you have chosen will work for the organism you are growing. Fertilizer formulations vary quite a bit, even with the same NPK rating, so it is probably advisable to test your selected fertilizer with a small culture before going on to bulk substrate.

Urea is a common source of nitrogen in the formulations for chemical fertilizers, and it probably can also be used by itself as a supplement without pressure sterilization.

If you want something more "organic" than artificial fertilizer (and there is good reason to avoid dependence on substances which require energy from petroleum for their manufacture), human urine and animal urine can also serve as supplements that don’t need pressure cooking. However, they must be kept relatively free of micro-organisms until use. Addition of peroxide provides one way to do this.

Calculating how much supplement to add

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How do you calculate how much of the various supplements to use? Calculations are only approximate, and you will ultimately need to make decisions based on your actual yield of mushrooms at various levels of supplementation. But you can get an idea of what you'll need by consulting Stamets's Growing Gourmet and Medicinal Mushrooms, where the appendices reveal that rice bran has an NPK rating of roughly 2-1.3-1. So, if substrate would ordinarily get supplemented with 5 to 20% rice bran, which Stamets suggests, then a 20-20-20 fertilizer, which has 10 times as much nitrogen as rice bran, would be added at one tenth the rate of rice bran, or 0.5 to 2% of the dry weight of substrate. If you were adding one pound of rice bran to a bucket of pellet fuel, then you would add 1.6 oz of 20-20-20 fertilizer instead, that is, one tenth of a pound. With the commercial supplements, you will need to find out from the manufacturer what percentage nitrogen the material contains, and divide that number into 2.0 to learn what fraction of the amount of rice bran you would add. Sylvan's Millichamp 3000, made from soy, is about 7.3% nitrogen, so it will need to be used at about one quarter the rate of rice bran.

You can also directly calculate the amount of the material needed to give 0.1% to 0.4% nitrogen in the final substrate, without reference to the amount of rice bran used by Stamets:

1) Divide the percent nitrogen in the supplement by the final percent nitrogen desired in the substrate.
2) Divide the previous number into the total weight of substrate to be supplemented to get the weight of supplement to be added.

Thus, to get 0.2% final nitrogen (which requires a supplementation rate of roughly 10% of the dry weight of substrate with rice bran), how much soy flour would we need to add? If the soy flour is 7.6% nitrogen, 7.6 divided by 0.2 gives 38. If the total weight of substrate is 6.5 pounds, then 6.5 divided by 38 gives 0.17 pounds, or 2.72 oz soy flour to be added.

A note on measuring pH of substrate

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I use ColorpHast strips with a pH 4 to 10 range to measure the pH of all my media and substrates, aiming for a pH at make-up in the 6-7 range in most cases. ColorpHast strips are inexpensive and convenient, and with a three-color comparison, I am usually confident of my reading. However, it is not a good idea to try to measure the pH of medium with a color indicator strip once peroxide has been added, as the peroxide may change the chemistry of the indicator. With agar cultures and spawn, you can easily measure the pH before sterilization. With pellet fuel, use a small scoop such as a measuring cup (one that has been rinsed with boiling water) to take a small amount of substrate out of the bucket after adding and mixing in the boiling water plus lime. You can then use your color indicator strips to measure the pH of the removed substrate. Be aware, however, that the reading will only give you a relative idea of the pH eventually experienced by the mushroom mycelium if you have added granular lime which dissolves only very slowly. Addition of Mason's lime (CaOH, available in large sacks as "builders’ lime" from construction supply stores) gets around the latter problem, as it dissolves and reacts much more quickly, but with some mushrooms there may be an advantage to providing a "delayed release" source of alkalinity such as granular lime provides. Then you will simply have to calibrate the amount of lime you used against the ultimate yield of mushrooms to determine the optimal dose.

Culture containers

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Traditionally, sawdust cultures have been grown in special plastic bags with microporous filter patches, to allow gas exchange without letting contaminants gain entry. With peroxide in your fruiting substrate, however, you should be able to use ordinary trash bags (at a savings of $.50-$.80 per bag) to grow your mushrooms. Evidently the process used to produce trash bags pasteurizes them to the point that they do not harbor significant live-organism contamination. If you do use plastic trash bags, I recommend using the kind that are made from high density plastic, 0.5 mil thickness or less. These bags are thin enough that oxygen can diffuse through them, so that the cultures can be grown to maturity with the bags sealed closed by twist ties. Also, the thicker, softer bags are apparently made from PVC, which can leave estrogenic residues in the mushroom cultures, and certain of these softer bags are impregnated with fungicides.

