[Psilocybin]Home Mushroom Cultivation With Hydrogen Peroxide

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GrowingMushroomstheEasyWay

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GrowingMushroomstheEasyWay HomeMushroomCultivation withHydrogenPeroxide VolumeI

R.RushWayne,Ph.D.

GrowingMushroomstheEasyWay HomeMushroomCultivationwithHydrogenPeroxide VolumeI Copyright ©2001 R.RushWayne.Ph.D. Allrightsreserved.Nopartofthisworkmaybereproducedorusedinanyformorbyanymeans withoutpermissionoftheauthor.

FirstpublishedasGrowingMushroomswithHydrogenPeroxide,December1996 andas GrowingMushroomstheEasyWay, HomeMushroomCultivationwithHydrogenPeroxide January1998 ThirdEditionrevisedNovember1999 RenamedVolumeI,August2000 RevisedSeptember2001

file://U:\temp\Vol1pnew.html

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VisittheUpdatesPageoftheGrowingMushroomstheEasyWayWebsite (http://mycomasters.com/Updates.html) forperiodiccorrectionsandupdatestothismanual,notesonsourcesofsupplies, andnewsabouttheperoxidemethod.

CONTENTS

Introduction

Preliminaries TheMushrooms Hypsizygusulmarius(Elmoyster) Pleurotuseryngii (King oyster) Hericiumerinaceus (Lion’s Mane) Agaricussubrufescens (Almond mushroom) Lentinulaedodes (Shiitake) Pleurotusostreatus (Oyster mushrooms) Ganodermalucidum (Reishi mushroom) Coprinuscomatus(ShaggyMane) Hypsizygustessulatus (Shimeji) Strophariarugosa-annulata (King stropharia) Agaricusbisporus/brunnescens (button mushroom, Portobello)

EquipmentYouWillNeed SpecializedSuppliesYouMayNeed

TheBasicsonPeroxide

Whatperoxidedoes Biologicaleffectofperoxide Advantagesofusingperoxideinmushroomculture Contaminantresistancetoperoxide

Whatperoxidedoesnotdo

Theneedforcautionwhenexposingmycelium Peroxideisnotasterilantatmushroom-growingconcentrations

Safetyandenvironmentalconsiderationsforhydrogenperoxide Peroxideandthespiritoforganicgrowing


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Lackofeffectofperoxideonsubstrateormushroomcultures

Stability Inpuresolution Athighertemperatures Inthepresenceofperoxide-decomposingenzymes Guardingthepurityofperoxidestocksolution

Variationsinperoxideconcentrationfromcommercialsources Measuringperoxideconcentration Calculatinghowmuchperoxidesolutiontouse

GrowingandMaintainingAgarCultures Preparingagarplates MYAmedium Usethelowesteffectiveconcentrationofperoxideinagar

No-pressureagarmedium Hazardsofagardripsandimportanceofcleanplates Reusingperoxideagarmedium

Acquiringmushroomcultures Importanceofstartingwithgoodstrains CloningMushrooms

Strainstorage . Distilledwatermethod Keepstorageculturesperoxide-free

Inoculatingandhandlingagarcultures Coolinghotscalpels Inoculatingfromstorageculturesorperoxide-freemedium Preventingoccultcontaminationwithbottominoculation:cleaningthemycelium Incubatinginoculatedplatesandstoringuninoculatedplates

MakingMushroomSpawn Economicadvantageofmakingyourownspawn Advantagesofsawdust-basedspawn

"Tenminute"no-autoclavesawdustspawn NitrogensupplementscompatiblewithTenMinuteSpawn ImportanceofcleancontainersinmakingTenMinuteSpawn

Pressure-sterilizedsawdustspawn


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Grainspawn Difficultiesofgrainspawnandpitfallsinspawnmaking

Spawncontainers Inoculatingspawn Agarchunkmethod Useperoxide-adaptedmyceliumforspawninoculation Incubatingandshakingthespawn Liquidculture

Colonizationofbulksubstrate Importanceofchoosingsubstrateslackingperoxide-decomposingenzymes Pasteurizablesubstratescompatiblewithperoxide Recipesforfruitingsubstrates

Woodchipsandsubstratedensity Preparingsupplementedsawdustwithperoxide

Nitrogensupplementsforbulksubstrate Calculatinghowmuchsupplementtoadd MeasuringpHofsubstrate

Culturecontainers Trashbagsasculturecontainers Excludingfungusgnats Plasticbucketsasanalternativetobags

Inoculatingsupplementedsawdust Breakingupspawnforinoculationintofruitingsubstrates Addingthespawntothesubstrate

Mushroomformation Generalproceduresforfruitingoyster-likemushroomsandHericium Protectingyourselffromspores Mushroomsneedingacasinglayer

Seasonalplanning Outdoorvs.indoorgrowing


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Harvesting Troubleshooting

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Conclusion AbouttheAuthor

Introduction BacktoContents WhenIfirsttookaninterestingrowingmushrooms,Icheckedoutawell-knownbookonmushroom cultivationfromthelibraryandeagerlyreadthroughit.Butmyinterestsoonturnedtogeneral discouragementasIreadaboutalltheequipmentandproceduresthebookinsistedwerenecessary togrowmushroomswithoutgettingtheculturescontaminated.Iwouldneedasterilelaboratory space with a laminar-flow hood fitted with electrostatic and HEPA filters and an ultraviolet light.Thisspacewouldneedasterileair-lockentrywaywithafootwash,andIwouldneedto havespecialclothing toenter it,sothatIcouldwash downthefloorswith chlorinebleach everyday.Myfruitingmushroomswouldhavetobegrowninaseparatebuildingaltogether,soas toavoidgettingsporesintothesterilelaboratory.Thesefruitingcultureswouldhavetobe growninspeciallydesignedplasticbagswithmicroporousfilterpatchesattached,sothatthe mushroommyceliumcouldgetoxygenwithoutlettingmoldsporesorbacteriagetin.Ofcourse,I wouldneedanautoclaveoratleastaspeciallydesignedpressurecookertosterilizethemedia thatwentintothesebags. Afterconsideringtheserequirementsbriefly,Iputasidethethoughtofgrowing mushrooms.I wasn’tabouttogetallthatequipment,andIfiguredIprobablywasn’tcutoutforthejob anyway.FromwhatIcouldgather,myhousewouldbeadeathtrapformushroomcultures.Neither mywifenorIarecarefulhousekeepers.Wehaveunabasheddustandclutter,andgreenandwhite fuzzy things can be found in and outside the refrigerator. Although I was skilled at sterile techniquefrommyyearsasagraduatestudentinbiochemistry,Ididn’tthinkthatwouldsaveme fromthelegionsofeagercontaminantsthatwouldsurelydogmyeverymovementshouldIattempt togrowanythingsodelectableasmushrooms. Still,thethoughtofgrowingmushroomsdidn’tdisappearentirely.Instead,ayearorsolater, itresurfacedagainintheformofanewidea.Ihadreadthatculturemediausedforgrowing orchidseedscouldberenderedfreeofcontaminantsifhydrogenperoxidewasaddedtothemedium. Whiletheperoxidekilledbacteria,yeast,andfungalspores,itlefttheorchidseedsviable becausetheycontainedenoughperoxide-decomposingenzymestoprotectthemselves.Thentheorchid seedscouldbegerminatedandtendedeasilybyrelativebeginnerswithouttheneedforstrict steriletechnique. Soherewasaquestion:couldaddedperoxidebeusedtokeepmushroom-growingmedia,likeorchidseed media, free of contaminants? If it could, then perhaps mushroom growing could be made accessibletobeginners,justlikeorchidseedgermination.SoIresolvedtotryitwithmushroom mycelium. Whatfollowedwasafairlycomplicatedandnon-linearprocessoflearningaboutgrowingvarious mushrooms,experimentingwithaddinghydrogenperoxide,tryingdifferentconcentrations,learning aboutdifferentculturemediaandhowtheyinteractedwiththemushroomsandtheperoxide,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,tighteningmyprocedures,andonandon,untilIdevelopedsomefairlyreliable guidelinesforwhatIwasdoing.ItalltookfarlongerthanIeverwouldhaveguessed.Butthe upshotofitwasthat,yes,mushroomgrowingcanbemadeaccessibletobeginnerswithouttheneed forsterilefacilities,airfiltration,orevenapressurecooker,ifoneaddshydrogenperoxide


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tohelpkeepthemushroomculturemediafreeofcontaminants.UsingthetechniquesIdeveloped, allthestagesofmushroomculturecan nowbecarriedoutbyrelativebeginners,with awide varietyofmushrooms,andwithoutinvestingasmallfortuneinequipmentandfacilities. Ihavewrittenthecurrentbookasaguideforthehomehobbyistinterestedinmushroomgrowing, 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 possibleforthefirsttimetoperformallphasesofgourmetmushroomcultivationinthehome withoutsterilefacilitiesandairfiltration,thecurrentbookgoesevenfurtherandpresents proceduresthatdonotrequirepressuresterilization. Ofcourse,notalloftheproceduresdescribedinthisbookwerecreatedbyme.Inparticular, anyproceduresnotspecificallyrequiredforusingtheperoxidetechnique,ornotspecifically madepossiblebytheperoxidetechnique,arelikelytohaveoriginatedelsewhere. Forindepthaccountsofthetraditionalmethodsofmushroomcultivation,aswellasthegrowth requirementsforawiderangeofmushroomspecies,pleaseconsultStamets,GrowingGourmetand MedicinalMushrooms,ChiltonandStamets,TheMushroomCultivator,oranotherbasictext.These booksarevaluablereferencevolumesforanyonewhoseriouslywantstopursuemushroomgrowingin detail.Also,asyouglancethroughthesebooks,withpageafterpageofdiscussionofelaborate sterile procedures and sources of contamination, you will enhance your appreciation for the simpletechniquescontainedinthismanual. Notethatsterileoraseptictechnique,whichtheproceduresinthisbookrequiretosomeextent, isalwaysbetterdemonstratedthandescribed.Itismyhopethatthereaderwillseekoutdirect instructioninthisregard.Yourlocalmycologicalsocietymaybehelpfultothatend. Also note that this book is not intended as a guide for commercial cultivation of mushrooms, althoughthemethodsdescribedheremaywellprovevaluabletosmallscalecommercialgrowersas wellastohomehobbyists.

Preliminaries TheMushrooms

BacktoContents Justaboutanymushroomthatiscurrentlycultivatedcanbegrowninthehome.However,someare easiertogrowthanothers,andsome,thougheasy,arenotasrewardingasothersthataremore difficult. Icurrentlyfocusonfourmushroomsinmyownhomegrowing.Theseare: Hypsizygusulmarius, 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 aggressivelyonsawdustorstraw,itrarelyhascontaminationproblemsfollowingthetechniques describedhere,anditdoeswellinavarietyofconditionsandtemperatures,fruitingeither verticallyorhorizontally.Whenwellcultivated,themushroomsarelargeandattractive,rather likestrangewhiteflowers,andtheyareinmyopinionthemostdeliciousoftheoyster-style mushrooms(notcountingP.eryngii). Pleurotuseryngii or King oyster: a member of the oyster mushroom family, but it does not have theusualoystermushroomappearanceorhabit.AnativeofEurope,itgrowsupfromthegroundin a regal stance, rather than on trees and logs. It is large and thick-fleshed. Its substrate requirementsaremorenarrowthanotheroysters,asareitstemperaturerequirements.Ihaveread thatitpreferssawdusttostraw,althoughIhavenotexperimentedenoughwithitonstrawto confirmordenythis.Itfruitsbestinfallorspringtemperatures,growingataglacialpacein