Unless you use traditional gussetted mushroom bags, you will need to put your bags inside containers of an appropriate size to provide a form for the substrate. Small boxes which will hold 5-6 pounds of substrate can often be scavenged from health food stores or the like, or you can buy plastic containers such as nursery pots.

Disposable bags create considerable waste for the landfill. An alternative is to use plastic buckets with lids, 2 or 3 gallon in size, preferably HDPE plastic, recycling number 2. (Five gallon buckets are easier to come by, but they are a little too big for your average sawdust culture). These can be cleaned with detergent and reused after rinsing with boiling water. . If the lids are left slightly loose during the spawn run to allow gas exchange, the buckets are excellent culture vessels for mushrooms species that fruit vertically, such as P. eryngii. H. erinaceus will also grow in them if you open the bucket and turn it on its side when fruiting time comes. (I fill the bucket only about a third to half way full with substrate, so the upper part of the bucket provides a moisture barrier). H. ulmarius would be a little cramped for space in one of these buckets, unless you were to fill substrate nearly to the top, so that the mushroom clusters could grow out above the rim. To get a second flush, then, you would need to take the round block out of the bucket and turn it upside down, since H. ulmarius doesn’t like to fruit from the same surface twice.

Inoculating supplemented sawdust

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Inoculating supplemented sawdust

I prepare spawn for inoculation in the traditional way:

1) The day before I want to use the spawn, I break up the spawn by whacking the jar against something hard protected by something soft.
2) When the particles are separated, I put the jar back on my spawn shelf and incubate overnight to give the mycelium a chance to put out some new growth. This makes a considerable difference in how fast the mycelium will surge into the new substrate. If I am working with grain spawn, it also gives me a chance to see bacterial contamination in the form of "wet" or greasy looking grain kernels that havenÕt acquired a new fuzz of mycelium. When your spawn has been grown using peroxide, the presence of two or three wet kernels will probably not interfere with the subsequent success of your colonization of bulk substrate, since the bacteria that are able to survive peroxide exposure are generally fairly benign organisms when present in small quantities. However, if you have quite a few more wet kernels than two or three, the bacteria will likely slow the colonization of bulk substrate substantially, which then may give a chance for mold to gain entry. So you will probably want to discard spawn with any significant quantity of wet kernels.
3) I inoculate the pellet fuel sawdust by breaking up the spawn briefly, then pouring it directly into the 5 gallon bucket with the substrate. I close the lid and mix everything together by rotating the bucket.
4) I pour the mix into bags. Each bag gets opened up and set inside of a box of the appropriate size to receive the substrate.
5) When I have filled the bag to the capacity of the box, I close the lid on the remaining inoculated substrate, and taking care not to touch the inside surface of the bag, I shift the bag a bit to fill any gaps, then twist the mouth of the bag closed and seal it with a twist tie.
6) Lastly, I compress the sawdust by pressing down on the bag, gently but firmly. I find that this speeds growth of some cultures, especially with a light sawdust like fir or cottonwood.

After labeling, the box is ready to incubate, and from here on out, I follow standard mushroom-growing procedures. You can use the resulting blocks of mycelium either directly for fruiting mushrooms, or the blocks can serve as spawn for inoculating logs or beds of fresh wood chips outdoors.

Growing Mushrooms on Straw

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A number of mushrooms will grow on straw, and straw is the traditional substrate for oyster mushroom cultivation. The standard way to prepare straw for mushroom growing is to steep it in water heated to 180 degrees F for an hour, then drain, cool it down and inoculate. However, without some specialized equipment, it can be rather awkward to heat any significant amount of straw in hot water, and it is often difficult to tell whether the straw has cooled sufficiently for inoculation unless you have a compost thermometer. So, I prefer to use a cold water hydrated lime soak. Hydrated lime (calcium hydroxide or Mason’s lime, available at builder’s supply stores) is a caustic powder which creates a strongly alkaline solution in water. The alkali both pasteurizes the straw and wets it by penetrating the waxy coating on the straw. When the solution is drained off, the liquid that remains behind on the straw gets converted by carbon dioxide in the air to ordinary calcium carbonate lime, which is alkaline but no longer caustic.

I have a large plastic tub that will take a quarter of a bale of straw. With the straw in place, the tub then takes 25 gallons of water to fill to the rim. I use a one half percent solution of hydrated lime, which means one pound of lime in 25 gallons of water. I mix a slurry of hydrated lime in a separate bucket, then pour it into the tub in stages as the tub is filling with water, to assure thorough mixing. (Caution: hydrated lime will burn skin and eyes. Be sure to wear gloves and eye protection when handling the powder or the solution. Also, store the powder in an airtight container to maintain its potency). I let the straw soak for about 16 hours, then drain it for two hours. You can probably get away with using half as much lime, but then it may prove wise to soak the straw somewhat longer, say 24 hours instead of 16.

I inoculate the straw by filling a plastic trash bag, draped inside a large cardboard apple box. I put in a layer of straw, then a sprinkling of spawn, then another layer of straw, then more spawn, and so forth. Finally I close up the bag and compress the straw by pushing the bag and its contents down into the box as far as I can get it to go. A quarter of a bale of straw fills three apple boxes worth. The bags get loosely twisted closed and covered with old bed sheets to incubate for about a month.

On hearing about the peroxide method, many mushroom growers have the thought that peroxide could be used to replace the cumbersome hot water steeping or steaming required to pasteurize straw for oyster mushroom cultivation. Unfortunately, peroxide cannot be used by itself to pasteurize straw. This is so because the many microscopic and macroscopic particles of live mold in straw are highly resistant to peroxide, and also because straw contains a large amount of active peroxide-decomposing enzymes.

Mushroom formation

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For most commonly cultivated mushroom species, mushroom formation begins soon after the cultures are shifted to cooler temperatures, given more light, and given more fresh air, provided the substrate has been thoroughly colonized. There is not much need for hydrogen peroxide during this phase, since the mycelium is well established.

The precise procedures for inducing mushroom formation differ from one species to another, and it is beyond the scope of this manual to review them all, but I will give guidelines for my favorite species. Two of the mushroom species most familiar to me are also among the easiest to fruit: Hypsizygus ulmarius (the White Elm mushroom) and Hericium erinaceus (the Lyon’s Mane or Pom Pom mushroom). Many oyster mushroom species follow similar procedures to that required by H. ulmarius. The other species most familiar to me, Pleurotus eryngii (King Oyster) and Agaricus subrufescens (almond mushroom) follow a different fruiting pattern. Shiitake follows yet another.

Most of the "easy" mushroom species are ready to fruit when the bulk substrate is thoroughly grown through. Often the blocks look white at this time, rather than the original brown of the substrate. How long it takes for a culture to reach maturity depends on the organism, the substrate, and the incubation temperature. Hericium can take as little as 2-3 weeks to form small white, globular fruiting initials on the upper surface of the block, but I like to wait until a month has elapsed before opening the bags. H. ulmarius takes about 5 weeks on fir sawdust or straw and six weeks on oak sawdust (at ordinary room temperature), after which small clusters of pinhead primordia will begin to form spontaneously. By cutting an "X" or a single slit with a clean knife through the bag on the side of the block, H. ulmarius and various Oyster mushroom species will usually form primordia at the site of the cut within a week or two, and mushrooms will soon develop. Provide mist spray when the mushrooms are about an inch high.

Hericium will also form mushrooms at the site of a cut in the bag, but in the winter I find it easier to grow large fruiting bodies by allowing the organism to fruit inside the bag. Simply turn the block on its side and open the bag a bit, allowing air exchange but still providing a moisture barrier. Fruiting bodies will form at random from the fruiting initials that have already developed.

If you grow only a few blocks of mushrooms, ventilation will not be much of an issue. But with more blocks, the need for ventilation to remove carbon dioxide increases. If your mushrooms are not getting enough air as they develop, they will become deformed. H. ulmarius and other oyster mushrooms, for instance, will grow long stalks and irregular caps when the carbon dioxide concentration is too high. When you have enough blocks to need a fan, then an automatic misting system canÕt be far behind.

Note that if you decide to grow H. ulmarius or another oyster species in your home, you will probably need to take precautions to protect yourself from the tremendous amount of spores produced by these organisms. Harvesting the mushrooms when young can help keep the spore load down. Covering the fruiting cultures with Reemay™ or other row cover material will keep the bulk of the spores within the covering, while allowing gas exchange sufficient for fruiting. However, if you or someone in your family is sensitive to the spores, you may need to acquire an air purifier to eliminate the spores from the air in your living space, or else keep the cultures in an out-building.