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thecold ofwinter,anddying backinthehotterpartsofthesummer.Itisoneofthemost delicious of cultivated mushrooms if cooked properly, sauteed rapidly in a wide pan, without beingallowedtostewinitsownjuice,thenlightlysalted. Hericiumerinaceus or Lion’s Mane, also called Pom Pom mushroom: a fungus that lacks the stalk andcapofatraditionalgrocerystoremushroom,insteadappearingasakindofsnowballcovered withwhiteicicles.Itgrowsrapidlyonsawdustsubstrates,andfruitseasilyoverarangeof temperatures.Ihaveheardthatitcanbegrownonstrawaswell,althoughIhavenevertriedit. Chefslovethismushroom,andindeedithasadeliciousgourmetflavorsometimestastinglike lobster. Agaricus subrufescens, or almond mushroom: a member of the family that includes the domestic button mushroomandthePortabellas.Itisdistinguished byitsunmistakableflavorofalmond extract.Likethedomesticmushroom,itpreferstogrowoncompost,butitcanalsobegrownon straw,woodchips,orsupplementedsawdust.Itisawarmweathermushroom,butitwillalsofruit indoorsinthewinterinaheatedroom,makingitagoodcandidateforyear-roundcultivation. This mushroom generally needs a casing layer--a layer of friable, soil-like mixture usually containingpeat--appliedtothesurfaceofthecultureinordertoformfruitingbodies. Someothermushroomstoconsiderinclude: Lentinulaedodes or Shiitake: ever popular, grown by many people and written about extensively elsewhere.Iamnotashiitakegrower,butthemethodsofculturehandling,spawnandsubstrate preparation described here will all work for shiitake as well as for the mushrooms that I normallygrow.Besuretogetashiitakestrainselectedforgrowthonsawdustifyoudecideto growthisspeciesusingpelletfuelasyourbulksubstrate.Warmweatherandcoolweatherstrains areavailable. 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.TheywerethefirstmushroomsIfruitedusingtheperoxidemethod.Strainsexistfor mosttemperatureranges.ThesporesofP.ostreatus,whicharereleasedingreatquantityfrom maturefruitingbodies,cancausehealthproblems. Ganodermalucidum or Reishi: a top flight medicinal mushroom with immune stimulating properties. Thismushroomgrowsonhardwoodsawdustinwarmtemperatures.ArelatedspeciesfromthePacific Northwest,Ganodermaoregonense,preferscoolertemperatures.Thewoodymushroomsarebrokenup andmadeintotea. Coprinuscomatus or Shaggy Mane: a mild tasting, short-lived mushroom that grows best on compost. Inevergotittofruitindoors,butafterIdiscardedtheculturesinmyyard,itappearedinmy gardenforacoupleofseasons. Hypsizygustessulatus, 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 initiatefruiting,soIdidn’texperimentmuchwithit,buttherearepresumablyothersthat willfruitwithoutthat. Strophariarugosa-annulata, or King Stropharia: a large mushroom that grows on beds of wood chips orstrawandrequiresacasingandwarmweathertofruit.Themyceliumgrowsslowly,andonlyone varietyiscurrentlyknowntofruitindoorswithoutregardstoseasonoftheyear. Agaricus bisporus/Agaricus brunnescens, or the white button mushroom, also the brown button mushroom,criminiand portobellos:so manybutton mushroomsarenow grownbylargecommercial farmsintheUS,andsoldsocheaply,thatthesecompaniescannolongermakeaprofit.Likethe almondmushroom,thepreferredgrowthsubstrateforbuttonmushroomsandportobellosiscompost. Preparationofqualitycompostisacomplicatedandlabor-intensiveprocessthatisbeyondthe scopeofthismanual.Butbuttonmushroomscanalsobegrownonstrawpreparedbytheperoxide method(seeVolumeII).Theyieldwillnotbeashighasoncompost,butstrawissomucheasier toprepareathomethatyouprobablywon'tmisstheextrayield.Aswiththealmondmushroom,a casinglayerisrequiredforfruiting,butbuttonmushroomsrequirecoolerconditions.Spawncan


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bepreparedwithpressurecookedgrain(thisvolume)orwithsteamedinstantrice(seeVolumeII, "TenMinuteGrainSpawn"). EquipmentYouWillNeed

BacktoContents

Somematerialsandequipment youmayneedfortheperoxidemethod.

Themethodsdescribedinthismanualrequireverylittleinthewayofequipmentforgrowingyour ownmushroomsathome.Handlingandmeasuringhydrogenperoxiderequiresonlyameasuringpipette (10mlvolume)andagraduatedcylinder(probably100or250mlvolume).Thesecanbepurchased fromscientificsupplystores.Tomeasuretheperoxideconcentrationinthebottlesyougetfrom the drug store, you will also need a small test tube with a lip, and a balloon. Preparing mushroomsspawnrequiresjarswithlids(pint,26oz,orquartjars),acoveredpotforsteaming bigenoughtoholdthejars,asmallscaleorbalanceforweighing,andsomeclearplasticfood storagebags.Preparingagarculturesrequiresinadditionasetofpetridishes.Irecommend reusable plastic petri dishes if you can find them. I purchased mine at my local scientific supplystore.Apressurecooker,although notabsolutelynecessary,willbeuseful.Thesecan often be found used at second hand stores that carry kitchen implements, or new in the kitchenwaresectionofhardwarestoresanddepartmentstores.Makesurethecookeryougetis tallenoughtoaccommodateyourjars.YouwillNOTneedaglovebox,HEPAfilters,ultraviolet lights,asterilelaboratory,laminarflowhoods,airlocks,footwashes,etc.etc. Preparingandhandlingbulkpelletfuelsubstraterequiresacoveredpotforboilingandcooling water,asecond potsuchasateapottoboilwaterfor pasteurizing containers,and alarger containersuchasafivegallonplasticbucketwithatight-fittinglid.Fivegallonbucketscan oftenbescavengedorpurchasedcheaplyfromicecreamfactoriesandvariousotherkindsoffood preparationestablishments(avoidbucketsthathavebeenusedforpaint,solvents,orothertoxic substances). Forgrowingoutthebulksubstrate,you’llalsoneedsomesmallboxes(usuallynobiggerthan about500cubicinches,or8"x8"x8")andsomefresh0.5milorlesshighdensitytallkitchen bags(avoidthethickersoftplasticbags),orasetof2to3gallonplasticbucketswithlids. Forhelpingthemushroomsalong,you’llneedahandmister,andacoolspace.Later,ifyouare growingalotofmushrooms,youmaywantafanandanautomaticmistingsystem. SpecializedSuppliesYouMayNeed

BacktoContents Formakingagarmedium,youwillneedagar,lightmaltpowder,and(ifyouplantopressurecook 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 althoughagarbyitselfismoreexpensivethanready-mixedMYAmedium,thelatterisonlyhalfor lessagarbyweight,soitisnotnecessarilyabetterdeal.Maltpowderisavailablefromhome brewsupplystoresorscientificsupplyhouses.Yeastextractflakesareavailablefromhealth


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foodstores. Forspawnmakingandbulksubstrate,youmayneedpaperfiberpelletsandwoodpelletfuel.The paperfiberpelletsaresold inmyareaasCrown ™AnimalBedding orGoodMews™CatLitter. (Checkgrocerystores,petsupplystores,farmandgardensupplystores,etc.)Inruralareasof the U.S. and Canada, wood fuel pellets can be found in grocery stores, hardware stores, farm supplystores,storesthatsellpelletstoves,etc.Inurbanareas,checkyourphonebookfor distributorsofwoodpelletstoves,orcheckwithyourruralfriends.Itmaytakeadriveoutof towntogetsome.Trytofindoutwhatkindofwoodtheyaremadefrom,andlookforhardwoodfor mostmushrooms(firpellets,however,willworkwellforP.eryngiiandA.subrufescens).

TheBasicsonPeroxide Whatperoxidedoes

BacktoContents Theperoxideradicalisareactiveformofoxygenwhichattacksvariousorganiccompounds.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,andsporesoffungi,includingmushroomspores.Itapparentlycanalsokill smallairborneparticlesoffungi,andthecontaminantsassociatedwithhumanskincells,which aresaidtobecontinuallyflakingoffofthemushroomcultivator.Hydrogenperoxidethusactsto someextentagainstallcommonly-encounteredairbornecontaminantsofmushroomculture,including mushroom spores themselves. By contrast, antibiotics generally act only against bacterial contamination,andfungicidesactonlyagainstyeastsandfungi. Thebeautyofperoxideisthatitdoesnotkillestablishedmushroommyceliumorinterferewith itsgrowthandfruiting.Despiteperoxide’swiderangeofactionagainstthecommoncontaminants ofmushroomculture,thereisarelativelywiderangeofconcentrationsatwhichperoxidewill 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 itselfagainstmuchhigherconcentrationsofperoxideradicalthancanisolatedspores,cellsor tinyfragmentsofmulticellularorganisms.Sowecanaddhydrogenperoxidetomushroomcultures, andthemyceliumwillgrowbutthesmallcontaminantswilldie. Thisarrangementhasanumberofadvantages.Mostobviousisthatitreducestheneedforcostly andelaboratefacilitiesandequipmentforenvironmentalcontaminantcontrol.Byaddinghydrogen peroxide to mushroom culture media, it becomes possible to perform all phases of traditional mushroomcultivation,fromisolationtofruiting,successfullyinnon-sterileenvironmentswith unfilteredair.Goneistheneedforspecialcleanrooms,HEPAfilters,pre-filters,laminar-flow hoods,UVlights,airlocks,gloveboxes,oranyotherequipmentassociatedwithenvironmental control of microbial contamination--even microporous filters on bags and jar lids become superfluous. Using peroxide, the equipment minimally required for contamination control comes downtosomemeasuringimplements,asourceofboilingwater,andalargepotforsteaming(ora pressurecookerforaddedsecurity)--littlemoreelaboratethanisfoundinmanykitchens.And whereas the traditional methods of mushroom culture required skillful sterile technique and immaculatepersonalcleanlinessforsuccesswithagarculturesandspawn,useofperoxideallows successwithonlymodeststeriletechniqueandonlyminimalattentiontopersonalhygiene.What’ smore,itbecomespossibletofruitmushrooms--eventhosethathavethehighestsporeload--in thesamebuildingusedtomaintainagarculturesandgrowspawn,withoutthefearthatspores released by the fruiting bodies will diffuse into the agar cultures and ruin them. Hydrogen peroxideuniquelywillkillthesporesoftheverysamemushroomswhosemyceliumitprotects. Docontaminantsdevelopresistancetoperoxide,thewaytheydotoordinaryantibiotics?Yesand no.Manyofthecontaminantsarealreadyresistanttoperoxide,andoncetheyhaveestablisheda colony, they will grow vigorously. Live Aspergillus (blue green) mold is very resistant to peroxide.Butevidentlyperoxideatsufficientconcentrationoverwhelmstheresistancemechanisms of the single-celled organisms and isolated spores, and those of very small, isolated multicellularorganismsaswell.


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Whatperoxidedoesnotdo

BacktoContents One thing peroxide does NOT do is eliminate all need for concern about sterile technique. To repeat,althoughaddedperoxidewillkillisolatedspores,yeast,andbacteriathatfindtheir wayintoyourcultures,andtheseareabigpartofroutinecontaminationproblems,peroxidedoes NOTkillestablishedlivemulticellularorganisms(suchasgreenmold)beyondacertainsize.It also will not do very well against high local concentrations of mold spores. Evidently multicellularorganismsandhighconcentrationsofgerminatedsporesareabletoproduceenough peroxide-decomposing enzymes to defend themselves against high concentrations of external peroxide.Andsincebothmulticellularorganismsandconcentrationsofsporescanbemicroscopic and reside on your hands or on particles of dirt or dust, you still have to take sensible precautionsto keepyourhandsandallnon-sterileparticulatematteroutofyourearly-stage cultures,evenwithperoxideadded.Althoughyoudon’thavetobeafraidtoleaveculturesopen totheairforbrieftimes,toperformmanipulationsorotherwisecheckonthem,you’llstill wanttousecommonsenseinavoidingcontamination.Youwouldn’twanttousethelidtoapetri dishafteryoudroppeditonthefloor,forinstance.Neitherwouldyouwanttoallowvisible, non-steriledebrisofanysorttofallintoyourcultures,orinsectsofanykindtoenterthem. Itisagoodideatoperiodicallywipethedustoffshelvesusedtoincubatecultures.Youwill still need to flame or otherwise sterilize whatever instrument you use to transfer chunks of myceliumfromoneculturetoanother.AndImakeitaregularpracticetowipemyfingerswith rubbingalcoholbeforeperforminginoculationsofspawnoragarcultures.Idothesamewithany countersurfacesIusetoperformmanipulationswithmypetridishcultures.Thisreducesthe chancesoflargerparticlesmakingitintotheculturesandhelpsprotecttheexposedmycelium. ItisalsoespeciallyimportanttoknowandrememberthatperoxidedoesNOTprotectthemushroom myceliumitselffromaerobiccontaminants.Themyceliumdecomposesperoxidethatcomesincontact 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 mediumorsubstratefromaerobiccontamination.Soyourmostcarefulprocedureshouldbereserved fortransferringmycelium,orperforminganyoperationwhichexposesmyceliumtounfilteredair. Andonceyourmyceliumiscontaminated,youwillneedtostartoverwithafresh,uncontaminated culture,orpurifyyourmushroomtissueinsomewaytofreeitofcontaminants.I’lldiscuss thismorelater. Finally, peroxide is not a sterilant except at concentrations too high to use for mushroom growth.Thatis,yougenerallycannotusehydrogenperoxidebyitselftosterilizeculturemedia ormushroomsubstrates.Attheconcentrationsthatarecompatiblewithmushroomgrowth,hydrogen peroxidewillnotkilllivemoldcontaminantsresidentinthemedium,andtheperoxideitself willberapidlydestroyedbytheperoxide-decomposingenzymesinnon-sterileorganicmaterials. Althoughsomesporesandbacteriamaybekilledbyaddingperoxidetonon-sterilemedium,there willbefarmorecontaminantsthatwilleasilysurviveandgrowwithinashorttime.Therefore the general rule is: all culture materials and containers must be pasteurized before adding peroxideorperoxide-containingmediumtothem;culturematerialsthatcontainraw,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 thatwouldnotbedestroyedbypasteurization. Safetyandenvironmentalconsiderationsforhydrogenperoxide

BacktoContents Therearenospecialsafetyprecautionsrequiredforhandling3%hydrogenperoxide.Itstoxicity isverylow,anditdecomposescompletelytowaterandoxygenwhenitisspilledoringested.It isodorlessanddoesnotstainorburn.Itisgenerallynotevenactiveasableachuntilit reaches 60oC, the temperature of very hot tap water. Every evidence suggests that it is environmentallybenign. Sincecommercialperoxideispreparedchemically,ratherthanextractedfromnaturalsources,it


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probablywouldnotbeconsideredcompatiblewithorganiccertificationstandardsfollowingthe criteria currently in vogue. However, I consider the use of peroxide to be in the spirit of organiccultivation.Sincetheperoxideaddedtomushroomculturesdecomposesentirelytowater andoxygenasthemushroommyceliumoccupiesthesubstrate,therecanbenotraceoftheadded peroxideleftinthemushroomcrop,beyondwhatisnaturallythereduetometabolicprocesses. Moreover,hydrogenperoxideitselfisfoundnaturallyinallaerobiclivingorganismsandina varietyofnaturalenvironments.Fromtimeimmemorial,honeybeeshavesecretedenzymeswhichadd 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 producetheirownperoxidetohelpbreakdownthewoodysubstratestheorganismsencounter.And peroxideisevenapartofthehealingdefensesofthehumanorganism.Indeed,aroundtheworld, thousandsofproponentsofasystemofhealingcalledoxygentherapyactuallyingestfood-grade peroxidesolutiononadailybasistocurevariousillsandpromotevitality,andsomepeople havedonesoformanyyears(Idonotnecessarilyrecommendthis,however).Finally,theuseof peroxide circumvents the need for resource-intensive equipment, facilities and supplies, simplifyingeverystageofthemushroomcultivationprocess. Thereissomequestionastotheeffectperoxideoxidationmayhaveonthemushroomsubstrate itself.Chlorine,whenitreactswithorganicmaterialslikepaperpulp,producessmallamounts ofdioxin,averydangerous,cancer-causingchemical.Hydrogenperoxidedoesnotproducedioxin, andasaresult,environmentalistsarecampaigningtogetpapercompaniestobleachtheirpaper fiberwithperoxideratherthanchlorine.Still,itisconceivablethatperoxidecouldproduce someotherharmfulsubstancewhenitreactswiththeorganicmaterialsinmushroomsubstrates.I havenotruledoutthispossibility,butIconsideritunlikely.Foronething,aerobicliving 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 reactionofwaterwithoxygeninresponsetotheultravioletraysinsunlight.Thismeansthat aerobicorganismsmostlikelyhavedevelopedmetabolicmachinerytodealsafelywiththevariety ofoxidation productsthatresultfrom thereactionofperoxidewithbiologicalmaterials.In addition,hydrogenperoxideischemicallyquitestableinsterilizedmushroomsubstrates,andthe concentrationofperoxidewe’reusingissolowthattheamountofsubstrateoxidationgoingon hastobeverylowindeed.Finally,Ihaveseenabsolutelynoevidenceofanymutagenicortoxic effectofperoxide-treatedmushroomsubstrateonthemyceliumorfruitingbodies.Agarcultures containing hydrogen peroxide give fine, healthy halos of mycelium, and the final fruiting culturesproducemushroomsasbeautifulasanygrownbytraditionalmethods. Stability

BacktoContents The3%hydrogenperoxidesolutionavailableatsupermarketsanddrugstores,withphosphoricacid stabilizeradded,isquitestableontheshelfwhenkeptrelativelycool.Whenaddedtoheatsterilizedandcooledmushroomculturemedia,hydrogenperoxideevidentlydecomposesonlyslowly. Preciselyhowlongitwilllastispresumablyacomplexfunctionofmediacomposition,peroxide concentration, and temperature. However, my experience so far is that peroxide continues to excludecontaminantsforlongenoughtoallowthemyceliumofavarietyofmushroomspeciesto safelycolonizetheirsubstrates. Ontheotherhand,hydrogenperoxideshouldgenerallynotbeaddedtohotculturemedia,unless youaregoingtoaddextratocompensateforlossesfromdecomposition.Sinceperoxidebecomes activeasableachabove60oC, it will decompose readily when in contact with complex organic materialsatthistemperatureandabove.Sowaituntilyourmediumhascooled--ifnottoroom temperature, then at least to a temperature that is comfortable to the touch--before adding peroxide. Incontrasttoitsbehaviorinpuresolutionorsterilizedmedia,peroxidebreaksdownrapidlyin thepresenceofperoxide-decomposingenzymes,ashappenswhenyouputperoxideonawound.The brokenskincellsandbloodvesselsofawoundcontainperoxide-decomposingenzymesinabundance, andtheyrapidlybreakdownperoxidesolutionandreleaseoxygenbubbles.Similarenzymes,known ascatalasesandperoxidases,arefoundinallkindsoflivingoronce-livingmaterial,unlessit hasbeenheattreatedorextensivelyprocessed.So,uncookedgrain,flour,sawdust,wood,etc. all will destroy peroxide in short order. This means that you will need to keep all such


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materialsoutofyourstockperoxidesolution.Italsomeansthatifyouwanttoincorporatesuch materialsintoaculturemedium,youhavetobesureeverythinginthatmediumgetsthoroughly heat-treated or cooked clear through to destroy peroxide-decomposing enzymes before you add peroxide. Itakeseveralmeasurestoguardthepurityofmystockperoxidesolution.WhenIamaboutto withdrawperoxide,Ifirstwipedownthelidandupperpartofthebottlewithrubbingalcohol, to keep out particles that might contain live contaminants. Then I either free-pour to a pasteurized measuringvesselorIuseaclean,pasteurizedpipettewith themouth-end plugged withcottontodrawupthesolution.Pipettesdonotneedtobeautoclaved,buttheyshouldbeat leaststeepedinboilingwater(filledsomewhatbeyondthetopgraduation,butbelowthecotton plug) for a few minutes, then cooled, before using them to withdraw peroxide. A one hundred millilitergraduatedcylindermakesaconvenientvesselforsteepinga10mlpipetteinboiling water.Theheatwillkillanyliveorganismsinthepipette,whiletheperoxideitselfwillkill remaining heat-resistant spores. I also take care never to set the peroxide bottle cap down unlessIamcertainitwillnotcontactcontamination. Variationsinperoxideconcentrationfromcommercialsources

BacktoContents Oneannoyingfactoflifewhenusingperoxideisthatthesolutionyougetfromthedrugstoreor supermarket,labeledascontaining3%hydrogenperoxidesolution,U.S.P.,mayormaynotactually containa3%solution.Theconcentrationcanvaryconsiderably,bothaboveandbelow3%.Youcan protectyourselfsomewhatfrombuying"wornout"peroxidebylookingfortheexpirationdateon thebottle,andgettingonewiththelatestdate,ifthereisadateatall.(Thebottlesof peroxideIgetlistonlythemonthofexpiration,nottheyear).However,eventheexpiration dategivesnoabsoluteassurancethattheconcentrationisreally3%.Itisimportant,therefore, tohaveawaytomeasuretheperoxideconcentrationinthesolution.Thiscanbereadilydoneby decomposingasampleoftheperoxideandmeasuringthereleasedoxygen,whichIdowithasimple balloontechnique. Hereismymethodforgettingaroughmeasurementofperoxide: BacktoContents Get a clean test tube (preferably one with a lip or screw cap), a small birthday-party type balloon,andaslice,smallenoughtofitintoyourtesttube,ofthestalkofanymushroomyou havehandy(forbestresults,useayoung,rapidlygrowingmushroomandtakeapieceofstalk, trimming off the natural skin to expose plenty of broken cells). If you don’t have any mushrooms,apieceofbananaorotherskinnedvegetableshoulddojustaswell).Youwillalso needyourperoxidesolution,arubberband,apasteurizedmeasuringpipette,a100mlgraduated cylinder,andapotofwater. 1)Withthepasteurizedmeasuringpipette,withdraw5mloftheperoxidesolutionfromthebottle andtransferitintothetesttube. 2)Placethesliceofmushroomintheupperpartofthetube(don'tletitslipintotheperoxide yet). 3)Makesuretheballoonisemptyofairandstretchthemouthoftheballoonoverthemouthof thetube(tiltthetubetokeepthesliceofmushroomfromslippingintothesolutionuntilthe balloonisinplace. 4)Putarubberbandaroundthemouthoftheballoononthetube,tokeepgasfromescapingas thepressurebuilds(Ihavefounditmosteffectivetouseabrokenrubberbandthatcanbewound tightlyaroundthethreadsofthetube,overthemouthoftheballoon). 5)Oncetheballoonissealedinplace,letthemushroomsliceslipdownintotheperoxide solution.Thesolutionshouldbeginbubblingoxygenimmediately. 6)Agitatethetube.Theperoxidesolutionshouldbelargelydecomposedinfivetotenminutes, dependingontheamountofcatalase/peroxidaseinyourmushroomslice. 7)Whendecompositionisalmostcomplete,you'llseethatthebubblingwillhaveslowedandthe bubbleswillhavebecomequitesmall.Meanwhile,theballoonshouldhavebecometautasitbegan tofillwithreleasedoxygen.


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Testingperoxideconcentration

Now,mycollegechemistrytrainingtellsmethat5mlsofa3%solutionofhydrogenperoxide shouldgenerateabout49mlsofoxygenwhentheperoxidedecomposescompletelyatroom temperatureandoneatmospherepressure.Tomeasuretheoxygenreleasedfromyourperoxide solution: 1)Fillagraduatedcylinderwithwaterandturnitupsidedowninapotofwater,makingsure allbubblesareout. 2)Twisttheballoononyourtesttubetotrapthereleasedoxygen,removetheballoonfromthe tubeholdingthetwisttightly,andputtheballoonunderthewaterinyourpot. 3)Carefullyreleasethegasfromtheballoonupintotheinvertedgraduatedcylinder,displacing thewaterinsideit. 4)Keepingtheopenendofthecylinderunderwater,readthevolumeofoxygenoffthegraduated cylinder.

Measuringreleasedoxygen inaninvertedgraduatedcylinder

ThefirsttimeIdidthis,Igot52mlsofgasinsidemygraduatedcylinderfrom5mlsof peroxidesolution.Giventhattheremaywellhavebeenabout3mlsofairintheflatballoon beforeIstarted,theperoxidesolutionprobablygeneratedprettyclosetothetheoreticalamount ofoxygenfor5mlsof3%solution. Here’showtocalculatetheamountofperoxidesolutionyouwillneed,ifyousolutiontests higherorlowerthan3%:


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BacktoContents 1)Dividethevolumeofoxygenexpectedfor5mlsof3%solution(49mlsiftheballoonis completelyemptytobeginwith,or52mlsintheaboveexample,countingthefewmillilitersof airinitiallytrappedintheballoon)bythevolumeofoxygenyouactuallygot. 2)Multiplythepreviousnumberbythevolumeofperoxidesolutionyouwouldaddtoyourmedium orsubstrateifitwerereallya3%solution(thisvolumeisgiveninappropriatesectionofthis manual,forinstance,inthesectiononagarculture,youwillfindthatyouwouldneedtoadd6 mlsof3%peroxidefor1literofpressure-cookedagarmedium). Knowingthepreciseconcentrationofperoxideismostimportantwhenyouaremakingagarplates (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 substratethanforspawn,butthereisstillsomeroomforvariationthere,aswell.Irecommend youdotheballoontesttocheckeachnewbottleofperoxidesolutionyouuseformakingagar plates,andchecktheperoxideyouuseformakingspawnandbulksubstrateatleastuntilyou knowhowreliableyourlocalproductis.Thatway,youwillknowforsurethatyouaregiving your cultures the protection you expect. Also, you may want to experiment with the peroxide sourcesinyourlocalareatoseewhosellsthemostreliableproduct.Paradoxically,cheapest maybebest,becausetherewillberegularturnoverofthestockwherethepriceislowest.If peroxideisnotreadilyavailableatlocalstoreswhereyoulive,youwillprobablywanttoorder itfromachemicalsupplyhouse.Theywilloftencarry30%or35%solution,whichcanbediluted. Swimming pool supply stores also may carry similar solutions. Note, however, that these concentratedsolutionsareconsiderablymorehazardousthanthestandard3%solution.Readall theprecautionsandwarningsonthecontainerandactaccordingly.

GrowingandMaintainingAgarCultures BacktoContents Thefirststageofmushroomgrowingisthepropagationandmaintenanceofmushroomtissue(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 inoculatethesecondstagecultures,thespawn. Preparingagarplates

BacktoContents There are many recipes for agar medium that can be used to grow mushroom mycelium on petri dishes.Ihavetriedseveralofthese,butIcurrentlyuseonlyone:maltyeastagarmedium,also knownasMYA.ThismediumhasworkedrespectablyforeverymushroomspeciesIhaveattemptedto grow.Itisnotsorichthatitcontaminatesinstantly,yetmoststrainsgrowacrossapetridish ofMYAintwoorthreeweeks.Inmyopinion,ifyouareusingperoxideinyourmedium,thereis notmuchpointtogrowingthemyceliumanyfasterthanthat,sinceitwilljustforceyoutomake upmoreagarplatessooner,tokeepthemyceliumfresh.Also,afterrepeatedlytransferringthe myceliumfromplatetoplate,somegrowersrecommendthatyoustartanewwithmyceliumfroma storageculture,toavoidproblemsofsenescence(aging)ofthemycelium.Thefasterthemycelium grows,accordingtothisview,thesooneronehastogobacktostorage.Ifthisistrue,Iwould justassoonhavethemyceliumgrowrelativelyslowly. Imaintainallmypetridishculturesonperoxide-containingmedium.Contaminationonperoxide platesisrare,aslongasafewprecautionsarefollowed,andyouwon’tneedtobuyalaminar flowhoodorbuildagloveboxtokeepcontaminantsout.Youcanpouryourplatesintheopenair inyourkitchen,andyoucanstoreandincubateyourplatesalmostwhereveryoulike,aslongas thespotisrelativelycleanandtheenvironmentiscompatiblewithmycelialgrowth.However,see myrecommendationsattheendofthissection. MYAMedium