The almond mushroom, as well as King Stropharia and Shaggy Mane, and sometimes King Oyster all need something called a casing layer applied to the mushroom culture to stimulate fruiting body formation. Casing is a mixture designed to imitate a moist, friable, loamy soil. It contains microorganisms that promote mushroom formation, and it provides a moisture reservoir for mushroom growth. It generally contains little available nutrition for the growth of mushroom mycelium, and this feature also sends a signal to the mushroom culture to begin mushroom formation. Most casing contains peat moss, and a simple formula I have used for almond mushrooms simply calls for one part peat mixed with one part garden soil, plus a handful of gypsum (calcium sulfate) for two or three gallons of mixture, all moistened until damp but not clumpy. The casing is applied to the top of the mushroom culture to a depth of two inches at the most. Be careful not to tamp it down, as the porous structure is essential for encouraging formation of mushroom primordia.

Peat bogs are an endangered habitat worldwide, so we need to find alternatives to the use of peat in casing. Unfortunately, I don’t have a complete answer for this problem yet. In some cases, soil alone may suffice, or soil plus vermiculite. Vermiculite by itself is a possible alternative (although it will not supply microorganisms). Almond mushrooms and King Oysters do not absolutely require a casing (the casing does tend to accelerate primordia formation with P. eryngii, however), so manipulation of conditions may lead to good fruitings without it for these organisms.

If you do apply a casing, you will then need to wait a week or two for the mycelium to grow up into the casing before mushrooms will begin to form. With the almond mushrooms, this is the time to warm up the cultures (I put my apple-box cultures of almond mushroom on an electric "cat warmer" to be certain they are warm enough). It is also the time to sprinkle the casing lightly with water every couple of days to keep it moist. (With P. eryngii, warming and sprinkling are not necessary). Mushrooms usually begin to form a few days after the mycelium begins to reach the surface of the casing.

Seasonal planning

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I you are only growing a few mushrooms, and you have a cool, insulated indoor space such as a lighted basement, you will probably be able to grow your favorite mushrooms all year. However, if your are growing outdoors, or you are growing a lot of mushrooms indoors (so that you need ventilation from outdoors), you may need to plan ahead to have the appropriate mushroom cultures ready in the right season. I fruit my mushrooms in a basement with open windows and a fan to bring in fresh air, so the mushroom growing gets harder in the hottest part of the summer and the coldest part of the winter. Closing the windows is not an option, as carbon dioxide will build up and inhibit mushroom formation. Heating or cooling the incoming air is certainly possible, but it runs up the energy bill too much for my taste. So all my mushrooms do best in fall and spring.

In the coldest part of the winter, both the temperature and the light levels fall. All the mushrooms take longer to finish colonizing the bulk substrate. P. eryngii fruits with difficulty, and H. ulmarius grows very slowly, producing longer stalks (and deformed caps, if the light and temperature levels are too low). H. erinaceus also grows slowly at this time, but this species still produces normal, but small, fruiting bodies even in very cold weather. Agaricus subrufescens needs warmth, but paradoxically it makes a good indoor mushroom for winter, because it will fruit in heated room and it doesn't need as much air or light as the other mushrooms.

In the hot parts of summer, there is plenty of light, but it can be a problem to initiate fruiting at the higher temperatures. Keeping humidity up can be difficult, too. However, I have had H. erinaceus blocks fruit on a dry compost pile in 90 degree weather; evidently the fresh air initiated fruiting in that case, since my indoor H. erinaceus blocks refused to fruit until the temperature came down substantially. A. subrufescens likes warm weather and tends to fruit as the peak temperatures pass. Ganoderma lucidum also prefers warm weather, as does Stropharia.

Outdoor growing vs. indoor growing

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I grow almost all of my mushrooms indoors. This allows me to grow them year round because of the more moderate temperature, and it also saves me from having to deal with slugs and snails, which love mushrooms and grow in great numbers in my area (actually, I still get a few slugs that manage to climb in my windows, travel down the cement basement wall and across the cement floor, and eventually enter my fruiting cultures). Deer may also eat mushrooms, and fungus gnats cannot be easily controlled in outdoor mushroom patches. So I recommend indoor growing. But if you think you can keep all the competitors away, and you don’t mind the seasonal restrictions, outdoor growing can offer considerably more physical space for your crops, and it avoids the problems associated with mushroom spores. You will just need a shaded area that can be kept moist.