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BacktoContents HereistherecipeIuseforoneliterofMYAmedium: 12gms(0.42oz)agar 12gms(0.42oz)lightmaltpowder 1gm((0.035oz)nutritionalyeastpowder 0.5gm(0.017oz)grainflour(Irotatebetweenwheat,rye,corn,rice,oats,andmillet) 0.5gm(0.017oz)rabbitfeed(orotheranimalfeedpellets) 4-5gmswoodfuelpellets(0.15oz--thenumberofwoodpelletscanbeincreasedforthosewooddecomposingspeciesthatdopoorlyonagar) 1litertapwater Ifyoupurchaseready-mixedMYAmediumfromamushroomsupplyhouse,itwillprobablyonly containthefirstthreeingredients:agar,malt,andyeast.(Youcanaddtheothers).Checkthe instructionstoseehowmuchofthepowderthemanufacturerrecommendsyouuseperliterof water.Usuallyitwillbesomethinglike40to50gms.Dependingontheproportionofagarto maltpowder,youshouldbeabletocuttherecommendedusageinhalfandgetamediumthatis actuallybetterforthelongtermhealthofyourmushroomcultures. Ipreparetheagarmediumforplatesasfollows:

1)Iaddalltheingredientstoajarwiththedesiredamountofwater.Thejarshouldholdabout twicethevolumeyouwillactuallyuse,tokeeptheagarfromboilingoverwhenitcooks. 2)IadjustthepHwithalittlebakingsoda(mywaterisacidic,butyoucouldusevinegarif yoursisalkaline.Also,seemy"NoteonMeasuringpHofSubstrate"belowinthesectionon preparingbulksubstrate). 3)Ithenusemyordinarykitchenpressurecookertomeltandsterilizethemedium.(Iusetap waterandhavenothadanyproblemswithit.Infact,whenIgrewmushroommyceliumonmedium preparedwithdistilledwater,growthwasnoticeablyslower).Iputlidslooselyinplaceand pressurecookat15psifornomorethan10minutes,allowinganinitialtenminutesofsteaming tomelttheagarbeforeputtingonthepressureregulator.(Ifyouareusingready-mixedMYA medium,theinstructionsmaysaytopressurecookformuchlongertimes,forexample,45minutes. Don'tdoit!20minutesisthemostyou Õllneed,andanylongerwillproducecarmelization productsinthemediumthatareharmfultothemycelium).Ialsosterilizeasetofpetridishes alongwithmymedium,placingthedishesinalargetomatocancoveredwithaluminumfoil(Iuse plasticreusablepetridishes,andaliterofmediumfillsupabout30plates). 4)Whenthecookingisfinished,Igentlyslidethecookerofftheburnerandallowthepressure todrop.Icarefullyremovethejarcontainingthemediumandletitcoolintheopenaironmy countertop.Thereisnoneedtoavoidentryofunsterilizedair,assumingthereisnotagreat dealofheavydust,sincetheperoxidewillkilltheairbornecontaminantswhenitisadded. 5)WhenIcanhandlethejarquitecomfortably,Iusuallyputthejarofagarinapotofwarm waterforthelastpartofthecoolingprocess,sincetheagarisclosetosolidifyingatthis temperature. 6)ThenIaddmyperoxidesolutionwithapasteurizedpipetteandquicklymixtheperoxideinto themediumbymovingthejarwithacircularmotion,reversingdirectionsacoupleoftimes(but doingmybesttoavoidmakingalotofbubbles,whichwillenduponthesurfaceoftheagar). 7)OnceI'veaddedtheperoxide,Igostraighttomypetridishes,whichIhavesetoutona cleancounter,andIfree-pourthemediumintothedishes,closingthelidsasIfinish.


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PouringmeltedagarintoPetridishes

8)Whentheagarhassolidified,Isetasidetheplatestodryforafewdaysinalightly coveredtray. Tobeonthesafesidewithmyplatecultures,IusethelowestconcentrationofperoxidethatI havefoundeffectiveinagarmedium,whichisabout0.018%,or6mlsperliterofmedium.(You canaddtwicethismuchwithoutvisibleharmtothemyceliumofthespeciesIhavegrown,but note that very slow-growing species such as Stropharia may be more sensitive to peroxide exposure.Theproductionofprotectiveperoxide-decomposingenzymesseemstoberoughlyparallel to the rate of growth of the organism). When the plate is inoculated, the concentration presumablybeginstodropslowlybelowtheinitiallevelastheperoxideisdecomposedbythe spreading mycelium. Eventually, the peroxide should disappear completely when the agar is overgrown,ifnotearlier.Oncethisstageisreached,coloniesofmoldmaybegintoappearat theedgeoftheagarplate.

Nopressureagarmedium

BacktoContents Ifyoudonotownapressurecooker,ordonotwanttouseone,youcanstillmakeserviceable agarplatesbyboiling/steamingtheagarmedium,providedyoualtertheaboverecipesomewhat. You will need to replace the ingredients that contain peroxide-decomposing enzymes with other ingredientsthatarefreeofthoseenzymes.Intheaboverecipe,agar,maltpowder,andpellet fueldonotcontainperoxide-decomposingenzymes,butyeastpowder,flour,andrabbitchowall do.Inordertouseperoxideinouragarmedium,weordinarilyhavetopressurecookthemedium todestroytheperoxide-decomposingenzymesintheseingredients.However,otheringredientscan beusedintheirplace.Theyeastpowderprovidesvitamins,sothisingredientcanbereplacedby abitofafreshsyntheticB-complexvitaminpill.Becauseitissynthetic,itwillnotcontain 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 substancesarefreeofperoxidedecomposingenzymes,substanceslikegelatin,soymilkpowder, non-fatmilkpowder,lowsodiumsoysauce,etc.Totestforthepresenceoftheenzymes,mixa littleofthesubstanceinquestionwithsome3%peroxidesolutionandwatchforevolutionof bubbles.Nobubblesmeansyouareintheclear. Herethenisarecipeforoneliterof"Nopressure"agarmedium: 12gmsagar 12gmslightmaltpowder 0.5gmprocessednitrogensource(rotatebetweengelatin,soymilkpowder,milkpowder,low sodiumsoysauce,etc.)


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5-7woodfuelpellets smallchip(0.1gm?enoughtoturnthesolutionslightlyyellow)fromsyntheticBvitamincomplex pill 1literclearwater,freeofparticulates 1)Letyourjarcontainingthismedium(andyourpetridishes,ifreusable)sitinboilingwater inacoveredpotwithaheavylidforaboutforty-fiveminutes,whichallowstimetomeltthe agarandkillanyliveorganismsinthemedium(itmaybeadvisabletosteamreusablepetri dishesevenlonger). 2)Thenremovethejarandletitcool,addingtheperoxideasinthefirstrecipe.Theperoxide will kill any spores remaining in the medium. I add slightly more peroxide to non-autoclaved plates,about8mlsperliterofmedium.IngeneralIfindthatnon-autoclavedperoxideplates contaminate more often than autoclaved peroxide plates, but they still do considerably better thanplatesmadewithoutperoxide. Watchoutfordripsofagarmediumthatlandontheoutsideofyourpetridishes.Iftheseare notcleanedoff,theywillgrowmoldwithinafewdays,andthesporeswilldiffuseintothe platesandstartgerminatingattheouteredgeoftheagar. IfyouareworkingwithreusablepetridishesasIam,cleanthemcarefullyafteryoutakeout theoldagar.Eventhesmallestamountofoldmediumleftinaplate,ifitisnotincontact withtheperoxideinthenewagarthenexttimeyouusetheplate,cangrowmoldandbecomea jumping-offpointforcontamination. A benefit of pouring plates when the agar is so cool, is that there is considerably less condensationontheinsideofthelids,thanifyoupourhot.Thisobviouslymeansthatyouwon’ thavetotakespecialmeasurestogetridofcondensation,suchasshakingoutthelids,or warmingtheplatestoevaporatethedroplets.Andyou’llbebetterabletoseewhatishappening inyourplates.However,theagarsurfacestillneedssomedrying,soIlettheplatessitat roomtemperatureforaday,lightlycoveredwithasheetofwaxpapertokeepdustoff,beforeI usethem.Plateswithagarmediumthathasbeensteamedwillbewetterthanplateswithmedium that has been pressure cooked, because of the lower cooking temperatures and shorter cooking time,sotheywillneedtobedriedforalongertime. Ifyouhaveextraagarmediumafteryourplatesarepoured,theexcesswillremainsterilestored intherefrigerator.Whenyougetaroundtousingit,youcanmelttheagaragain,butnotethat youwillneedtoaddperoxideagain,becausetheheatofmeltingwillhavedestroyedwhatyou addedthefirsttime. Acquiringmushroomcultures

BacktoContents Thereareseveralwaystoacquireacultureofmushroommyceliumtogrowoutonyouragarplates. Sporesfromamushroomcanbegerminatedinnutrientmedium.Tissuecanbecutasepticallyoutof afreshmushroomandcoaxedtosproutmyceliumonnutrientagar.Oramushroomtissueculturecan bepurchasedfromacommercialsupplier,usuallyintheformofanagartubecultureorapetri dishculture. Since peroxide-containing nutrient medium kills mushroom spores, I have not worked with germinatingsporestoacquiremushroomcultures.Instead,Iprefertopurchasetissuecultures fromareputablesupplier.ThewayIseeit,thesupplierhasalreadygonetothetroubleof isolatingamushroomstrainwithdesirablecharacteristics,andbypurchasingatissueculture,I am relatively assured of obtaining a mycelial isolate carrying the same desirable characteristics.Bycontrast,ifyoutrytogrowamushroomstrainyou’veisolatedfromspores orcloninganditdoesn’tfruit,youwon’tknowwhetheritistheconditionsyouareproviding, orthestrainthatiscausingtheproblem.(Sporesarelikeseeds:theymayormaynothavethe samegeneticcharacteristicsastheparent).Andyoucanwasteenormousamountsoftimetryingto fruitaworthlessstrain.Moreover,onceyouworkouttheconditionsforgrowinganisolatein yoursituation,ifyoueverlosetheculture,unlessyoupurchasedfromacommercialsupplier,


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youcan’tgobacktothesamesupplierandgetanother"copy."You’llhavetostartallover andworkouttheconditionsagainforanewstrain. Whenyoupurchaseatissueculturefromacommercialsupplier,itisgenerallyunderstoodthat you will use that culture to grow--and sell if you choose to--spawn, fruiting cultures, and fruitingbodiesofthatmushroomstrain.Itisalsopresumedthatyouwillnottakethatculture anduseittoestablishyourowncommercialstrainbank,sellingagarculturestoothers.Ifyou wanttosellagarcultures,theethicalrouteistoisolateyourownstrainsbycloningfromthe wildorgerminatingspores. Cloningmushrooms

BacktoContents Itcanbefun,regardless,tocloneyourownmushroomculturefromaspecimenyoucollectinthe wild.Perhapsitwillprovideyouwithafirst-ratefruitingstrain.Ifyou’dliketotryit, youwillneedsomenutrientagarplatescontainingperoxide(seebelow),ascalpel,anethanolfueledalcohollamp,andafreshmushroom. Tocloneamushroom: 1)Cleanofftheexternalsurfacesothatthereisnoloosedebris. 2)Breakopenthemushroomcap(orbaseofthestalk)ascleanlyasyoucan. 3)Lightyouralcohollampandflameyourscalpelblade.Thencutoutasmallpieceofclean tissuewithinthemushroomthatdoesnotcontactanyexternalsurface.Thiswillobviouslybe easierwiththick,fleshymushroomsthanwiththinones. 4)Whenyouhavepieceofmushroomonyourscalpel,transferittothemiddleofoneofyour nutrientagarplates.Sincethechancesoffailurearehigh,takeafewmorepiecesifyoucan andtransfereachonetoitsownagarplate. 5)Finally,stacktheplates,wraptheminaclearplasticbag,andsettheminaconvenient placetoincubateatroomtemperature. 6)Mycelialgrowth,ifitoccurs,shouldbecomevisibleinanumberofdays,spreadingoutfrom thechunkofmushroomtissue.Moldorbacteriamaygrowaswell,inwhichcaseyoumayhaveto cutoutabitofcleanmyceliumandtransferittoafreshplate.Ifyoudecidetosubclonethe myceliumawayfrommoldcontaminantsinthisway,besureyoudoitbeforethemoldhasmatured enoughtodarkenincolorandformspores.Otherwiseyouwillsimplybetransferringmoldwith themycelium. Ifyouaretryingtoclonemushroommyceliumfromthewild,rememberthathydrogenperoxidein yourmediumwillnotbyitselfcleanyourmyceliumofresidentcontaminants.Ifyourmaterialis dirty, and you can’t get a piece of clean tissue by breaking open the stalk or cap of the mushroomyouwanttoclone,peroxideintheagarwillnotimprovematters.Itisnotasterilant. However, if your material is basically clean, peroxide in your agar will at least reduce the incidenceofbackgroundcontaminationonyourcloningplates. Strainstorage

BacktoContents Onceyouhaveacquiredamushroomculture,youwillneedawaytosafelypreservesamplesofit forlongperiods,sothatyoucangobacktothesepreservedsamplesifanythinghappenstothe activecultureyou useeveryday.Thestoragemethod Iusesimplyrequiresscrapingabitof myceliumoffanagarplateandtransferringittoascrewcaptubeofsteriledistilledwater (thankstoJoeKishforbringingthistechniquetomyattention).Onceinthedistilledwater, the mushroom mycelium goes dormant and will last indefinitely for some strains (oyster-type mushroomslastonlyaboutayearinmyexperience).Refrigerationisnotevenrequired. Althoughagarslantsarethe"traditional"waytostoreculturesforthosewhodon’thaveliquid nitrogen,slantsdonotpreservestrainsverylong--sixmonthsatbest.


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Now,whenyoumakestoragepreparationsofstrainsyouwanttopreserveforlongtermstorage,I recommendthatyoupreparethesewithoutresorttohydrogenperoxide.Thereasonforthisisthat I don’t really know what long term effects peroxide exposure may have on mushroom storage cultures.Coulditacceleratesenescence?Doesitweakenthestrainsgradually?Aretheregradual geneticchanges?Iamsimplynotinapositiontoruleoutalltheproblemsthatcouldoccurwith all the different species you may want to store. In addition, actively growing cultures are betterabletodefendthemselvesagainstaddedperoxidethandormantstoragecultures,whichmay bemoresubjecttodamage.So,althoughslantsanddistilledwaterstoragetubescaneasilybe preparedwithperoxide,peroxide-freestorageofstrainsisthesafestcourse.Besides,itisso easytopreparegoodcleandistilledwaterstoragetubesbydispensingthedistilledwaterinto thetubes,lightlyscrewingonthecaps,thenpressurecookingforhalfanhour(ifyoudon't haveapressurecooker,Iwouldtryboilingforanhourwithafewdropsof3%peroxide;the peroxide will kill the heat-resistant spores, and then the lengthy boiling will destroy the peroxide).Andunlikepetridishes,screwcaptubescanbeflamedonopeningandclosing,making iteasiertokeepthemsterilewithoutairfiltrationwhileinsertingmycelium.Iwrapthefinal storagetubes,containingmycelium,inclearplastic"foodstorage"bagsbeforeputtingthemina safespotinmybasement. Inoculatingandhandlingagarcultures

BacktoContents Iinoculateagarplatesandslantsbysterilizingascalpelwiththeflameofmyalcohollamp, thentransferringtoeachfreshplateorslantasmallchunkofmycelium-impregnatedagarcut fromaplatethathasahealthyhaloofmyceliumgrowingacrossit.

Transferingmyceliumbywayofanagarchunk.

Whenusingaflamedscalpeltocutoutagarchunks,Ifirstcoolthescalpelbyplungingitinto theagaroftheplatecontainingthecultureIwanttotransfer.(Traditionally,youwouldcool thescalpelbyplungingitintotheagarofthenew,unusedplate.Butthehotscalpelmay decomposesomeperoxideintheplateatthesiteofthecut.Inunfilteredair,thisspotthen mightbecomealocusforcontaminantgrowth,sinceitislessprotected.Thisisnotaproblem withtheplateIinoculatefrom,sinceitwillbediscarded.Butitmaybeaconcernwiththenew plate.SoIcoolthescalpelintheoldplate). 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 mycelialfragmentspickedupbyaninoculatinglooparenotenoughtoestablishcoloniesreadily in the presence of the concentrations of peroxide that are effective against contaminants, especiallywhentheculturehasnotbeengrowingonperoxidepreviously.Themyceliumhasamuch 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 othersharpsterileimplement.(Admittedly,itissomewhatawkwardandsometimesfrustratingto


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digpiecesofagaroutofaslantwithascalpel). Culturesthathavenotbeenexposedtoperoxidemediumpreviouslywilloftenlagatfirst,asthe myceliumadjuststothisnewfeatureofitsenvironment.Sometimesthemyceliumwillappeartobe trying to grow away from the peroxide agar at first. (You may observe similar behavior when transferring from aplatethatoriginallycontainedperoxidebutthathasbeen overgrownwith myceliumforafewdayssothatalladdedperoxidehasdecomposed).Soonerorlater,however,the myceliumwillsettledownandgrownormallyoverthesurfaceofthenewmedium. IhaveneverobservedanyproblemwithmystrainswhichIcouldattributetocontinuousperoxide exposure.Typically,Itransfermystrainsabouttentimesonperoxide-containingmediumbefore returningtoperoxide-freestoragecultures,althoughthechoiceoftentransfersisanarbitrary one,andreturningtostorageculturesmaynotbenecessaryatall. Notethatperoxideprotectsonlytheportionofanagarplatethatdoesnothavemyceliumgrowing on it. The mycelium itself is unprotected, since it decomposes the peroxide as it grows. Therefore,olderplateculturesthathavebeenovergrownwithmyceliumformorethanafewdays haveanincreasedlikelihoodofharboringcontaminants. Preventingoccultcontaminationwithbottominoculation

BacktoContents Itisalsopossibleforcontaminantstoaccumulateonmyceliumifyoutransferitrepeatedlyin unfilteredair,evenifthereisalwaysperoxideintheagarandthemyceliumnevercoversthe entireplate.Althoughyoumayneverseecontaminantsgrowingonthemushroommyceliuminyour plates,invisiblecontaminantswillslowlybuildup.This"occultcontamination"canbeaproblem 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 chancethattheoccultcontaminantscouldbloomwhentheyentertheunprotectedmedium. Toguardagainstthepossibilityofsuchoccultcontamination,Iuseasimpletrick:Iregularly inoculatethebottomoftheagarwhenIdomytransfers(Howoftenyoudothisdependsonhowyou storeyourplates,and for howlong.Thesafestcourseisto perform thisoperation atevery transfer,atleastwiththoseplatesusedfortransferringmyceliumtosubsequentplates.Butyou maybeabletogetawaywithputtingitofffortwoorthreetransfersbeforeitstartsaffecting yoursuccessrate).Iperformthebottominoculationasfollows: 1)Turntheplateupsidedown. 2)Liftonesideoftheplatebottomasifitwerehingedtothetop,andgentlyprytheagar diskoutintothelidoftheplatewithaflamedscalpel.(Ifyouragarregularlytearsorbreaks atthisstep,youwillneedtoincreasetheamountofagaryouaddformakingyourmedium.) 3)Closetheplatebackupuntilyou'reready,thentransferachunkofmyceliumtotheexposed undersideoftheagarwithaflamed,cooledscalpel. 4)Finally,afterinoculatingtheunderside,closetheplate,turnitrightsideupagain,and gentlypry(againwithaflamedscalpel)theagardiskbacktoitsusualpositioninthebottom oftheplate,nowsittingontopofthechunkofmycelium. Thisarrangementforcesthemyceliumtogrowupfromthebottomoftheagarthroughthemediumto thesurfaceoftheplate,leavinganyaccumulated contaminantsbehindin theprocess.Certain strainsmaynotrespondwelltothisarrangement,butsofarIhavenothadanyproblemaslong asthestrainIwasusingwasabletogrowvigorouslyonthemedium.However,becauseofthe amount of manipulation involved, this procedure does carry an increased risk of contamination comparedtosimpletransfers.WhereasIrarelyseecontaminationonperoxideplatesinoculatedin theusualwayuntiltheyareold,Iloseperhapsoneinfiveplatesinoculatedontheunderside oftheagar.Wipingdownyourcountersurfacesandyourfingerswithrubbingalcoholbeforeyou beginmayhelpcutdownonsuchfailures. Atrickybutimportantpointinthebottom-inoculationprocedureistoavoidscrapingbitsof agarontotherimofthepetridishbottomwhenyouclosetheplateafterinoculatingthebottom oftheagar.Bitsofagarthatgetontherim,oroutsideofit,tendtosproutcontaminants


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becauseoftheirproximitytoambientair.Itisalso advisabletouseplatesthathavebeen driedsufficientlytoeliminateobvioussurfacedrips.Iftheagarisstillverywetwhenpried intothelid,itmayleaveenoughmediumbehindinthelidtocausetroublesattheedgesofthe platelateron. 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 procedurebybringingalongtheoccultcontaminantswe’retryingtoconfinetothebottomofthe plate.Toleavethesecontaminantsbehind,excisewedgesonlyfromthetopoftheagar. Oncemyagarplatesareinoculated(Ikeepfouratatimeforeachstrain),Iplacetheminside freshclear-plasticfood-storagebags,whichIclosewithtwistties.Theclosedbagprovidesa still-airenvironmentandhelpskeepoutmaraudingfungusgnatsandmites.Iputthreeorfour petri dishes in a single bag. They then can be incubated anywhere that is convenient--on a bookshelf,inacloset,onacountertop,etc.However,Idonotrecommendstoringplatesinthe refrigerator,becauseofthecondensationthatisproduced,andIalsodon'trecommendincubating platesonashelfaboveaheater,becausetheon-offcyclesofheatingandcoolingwillcause contaminantstobedrawnintoyourplates. Beingabletostorefresh(uninoculated)plateseasilyisoneofthebenefitsofperoxide.Ikeep asetoffreshplatesstoredinacoolspot--again,nottherefrigerator.Liketheinoculated plates,Ikeepthesewrappedinplasticfoodstoragebags.EachtimeIuseoneofmygrowing cultures to inoculate spawn, I also take out one of these fresh plates and inoculate it to replacethecultureI’veused.Atthesametime,Ireplaceanyculturesthatmayhavedeveloped moldcoloniesattheedge.Thisway,thenumberofplatesIhavegrowingremainsconstant,andI rarelyfindmyselfshort.

MakingMushroomSpawn BacktoContents Production of spawn is the second stage in mushroom growing. Spawn is the "starter" used to inoculatebulkfruitingsubstrates,ortomakemorespawn.Traditionally,spawnmakingwasbest left to commercial spawn suppliers, who had the sterile facilities to keep spawn free of contaminants.Withthedevelopmentoftheperoxidemethod,however,spawnmakingisjustanother stepinthemushroomgrowingprocess,andaneasyoneatthat. Beingabletomakeyourownspawnwithoutasterilefacilityhasasignificanteconomicbenefit 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,grainwillcostyouaboutadollarortwo,andtheperoxide--tencents.(Sawdust,if notfree,willcostquiteabitlessthangrain).Andyouwon’thavetospendasmallfortune buildinganairfiltrationset-uporspecialcleanroomsforincubatingthespawn.


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Jarsofperoxide-treated sawdustspawnonabookshelf

Formyownmushroomgrowing,Ihaveswitchedalmostentirelytousingsawdust-basedspawn.With mycurrentmethods,sawdust-basedspawnmediumcanbepreparedmorequicklyandeasilythangrain 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,withalowerincidenceofmoldcontamination,thangrainspawn.Andcontaminationof 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 nutritiontothebulksubstrateasgrain,butitisnotparticularlydifficulttoaddthemissing nutritiondirectlytothesubstratefromothersourcesthatdonotrequirepressurecooking. For most mushroom species, grain spawn is recommended for straw, since the grain adds to the nutritionalbaseofthesubstrate.Andthatgrainhastobepressuresterilized.Still,thereare two good species that will grow well on straw using sawdust-based spawn instead of grain: Hypsizygusulmarius(theelmoyster)andHypsizygustessulatus(Shimeji).SinceH.ulmariusgrows every bit as easily and tastes considerably better than traditional oyster mushrooms of the Pleurotusfamily,Icanseenoreasontoincurtheaddeddifficultyofgrain-spawnmakingsimply togrowoystermushroomsonstraw. TenMinuteSpawn

BacktoContents My own procedure for preparing sawdust-based spawn originally required sterilizing separately enoughwatertoadddilutedperoxidetothespawnafterithadbeenpressure-cookedandcooled. Thisisanawkwardprocedureatbest,soIsoughtalternatives.Mysearchleadtothedevelopment of "10 minute spawn," a form of sawdust-paper pellet spawn that only requires a ten minute steamingandnopressurecookingatall.Thisisprobablythefastestmethodyetinexistencefor preparingsawdust-typespawn.Inthis"onestep"procedure,allthesolidingredientsareplaced inajar,thenperoxideisaddedwithallofthewater,andthespawnmediumisbrieflysteamed and cooled. Enough peroxide evidently survives the brief steaming to keep the spawn contamination-free. Here'stherecipeformaking"tenminutespawn:" Woodfuelpellets-1.5oz(42.5gm,orabout4tablespoons) Paperfiberpellets-3oz(85gm,orabout1/2cupplus1tbs) Groundlimestone-0.015oz(0.4gm,orabout1/4teaspoon) Gypsum(optional)-0.015oz(0.4gm,orabout1/4teaspoon) Nitrogensupplement-2%byweight(seebelow)-(usuallyabout3/4tablespoontotal) Hotwater-150mls,mixedwith3%hydrogenperoxide-20mls Jarwithlidcontainingfittedcardboarddisk(seebelowunder"SpawnContainers") 1)Placeintoa26ozorsimilarsizejarthewoodfuelpellets(ordinarysawdustwillNOTwork), paperpellets(Crown™AnimalBeddingorGoodMews™Catlitter,etc.),lime,gypsum(optional) andnitrogensupplement(seebelow).Thewoodfuelpelletsmustbemadeofarelativelylight wood,suchascottonwoodorfir,sothattheydisintegrateeasilyaswellasheatupandcool downquickly.Freshgroundoystershelllimewillsubstituteforlimestone. 2)Addthehotwaterwithperoxidetothejar,andmixslightly. 3)Afterallowingafewminutesfortheliquidtobeabsorbedandthewoodfuelpelletsto disintegrate,shakethejarwithatemporarylidinplacetomixtheingredients,thenknockthe jaronapaddedsurfacetodislodgesubstratefromtheupperpartofthejarbackdownintothe restofthesubstrate. 4)Slightlymoistenthecardboarddiskinthefinallidandputthelidlooselyinplaceonthe jar. 5)Placethejarinasteamerpotcontainingaboutahalfinchtoaninchofwater,coverthepot withafittedlid,bringthepottosteamingtemperature,andletitcookforjusttenminutes overarollingboil.(Iwantthepottoreachsteamingtemperaturequickly,soIstartwithhot tapwater.Thejarssitonarackthatelevatesthemslightlyfromthebottom). 6)Whenthetenminutesareup,withdrawthejarandletitcoolrapidlytoroomtemperature.