Harvesting

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Knowing when to harvest mushrooms is largely a matter of knowing how large they grow and what changes they go through as they mature. With P. eryngii and H. ulmarius, the uncurling of the margin of the cap is usually a sign that the mushroom has reached maturity, but you will need to correlate this change with the size of the mushroom to be sure. With A. subrufescens, the cap opens and the gills begin to turn reddish in color. With H. erinaceus, small "icicles" form and the mushroom softens.

Most mushrooms are said to be tastier if harvested before they start releasing many spores, although they may still gain more mass if left to grow further. It is certainly true that H. ulmarius is tastier when young, but I haven’t personally made taste comparisons with the other mushroom species I grow.

Trouble shooting

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Despite my use of hydrogen peroxide to protect my mushroom cultures, there have been many occasions when things did not go as I had planned and contamination appeared. Each time, I had to track down the problem and correct my procedure, and each timed I was relieved to learn that the use of peroxide itself was not flawed. The procedures I have described here to the best of my knowledge incorporate everything I have learned from my mistakes and should cover the key points required to produce contamination-free mushrooms with peroxide-based culture. Nevertheless, troubleshooting is an unavoidable part of mushroom culture, and you will have to do it sooner or later.

I always find it discouraging to read through lists of things that can go wrong, so instead, I have created a list of questions that draw attention to different aspects of the culture process for the purposes of troubleshooting contamination problems.

If you are adding peroxide, and you still suffer significant contamination, you might ask yourself some of the following questions:

Is the concentration of peroxide in your stock solution what it should be? Has it been over a month since you measured it?

Is your pressure-cooking equipment functioning properly?

Is steam able to enter your jars and equilibrate (are the lids loose enough? If pressure cooking, do you allow five minutes for steam to equilibrate before putting on the pressure regulator?)

Is your stove element (if electric) heating consistently?

Are you cooking at a high enough temperature and for a long enough time to eliminate resident contaminants and, if necessary, peroxide-decomposing enzymes?

Is your substrate moist enough for steam to penetrate?

Has your substrate or supplement spoiled before use?

Is your peroxide getting distributed evenly throughout the medium?

Is your pH reading accurate? (Peroxide is apparently most stable around neutral pH).

If your fruiting substrate is getting contaminated, is your spawn clean?

If your spawn is getting contaminated, is your inoculum clean?

If you are free-pouring diluted peroxide into your cultures, are there drips that run over unsterilized surfaces before falling into the culture?

Are your petri dishes free of traces of old medium?

If your agar plates are getting contaminated, is the contamination on the surface or within the agar? If it is on the surface, look for a source of contamination external to the agar medium; if it is in the agar, contamination is getting in before or during pouring.

Are you letting your medium or substrate cool sufficiently before adding peroxide?

Is your water clean and free of particulates?

Have you overlooked some source of unsterilized or unpasteurized material that can get into your cultures?

Are you mushrooms getting enough light (but not direct sunlight), fresh air, and humidity to grow to a good size?

Conclusion

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As I reach the end of this manuscript, I am forced to pause for a moment of self examination. I called this volume Growing Mushrooms the Easy Way, and now I have to ask myself whether I wasn't indulging in just a bit of self-serving exaggeration when I chose that name. After all, there are still far more ways for things to go wrong in mushroom cultivation than for them to go right. And I sometimes think it is a wonder indeed that we ever get any of these organisms to respond to our coaxing and produce their delectable fruiting bodies.

Well, it is a wonder. And even with peroxide maintenance of mushroom cultures, the process is far from fool-proof. But I feel gratified that the procedures described here do make it possible for hobbyists with at least a minimal degree of comfort with sterile technique to perform all the steps of gourmet mushroom growing and mushroom culture in an ordinary household, more easily than ever before, with no more special equipment needed for contaminant control than a steaming pot and a measuring pipette. And with the stress taken off of battling contaminants, home growers should be freed at last to focus on the thing that attracts us all to mushroom growing in the first place, the quest for ever more of those beautiful and delicious fungi.



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About the Author

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Rush Wayne holds a Masters degree in Biochemistry and Molecular Biology from Harvard University and a Ph.D. in Biochemistry from the University of California at Berkeley. He was first exposed to the elements of mushroom growing during his graduate work in the 1970’s but did not begin growing mushrooms in earnest until he began to implement the innovations contained in this manual in 1993. Instructions for his peroxide method of growing mushrooms are now in the hands of mushroom growers in over 65 countries around the world.




(Message edited by admin on January 02, 2004)

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