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7)Wetthecardboarddiskinsidethejarlidwithsome3%peroxidebyfree-pouringalittle peroxideintothelidandwigglingthelidtospreadtheliquid.Pouroffanyexcess. 8)Thespawnisthenreadytoinoculate. Intheaboveprocedure,Imixonepartsawdustwithabouttwopartspelletizedpaper.Thepellets allow the spawn to break up on shaking in jars after the mycelium has grown through the substrate.Ofcourse,youcanalsoprepareyoursawdustspawninheat-resistantplasticbags,and thenyouwon'tneedpaperpellets,sinceyoucanbreakupthespawnbymanipulatingthebag. Agar colonizes sawdust by itself with difficulty, which is why I have added an additional nitrogensourcetomysawdustspawnintheprocedure.Thestandardrecipecallsforonepartbran foreveryfourpartssawdust,butifyouuseanysuch"raw"supplementasbran,youwillhaveto pressuresterilizeyourspawnafterall,toeliminateperoxide-decomposingenzymes.Therefore,I haveidentifiedseveralnitrogensupplementsthatdonotrequirepressuresterilization. Tworeadilyavailablechoicesarepowderedsoymilkandpowderedcowsmilk.Ihaveusedeachof thesesubstancessuccessfullyintheaboverecipefor10minutespawnbyadding 0.3oz(ora littlelessthanatablespoon)tothespecifiedamountsofpaperfiberpelletsandwoodpellet fuel. Sylvan Corporation sells two processed supplements, one based on denatured soy protein (Millichamp3000),andtheotherbasedoncorngluten(CG60),andtheseservethepurposequite well (I add 0.30 oz in the above recipe for "10 minute spawn"). Neither of these commercial supplementsdecomposesperoxidewhenthesupplementisfresh,althougholderMillichamp3000and athirdsupplementsoldbySylvan,CS36,does. Artificialfertilizercanalsoprovideaworkablenitrogensource(forexample,about0.1ozof "SchultzInstant"brand20-30-20fertilizerworkswellintheaboverecipe).Ihaveusedthis successfullywithbothP.eryngiiandH.ulmarius.However,beforewarnedthatmushroommycelium takessometimetoadapttochemicalssuchasthese,sothegrowthwillstartoffquiteslowly. Perhapsyoudon'tliketheideaofusingartificialfertilizer.Well,sincehumanurinecontains nitrogenprimarilyintheformofurea,itcanbeusedasanorganicsupplementinplaceofthe fertilizer.Inthatcase,youcouldreplaceroughlyhalfofthewaterrequiredwithfreshurine. Touseothersupplements,theideaistoaddenoughtobringthepercentnitrogeninthespawn mediumtoabout0.4,orthepercentproteintoabout2.5.Seethesectiononsupplementsunder bulksubstratepreparationfordetailsofmakingthiskindofcalculation. Twofinalnotesonthisten-minutespawnprocedure:first,becarefultousecleancontainersand implements, use only clear water, free of particulate matter, and if you are working in a kitchen,makesureyou don'tgetflour,crumbs,orotherorganic matterinto thejarsorthe containersyouuseforweighingoutthemedium.Also,makesurenoneofyouringredients(oryour cardboarddisksforthelids)issooldithashadachancetogetmoistandstarttodecompose. This will introduce live contaminants containing active peroxide-decomposing enzymes. The procedureworksbecausetherearenoperoxide-decomposingenzymesinanyoftheingredients,so youneedtoensurethatthisremainsthecase. Second,theprocedurealsoworksbecausethesmallamountofmaterialIamusingfora26oz.jar canbeheatedandcooledquickly,sothatsomeintactperoxideremainsafterthesteamtreatment. Largerquantitiesofspawnwillbothtakelongertoheatthroughandlongertocool,sotheywill likelyrequiretheadditionofgreateramountsofperoxidetoassurethatanysurvives.Youwill havetoperformyourownexperimentstodeterminetheamountofperoxidetoadd. Pressure-sterilizedsawdustspawn

BacktoContents Ifyouarenotgoingtousewoodpelletfuelasasourceofsawdust,orifyouwanttousean 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


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enoughwaterseparatelytodilutetheperoxideinaboutone-thirdtoone-halfthetotalwater addedtothesubstrate.Aftermeasuringoutthedilutedperoxideyouneed,pouritintothespawn mediumandthenshakewelltodistributetheliquid. Here'stheprocedureasIusedtodoit: 1)Addroughlyhalfthetotalwateryou'llneedtothespawnmediuminasmanycontainersasyou wanttoprepare. 2)Measureoutandsterilizeenoughwatertoaddtheotherhalfofthetotalwater,with peroxide,toeachofthecontainerslater. 3)Diluteyourperoxideintothesterilewaterwhenitiscool,tomakea1to10dilution(that is,addavolumeof3%peroxidethatisroughlyatenthofthetotalvolumeofthewater).4) Measureouttheindividualamountsofwaterforeachspawncontainerinagraduatedcylinder pasteurizedwithboilingwater. 5)Free-pourthemeasuredwaterintoeachspawncontainer(resultinginanadditional1to2 dilution,sincethecontainersalreadycontainedhalfofthewater)makingsuretowipeoffdrips runningdowntheoutsideofthecylindersotheywon'tfallintothespawnduringpouring,and miximmediately.Thetotaldilutioncomestoabout1to20,oraperoxideconcentrationof roughly0.15%,sameasforgrainspawn. GrainSpawn

BacktoContents Now,ifyouhavedecidedthatyouneedgrainspawn,Ihavetocautionyou--especiallyifyouhave never madegrain spawn before--thatmaking grain spawn can provedifficulteven with peroxide addition.Thisisbecausethegrainavailabletoyoulocallymaycarryahighloadofendogenous contaminantsthatcannoteffectivelybeeliminatedbypressurecooking. So,althoughIhaveemployedlengthysterilizationperiodsandplentyofperoxide,Ihavenot beenabletoconsistentlymakecontamination-freeryespawnwiththeryegrainIgetlocally. Fortunately,Ihavebeenabletosubstituteagraincalledsoftwhitewheat.Ithasamuchhigher initialmoisturecontentthanryeberries(30%vs8%),butforwhateverreasonitismuchcleaner thantheryeIcanget.SoftwhitewheathasworkedwellformewhenIhaveaddedameasured amountofhotwaterandletthegrainstandovernightbeforepressurecooking,orwhenIhave steeped thegrain with excesshotwater.Igetcontamination-freegrainspawn virtuallyevery time with this grain. Unfortunately, soft white wheat is sometimes unavailable, and store personnelarepronetomixitupwithhardredwheat,alowmoisturegrainwhichgivesmethe sameproblemsasrye. Whatevergrainyouchoose,you'llwanttobesurethat1)yoursubstrateiscompletelysterilized beforeyouaddperoxide,and2)youhaveremovedalltracesofmediumontheoutsideofyour containers. Ofcourse,theproblemofthoroughsterilizationalsoexistsinpreparingspawninfiltered-air environments.Iftherearemoldsporesorbacteriainsidethegrainkernelsorothersubstrate particles,andthesearenotkilledbyautoclaving/pressurecooking,theycangerminateandspoil 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 pocketsofmediumthatareunprotectedbyperoxide. Thesecondproblemalsoexistsinconventionalcultivationpractice.Iftracesofculturemedium getontheoutsideofculturecontainers,thesebitsofmediumcanbecomelociforcontaminant growthandsporedispersal.Ifthishappenswithperoxide-protectedsubstrate,theculturewill 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 containerswithrubbingalcoholafterthespawnhasbeeninoculated. Here'showImakesoftwhitewheatspawn:


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1)Iweighout7ozofgrainintoa26ozjar. 2)Ithenaddanexcessofhottapwaterandatinyamountofbakingsodatooffsettheacidity ofmytapwater. 3)Next,Isteepthegrainatnear-boilingtemperaturesforanhourortwotoswellthekernels, drainingofftheexcesswaterwhenthegrainhasaboutdoubledinvolume. 4)Finally,Isterilizethejarofgraininapressurecookerforanhour.Theexactlengthof timeyouusewilldependonyourgrainandpressurecooker. 5)Whenthejarhascooled,Iadd10mls3%hydrogenperoxide(or20mlsperoxideforevery16oz graininitiallyadded),thenshakewelltocoatthegrain. Onegroweraddsfoodcolortohisperoxide,sothathecanseewhetherhe'sdoneathoroughjob of distributing the peroxide over the grain. (If your grain clumps significantly, it will be difficulttocoatthekernelscompletely,sotakecaretoadjustyourwatercontent,anddon’t soakorcookyourgraintoolong).Thefinalperoxideconcentrationishigh,about0.15%,but mushroommyceliumstillgrowswell,ifsomewhatmoreslowlythanwithoutaddedperoxide.(Ifyou makeyourspawnbyaddingameasuredamountofwater,remembertosubtractthevolumeofperoxide fromthewateryouadd,toachievethecorrectmoisturecontent).Youmaywellbeabletoget awaywithaddinglessperoxide,butifyouaddlessthan20mls3%solutionforeach16ozof grain,youwillmostlikelyneedtodiluteyourperoxideintoalargervolumeofsterilewater beforeaddition,toassurethoroughcoatingofthegrainbythesolution.Ontheotherside,you canaddupto40mlsperoxidewithoutseriouslyaffectingmycelialgrowthinmostcases. Spawncontainers

BacktoContents Igrowmyspawnin26ozpastasaucejars,sinceIcangetthesereadily.Theyhaveone-piece lids.Quartcanningjarswillworkjustaswell,especiallyifyouhaveone-piecelids,buta two-piecelidcanbeserviceableifyouputaslightlyoversizecardboarddiskinsidethelid,so thatitholdsthetopofthelidwithintheband.Besuretokeeptheinsideofthelidsclean foreachuse,aswellasthelidthreadsonthejars.Tracesofoldmediumaroundthemouthof thejarorinthelidcancausemajorproblems.Rustyspotsontheinsidesofthelidscanalso catchsuchtracesofmediumandprovideaplaceformicrobialgrowth. Notethatperoxideadditionmakesunnecessarytheuseoflidsfittedwithmicroporousfiltersas were traditionally required. However, the jar lids are a vulnerable area, even with peroxide addedtothemedium,sinceyouwillbeshakingthejarstodistributemycelium,andtheshaking canbringairbornemoldsporesthathavediffusedinsidethelid(orbitsofmoldthathavegrown in the crevices of a poorly cleaned lid) into contact with the mycelium (which itself is unprotected).Tocompensateforlidvulnerability,Idothefollowing: 1)Iprepareasetofthincardboarddiskscuttofitinsidemyjarlids(cerealboxcardboard workswell;justtraceacirclearoundthelidontothecardboardwithapen,thencutslightly insidethetracedcirclewithascissors). 2)For"10minutespawn,"Imixtheingredientswithaseparatelid,thenIputthelidswith cardboarddisksinplacejustbeforesteaming. 3)Asthespawncools,Iopenthelidsandwetthecardboarddiskswith3%peroxidebyfreepouringacouplemillilitersintoeachlid.Theperoxide-moisteneddisksthenformabarrierto airbornecontaminantentry. Forgrainspawnorotherspawnthatrequirespressuresterilization,Iwrapthedisk-containing lidsindividuallyinaluminumfoil,sterilizingthemseparatelyfromthejarsofspawn,whichI sterilize with temporary lids in place. Then, after adding peroxide to the sterilized spawn substrateandshakingitin,Iremovethetemporarylidandputinplaceoneofthesterilelids withacardboarddisk.Ithenwetthediskwith3%peroxidesolution. Inoculatingspawn

BacktoContents


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Inoculatingjarsof"TenMinuteSpawn" bytheagarchunkmethod

Sterilecontainersofspawnmediumcanbeinoculatedinacoupleofways.Youcancutchunksof myceliumoutofagarcultureswithasterilescalpelanddropthechunksintothecontainer.(If youdothis,firsttipthejarorbagtomakethesubstrateslopetooneside,soyoucangetthe chunksofagarawaysdownintothesubstrate,butstillatthesideofthecontainerwhereyou cancheckforgrowth).Oryoucanshakethecontainerafteraddingthechunks.Iprefernotto shakethecontainer,becausethechunksoftenendupstickingabovethemedium,unprotectedby peroxide,andtheyaredifficulttodislodgebyfurthershaking.Withperoxideadditionaswell, there is no clear benefit from shaking the chunks with the substrate. The small fragments of myceliumthatarebrokenoffthiswayseemtobetoosmalltoeffectivelyrecoverandgrowinthe presenceofperoxideatthehighconcentrationusedinspawnmedium.Therefore,Idroptheagar chunks (three pieces has been adequate for slow growing strains) down into the substrate and closethecontainer.WithsawdustspawnofH.erinaceus,Ithentapthejaronmycountertopack down the spawn medium around the agar chunks, since the organism seems to prefer a dense, closely-packedsubstrate. Notethatperoxide-treatedspawnmediumshouldonlybeinoculatedwithperoxide-adaptedmycelium, that is, mycelium that has grown out on peroxide-containing agar. Otherwise, the unadapted myceliummaydieoffortakeaverylongtimetoinitiatenewgrowthwhenconfrontedwiththe relativelyhighconcentrationofperoxideIhavesuggestedforspawnmaking.(Peroxide-treated bulksubstrate,however,containsamuchlowerconcentrationofperoxide,soitcansafelybe inoculatedwithspawnthathasnotbeenadaptedtoperoxide.) Myoriginalprocedurewastoputmyinoculatedjarsinsidefreshplasticfoodstoragebagstied closed.(Iwoulddothisimmediatelyafterwipingdownthejarswithrubbingalcohol).Iusedthe plasticbagstoprovideastill-airenvironment,andtokeepoutstrayfungusgnats.(Thebags canbereused,providedtheyarestillclean).Lately,however,Ihavebeenincubatingthespawn jarswithoutenclosingtheminfoodstoragebags,andthisappearstoworkalmostaswell. Finally,ImakesurethejarsaresealedcompletelyandIletthemyceliumgrowoutfromtheagar forseveraldays.Decompositionoftheaddedperoxideprovidesoxygentosupportmycelialgrowth uptothisstage,andcarbondioxidelevelsarenotyetveryhigh.WhenIhaveahaloofgrowth aboutacentimeterwide,Ithenshakethespawn,andthisnowresultsinnewgrowthappearingat manynewplacesinthemediumwithinafewdays.(Don’twaittoolongtoshakeyourspawn,as theamountofperoxidelefttoprotectthemyceliumsteadilydeclinesasthehaloofmycelial growthfromtheagarchunksgrowswider).Iusedtoloosenthelidtothejarslightlyafter shaking,toallowgasexchange,butInowfindthistobeunnecessary.Thecardboarddisk evidentlyallowsenoughgasexchangeevenwiththelidtighteneddown. The spawn is ready to use when the mycelium has grown through the spawn medium lightly but completely.Iusuallywaituntilthemyceliumhasstartedtoextendaboutahalfacentimeteror moreabovethetopsurfaceofthemediumbeforeIuseaspawnjarforinoculation.


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Ifyouareusingspawnbags,ratherthanjars,theprocedureisessentiallythesame.Youwon’t havetoworryaboutcontaminantsenteringthebagsastheycool--anythatdoenterwillbekilled bytheaddedperoxide. Whataboutusingperoxidetomakeliquidcultures?Ihavenotpursuedthispossibility,fortwo 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 secondreasonisthat,evenassumingthefirstproblemcouldbeovercome,Iwouldstillexpect theperoxideconcentrationtodeclinerapidlyinaliquidcultureastheintactfungalmaterial with its internal peroxide-decomposing enzymes circulates throughout the liquid. (With solid substrates,themyceliumisconfinedtoonearea,andtheperoxideconcentrationintheremaining 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 concentrationinverydilutesolutions.

Colonizationofbulksubstrate BacktoContents Colonizationofbulkfruitingsubstratesisthethirdstageofmushroomgrowing,leadingdirectly totheproductionofediblemushrooms. 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 mushroomswithouthavingtoautoclaveorpressure-cookthesubstrate.Fortheproceduresinthis volume, however, the substrate chosen has to be one that is devoid of peroxide-decomposing enzymes.Peroxidewillprovidelittleornobenefitwithsubstratesthatstillhaveagreatdeal ofbiologicalactivity,suchascompost,orpasteurizedstraw,orfreshwoodchipsthathavebeen treatedwithboilingwater. ThefirstmaterialIfoundtobeidealforusewithperoxidewaspelletfuelforwoodpellet stoves.Thissubstratecomespreviouslyheat-treated,soitwillnotcauseperoxidetodecompose, evenwithoutautoclaving.Asaresult,pelletfuelcanbeconvenientlypasteurizedforuseasa fruitingsubstratebyaddingboilingwater,whichbecomespartoftheprocessofbringingthe substratetothepropermoisturecontent.(Whenyouaddboilingwatertofuelpellets,theyturn backintothesawdusttheywereoriginallymadefrom).Hardwoodfuelpelletsaregenerallythe bestbetformostwood-decomposing mushrooms,butpelletsmadeprimarilyfrom Douglasfirmay workforyourstrainstoo.(Isuspectthattheheatandpressureusedincreatingthepelletfuel maybreakdownsomeofthemushroom-inhibitingresinsinthefir).Lookforabrandofpellets thatdoesnothaveanyadditives--thatis,plasticbinders.Mostdonot. AnothersubstratethatIhaveusedwithperoxideisrecycledpelletizedpaperfiber.Inmyarea, thisissoldasCrownAnimalBedding™,andasGoodMews™CatLitter.Theseproductshavebeen sanitizedbyadoubleheattreatment(according tothepromotionalmaterial).Thepelletsare stillabout30%moisturebeforeaddinganywater.Aswiththepelletfuel,thematerialhasno residual peroxide-decomposing activity. The drawback here is cost, as the animal bedding is usuallypricedaboutthreetimeshigherthanpelletfuelonadryweightbasis. Ifnopelletfuelorpaperfiberpelletsareavailableinyourpartoftheworld,youshouldplan to use one of the procedures presented in Volume II for your substrate preparation. Those procedures allow a greater variety of possible materials as substrates, including some that containperoxide-decomposingenzymes.Indeed,oneoftheonlymaterialsthatwillNOTworkwith anyofmyperoxideproceduresisrawsawdust,thatis,thesawdustproducedbythemillingofraw logs. Ifyouhaveanothersubstrateyou’dliketousewithperoxide,saypaperwasteorcardboard,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 amountofthesubstrateinacupandaddingsomefresh3%peroxidesolution.Ifnothinghappens


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right away, let it sit for a while. When peroxide-decomposing enzymes are present in the substrate,themixturewillbubbleandfroth.Iftheenzymesareallgone,themixturewilllook nodifferentfromsubstratemixedwithwater. Recipesforfruitingsubstratesvaryfromonemushroomspeciestothenext.Forwood-decomposing mushrooms,mostrecipesincludesawdust(whichwewillnowderivefrompelletfuel),atleast1% powderedlime,watersufficienttogiveafinalmoisturecontentofabout60to65%,andfrom520%ofthedryweightassomekindofnitrogenrichsupplementlikericebran(whichprovides about0.1%to0.4%nitrogenoverall). Highernitrogenlevelsinsupplementedsawdustgenerallytranslatetohigheryieldsofmushrooms, buttraditionally,highnitrogenhasalsotranslatedtogreaterriskofcontamination.Withthe peroxidemethod,thedangerofcontaminationmaynotincreaseappreciablywithhighernitrogen levels.However,tobeonthesafeside,Iseldomraisethenitrogenlevelabove0.4%. Woodchipsandsubstratedensity

BacktoContents Traditionalrecipesoftencallforwoodchips,butIhaveneverincludedtheminmysubstrate sinceitwouldrequiretheinconvenienceofpressure-cookingthechipsseparatelyandaddingthem tothepasteurizedbulksubstratelater.Somegrowersbelievewoodchipsarecrucialforgood growthofshiitake.IhavenotfoundthemnecessaryforH.ulmarius,P.eryngii,orH.erinaceus. However,forH.erinaceus,Ihavefounditbeneficialtogentlybutfirmlycompressthesawdust inmyculturesafterinoculationbypressingwithmyhandsontheoutsideofthebag,removing theairspace(butnottheabsorbedwater)inthesubstrate.Thismayservesomethingofthesame purposeasaddingwoodchips,bycreatingasubstrateofgreaterdensity.Icanwellimaginethat 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.Intraditionalmethodswithoutperoxideinthesawdust,itwouldnothavebeenadvisable tocompressthesubstrate,becauseofthedangerofcreatinganaerobicconditionsfavorableto deleterious organisms. With peroxide in the substrate, however, decomposition of the peroxide providesabeneficiallevelofoxygenevenincompressedsubstrate,thusmakingitpossibleto providethedensitysomeoftheorganismspreferwithoutinducinganaerobiosis. Preparingsupplementedsawdustwithperoxide

BacktoContents Sohere’swhattodowiththepelletfuel: 1)First,findacontainersuchasafivegallonplasticbucketwithatight-fittinglid,and cleanitthoroughly.(Formyroutinecleaning,Iwipedowntheinsideofthebucketwithascrub spongeandbiodegradabledishdetergent,thenrinsethisout.) 2)Nextrinsethecontaineranditslidwithboilingwater--ateakettle-fullshoulddo.From hereonout,youwillneedtoavoidtouchingtheinsideofthebucketortherim. 3)Placetheloosely-closedbucketonascaleandscoopinabout8.0poundsdryweightofpellets ifyouhaveoak,or6.0to6.5poundsifyouhavealighterwoodlikefir(sixpoundsisroughly agallonofpellets,ifyouprefertomeasurebyvolume.IuseaonequartglasspotthatI Õve boiledsomewaterinonthestovetodomyscooping,butitdoesn'treallyneedtobe pasteurized).Youwillprobablyhavetomakeyourownadjustmentsforyourlocalpelletfuel, settingtheweightyouuseaccordingtotheamountofsawdustyoucanfitinyourbucketalong withyoursupplementsandspawn,whilestillleavingenoughroomtomixthecontentsofthe bucketefficiently. 4)Ifyouareusingasolid,denaturednitrogensupplementlikeSylvan'sCG60orMillichamp3000, itcanbeaddedtothepelletfuelatthisstage. 5)Addyourlimetothepelletfuel.Iusepowderedoystershelllime,butbeforeIuseit,I bakemysupplyat400degreesFforacoupleofhourstoeliminateanyperoxide-decomposing enzymesresultingfrommicrobialgrowthontheshells.Crushedlimestoneisalsoagoodchoiceif youcangetit.Donotusedolomitelime,whichcontainsmagnesiumthatcaninhibitmushroom development.Formushroomsgrownonoakpelletfuel,Iuse2ozoflime,orhalfthatmuchfor lighterpelletfuelsuchascottonwood.


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6)Boilinacoveredpothalftheamountofwateryouwanttoaddtothepellets.(Yourwater shouldbeclearandfreeofanyobviousparticulates.Insomecasesthismaynecessitate filtering)..Forthisstep,Iboilabout3.5quarts(orabout3.5liters)for9.0lbsoftheoak fuelpellets.(Ifyouareusingasolublenitrogensupplementsuchasartificialfertilizer,it canbeaddedtothewaterbeforeboiling).Firpelletfuelsawdustislessdense,soIuseonly about6.5lbsofit,boiling3quartsofwater.Youmaywanttoexperimentwithdifferent moisturecontentsforthespeciesyouaregrowing.Oneadvantageofaddingperoxidetoyour culturesisthatyoucanaddmorewaterthanyoucouldotherwise,withoutdevelopinganaerobic areasinyoursubstratethatmightleadtocontamination.However,thepelletfuelsawdusttends toclumpasmorewaterisincorporated,whichmakesithardertopourintothebagslateron withoutspilling). 7)Whenthewaterhasboiledforaminute,setthelidofthebucketcarefullytoonesideand pourtheboilingwateroverthesubstrate.Sealthelidandmixthesubstratebyturningthe bucketforacoupleofminutestodistributethewater. 8)Boilinaseparatecoveredpottheotherhalfofthewateryouwanttoaddtothepellets (e.g.,another3.5quarts/litersofwater).Whenthewaterhasboiledforaminute,turnoffthe heatandsetthispotofwaterasidetocoolwithitscoverinplace.You'llusethiswaterto addperoxidelater. 9)Setthebucketofsubstrateasidetocool,withthelidinplace.Coolingusuallytakes severalhours.Thebottomofthebucketcanstillbesomewhatwarmtothetouchatthetimeof peroxideaddition. 10)Withaboiling-water-rinsedmeasuringcup,addabout1/2cupof3%peroxidesolutiontothe potofcooled,boiledwateryou'vesetaside. 11)Pourtheperoxidemixtureintothecooledbucketofsubstrateandmixthoroughlybyturning thebucket.Thisgivesafinalperoxideconcentrationofabout0.03%,oraonetoonehundred dilution. 12)Letthesubstratefinishcoolingtoroomtemperature.Itisnowreadytouse. Perhapsyouarewonderingatthispointwhetherthisprocedurecanbesimplifiedalongthelines oftheTenMinuteSpawnprocedure.Iftheperoxideconcentrationwereraisedtocompensatefor 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 peroxideconcentration.However,thesubstratetakesmorethantwiceaslongtocoolwhenallof themoistureisaddedinitiallyasboilingwater.Undertheseconditions,Isuspecttheperoxide willhaveadifficulttimesurvivingtheexposuretoheat,eveniftheinitialconcentrationis raisedseveralfold. Nitrogensupplementsforbulksubstrate

BacktoContents 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 pasteurizedsubstrate.Becarefultowipedripsofftheoutsidesofthejarsbeforepouring. Most of the traditional nitrogen supplements for mushroom culture require pressure cooking to eliminatetheendogenousperoxide-decomposingenzymesbeforepasteurization.(Theseenzymesare remarkablystable,andstandardpasteurizationproceduresaregenerallynotenoughtoinactivate them,evenwithdelicatesupplementslikebran).However,asIdiscussedinthesectiononmaking sawdustspawn,Ihavenowfoundafewsupplementsthatarefreeofenzymesandsocanbeadded withoutpressuresterilization,inthiscasemixedinwiththewoodpellets.Twooftheseare commerciallymanufacturednitrogensupplementsalreadyinuseintheAgaricusmushroomindustry (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. Thesesupplementsareanexcellentvalue,buthomehobbyistsmayhavedifficultyobtainingthem. Also,caremustbetakentokeepthemfromspoilinginstorage,especiallywithMillichamp3000. Somemoreexpensiveformsofprocessed protein aremorereadilyavailable,suchasTexturized VegetableProtein,powderedsoymilk,orpowderedcowsmilk. Anothertypeofsupplementthatcanbeused withoutpressuresterilizationissimplychemical fertilizer, such as a standard 20-20-20 fertilizer. Since these fertilizers do not come from


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livingorganisms,theycontainnoperoxide-decomposingenzymes.Nevertheless,forthemostpart thenutrientstheycontaincanbeutilizedbymushroommyceliumafteraperiodofadaptation.If youaregoingtotrythismethodofsupplementation,Irecommendthatyoupreparesawdustspawn usingthesamesupplement,sothattheadaptationperiodwillhavealreadytakenplacebythe timeyouinoculateyourbulksubstrate.Also,youwillgetachancetoseehowtheparticular fertilizeryouhavechosenwillworkfortheorganismyouaregrowing.Fertilizerformulations vary quite a bit, even with the same NPK rating, so it is probably advisable to test your selectedfertilizerwithasmallculturebeforegoingontobulksubstrate. Ureaisacommonsourceofnitrogenintheformulationsforchemicalfertilizers,anditprobably canalsobeusedbyitselfasasupplementwithoutpressuresterilization. If you want something more "organic" than artificial fertilizer (and there is good reason to avoiddependenceonsubstanceswhichrequireenergyfrompetroleumfortheirmanufacture),human urineandanimalurinecanalsoserveassupplementsthatdon’tneedpressurecooking.However, theymustbekeptrelativelyfreeofmicro-organismsuntiluse.Additionofperoxideprovidesone waytodothis. Calculatinghowmuchsupplementtoadd

BacktoContents 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 mushroomsatvariouslevelsofsupplementation.Butyoucangetanideaofwhatyou'llneedby consultingStamets'sGrowingGourmetandMedicinalMushrooms, where the appendices reveal that rice bran has an NPK rating of roughly 2-1.3-1. So, if substrate would ordinarily get supplementedwith5to20%ricebran,whichStametssuggests,thena20-20-20fertilizer,which has10timesasmuchnitrogenasricebran,wouldbeaddedatonetenththerateofricebran,or 0.5to2%ofthedryweightofsubstrate.Ifyouwereaddingonepoundofricebrantoabucket ofpelletfuel,thenyouwouldadd1.6ozof20-20-20fertilizerinstead,thatis,onetenthofa pound. With the commercial supplements, you will need to find out from the manufacturer what percentagenitrogenthematerialcontains,anddividethatnumberinto2.0tolearnwhatfraction oftheamountofricebranyouwouldadd.Sylvan'sMillichamp3000,madefromsoy,isabout7.3% nitrogen,soitwillneedtobeusedataboutonequartertherateofricebran. Youcanalsodirectlycalculatetheamountofthematerialneededtogive0.1%to0.4%nitrogen inthefinalsubstrate,withoutreferencetotheamountofricebranusedbyStamets: 1)Dividethepercentnitrogeninthesupplementbythefinalpercentnitrogendesiredinthe substrate. 2)Dividethepreviousnumberintothetotalweightofsubstratetobesupplementedtogetthe weightofsupplementtobeadded. Thus,toget0.2%finalnitrogen(whichrequiresasupplementationrateofroughly10%ofthedry weightofsubstratewithricebran),howmuchsoyflourwouldweneedtoadd?Ifthesoyflouris 7.6%nitrogen,7.6dividedby0.2gives38.Ifthetotalweightofsubstrateis6.5pounds,then 6.5dividedby38gives0.17pounds,or2.72ozsoyflourtobeadded. AnoteonmeasuringpHofsubstrate

BacktoContents IuseColorpHaststripswithapH4to10rangetomeasurethepHofallmymediaandsubstrates, aimingforapHatmake-upinthe6-7rangeinmostcases.ColorpHaststripsareinexpensiveand convenient,andwithathree-colorcomparison,Iamusuallyconfidentofmyreading.However,it isnotagoodideatotrytomeasurethepHofmediumwithacolorindicatorstriponceperoxide hasbeenadded,astheperoxidemaychangethechemistryoftheindicator.Withagarculturesand spawn,youcaneasilymeasurethepHbeforesterilization.Withpelletfuel,useasmallscoop suchasameasuringcup(onethathasbeenrinsedwithboilingwater)totakeasmallamountof substrateoutofthebucketafteraddingandmixingintheboilingwaterpluslime.Youcanthen


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useyourcolorindicatorstripstomeasurethepHoftheremovedsubstrate.Beaware,however, that the reading will only give you a relative idea of the pH eventually experienced by the mushroommyceliumifyouhaveaddedgranularlimewhichdissolvesonlyveryslowly.Additionof Mason's lime (CaOH, available in large sacks as "builders’ lime" from construction supply stores)getsaroundthelatterproblem,asitdissolvesandreactsmuchmorequickly,butwith somemushroomstheremaybeanadvantagetoprovidinga"delayedrelease"sourceofalkalinity suchasgranularlimeprovides.Thenyouwillsimplyhavetocalibratetheamountoflimeyou usedagainsttheultimateyieldofmushroomstodeterminetheoptimaldose. Culturecontainers

BacktoContents Traditionally,sawdustcultureshavebeengrowninspecialplasticbagswithmicroporousfilter patches,toallowgasexchangewithoutletting contaminantsgainentry.Withperoxideinyour fruitingsubstrate,however,youshouldbeabletouseordinarytrashbags(atasavingsof$.50$.80 per bag) to grow your mushrooms. Evidently the process used to produce trash bags pasteurizesthemtothepointthattheydonotharborsignificantlive-organismcontamination.If you do use plastic trash bags, I recommend using the kind that are made from high density plastic,0.5milthicknessorless.Thesebagsarethinenoughthatoxygencandiffusethrough them,sothattheculturescanbegrowntomaturitywiththebagssealedclosedbytwistties. Also,thethicker,softerbagsareapparentlymadefromPVC,whichcanleaveestrogenicresidues inthemushroomcultures,andcertainofthesesofterbagsareimpregnatedwithfungicides. Unless you use traditional gussetted mushroom bags, you will need to put your bags inside containersofanappropriatesizetoprovideaformforthesubstrate.Smallboxeswhichwill hold5-6poundsofsubstratecanoftenbescavengedfromhealthfoodstoresorthelike,oryou canbuyplasticcontainerssuchasnurserypots.

Fillingaplastictrashbag,proppedinside abox,withpelletfuelsubstrate

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 gallonbucketsareeasiertocomeby,buttheyarealittletoobigforyouraveragesawdust culture).Thesecanbecleanedwithdetergentandreusedafterrinsingwithboilingwater. thelidsareleftslightlylooseduringthespawnruntoallowgasexchange,thebucketsare excellentculturevesselsformushroomsspeciesthatfruitvertically,suchasP.eryngii.H. erinaceuswillalsogrowinthemifyouopenthebucketandturnitonitssidewhenfruiting timecomes.(Ifillthebucketonlyaboutathirdtohalfwayfullwithsubstrate,sotheupper partofthebucketprovidesamoisturebarrier).H.ulmariuswouldbealittlecrampedforspace in one of these buckets, unless you were to fill substrate nearly to the top, so that the mushroomclusterscouldgrowoutabovetherim.Togetasecondflush,then,youwouldneedto taketheroundblockoutofthebucketandturnitupsidedown,sinceH.ulmariusdoesn’tlike tofruitfromthesamesurfacetwice.


. If

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Inoculatingsupplementedsawdust

BacktoContents Ipreparespawnforinoculationinthetraditionalway: 1)ThedaybeforeIwanttousethespawn,Ibreakupthespawnbywhackingthejaragainst somethinghardprotectedbysomethingsoft. 2)Whentheparticlesareseparated,Iputthejarbackonmyspawnshelfandincubateovernight togivethemyceliumachancetoputoutsomenewgrowth.Thismakesaconsiderabledifferencein howfastthemyceliumwillsurgeintothenewsubstrate.IfIamworkingwithgrainspawn,it alsogivesmeachancetoseebacterialcontaminationintheformof"wet"orgreasylooking grainkernelsthathavenÕtacquiredanewfuzzofmycelium.Whenyourspawnhasbeengrownusing peroxide,thepresenceoftwoorthreewetkernelswillprobablynotinterferewiththe subsequentsuccessofyourcolonizationofbulksubstrate,sincethebacteriathatareableto surviveperoxideexposurearegenerallyfairlybenignorganismswhenpresentinsmallquantities. However,ifyouhavequiteafewmorewetkernelsthantwoorthree,thebacteriawilllikely slowthecolonizationofbulksubstratesubstantially,whichthenmaygiveachanceformoldto gainentry.Soyouwillprobablywanttodiscardspawnwithanysignificantquantityofwet kernels. 3)Iinoculatethepelletfuelsawdustbybreakingupthespawnbriefly,thenpouringitdirectly intothe5gallonbucketwiththesubstrate.Iclosethelidandmixeverythingtogetherby rotatingthebucket.

Inoculatingabucketofpellet fuelsubstratewithspawn

4)Ipourthemixintobags.Eachbaggetsopenedupandsetinsideofaboxoftheappropriate sizetoreceivethesubstrate. 5)WhenIhavefilledthebagtothecapacityofthebox,Iclosethelidontheremaining inoculatedsubstrate,andtakingcarenottotouchtheinsidesurfaceofthebag,Ishiftthebag abittofillanygaps,thentwistthemouthofthebagclosedandsealitwithatwisttie.


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Bagofinoculatedsubstrate sealedwithtwisttie

6)Lastly,Icompressthesawdustbypressingdownonthebag,gentlybutfirmly.Ifindthat thisspeedsgrowthofsomecultures,especiallywithalightsawdustlikefirorcottonwood. Afterlabeling,theboxisreadytoincubate,andfromhereonout,Ifollowstandardmushroomgrowingprocedures.Youcanusetheresultingblocksofmyceliumeitherdirectlyforfruiting mushrooms,ortheblockscanserveasspawn forinoculating logsorbedsoffresh woodchips outdoors.

Mushroomformation BacktoContents Formostcommonlycultivatedmushroomspecies,mushroomformationbeginssoonafterthecultures are shifted to cooler temperatures, given more light, and given more fresh air, provided the substratehasbeenthoroughlycolonized.Thereisnotmuchneedforhydrogenperoxideduringthis phase,sincethemyceliumiswellestablished. Thepreciseproceduresforinducingmushroomformationdifferfromonespeciestoanother,andit isbeyondthescopeofthismanualtoreviewthemall,butIwillgiveguidelinesformyfavorite species.Twoofthemushroomspeciesmostfamiliartomearealsoamongtheeasiesttofruit: Hypsizygusulmarius(theWhiteElmmushroom)andHericiumerinaceus(theLyon’sManeorPomPom mushroom). Many oyster mushroom species follow similar procedures to that required by H. ulmarius.Theotherspeciesmostfamiliarto me,Pleurotuseryngii(King Oyster)andAgaricus subrufescens(almondmushroom)followadifferentfruitingpattern.Shiitakefollowsyetanother. Most of the "easy" mushroom species are ready to fruit when the bulk substrate is thoroughly grownthrough.Oftentheblockslookwhiteatthistime,ratherthantheoriginalbrownofthe substrate. How long it takes for a culture to reach maturity depends on the organism, the substrate,andtheincubationtemperature.Hericiumcantakeaslittleas2-3weekstoformsmall white,globularfruitinginitialsontheuppersurfaceoftheblock,butIliketowaituntila month haselapsed beforeopeningthebags.H.ulmariustakesabout5weekson firsawdustor strawandsixweeksonoaksawdust(atordinaryroomtemperature),afterwhichsmallclustersof pinheadprimordiawillbegintoformspontaneously.Bycuttingan"X"orasingleslitwitha cleanknifethroughthebagonthesideoftheblock,H.ulmariusandvariousOystermushroom specieswillusuallyformprimordiaatthesiteofthecutwithinaweekortwo,andmushrooms willsoondevelop.Providemistspraywhenthemushroomsareaboutaninchhigh. Hericiumwillalsoformmushroomsatthesiteofacutinthebag,butinthewinterIfindit easiertogrowlargefruitingbodiesbyallowingtheorganismtofruitinsidethebag.Simply turntheblockonitssideandopenthebagabit,allowingairexchangebutstillprovidinga moisture barrier. Fruiting bodies will form at random from the fruiting initials that have


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alreadydeveloped. Ifyougrowonlyafewblocksofmushrooms,ventilationwillnotbemuchofanissue.Butwith moreblocks,theneedforventilationtoremovecarbondioxideincreases.Ifyourmushroomsare notgettingenoughairastheydevelop,theywillbecomedeformed.H.ulmariusandotheroyster mushrooms, for instance, will grow long stalks and irregular caps when the carbon dioxide concentrationistoohigh.Whenyouhaveenoughblockstoneedafan,thenanautomaticmisting systemcan befarbehind. NotethatifyoudecidetogrowH.ulmariusoranotheroysterspeciesinyourhome,youwill probably need to take precautions to protect yourself from the tremendous amount of spores producedbytheseorganisms.Harvestingthemushroomswhenyoungcanhelpkeepthesporeload down.CoveringthefruitingcultureswithReemay ™orotherrowcovermaterialwillkeepthebulk ofthesporeswithinthecovering,whileallowinggasexchangesufficientforfruiting.However, if you or someone in your family is sensitive to the spores, you may need to acquire an air purifiertoeliminatethesporesfromtheairinyourlivingspace,orelsekeeptheculturesin anout-building. Thealmondmushroom,thewhitebuttonmushroom,aswellasKingStrophariaandShaggyMane,and sometimesKingOysterallneedsomethingcalledacasinglayerappliedtothemushroomcultureto 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 moisturereservoirformushroomgrowth.Itgenerallycontainslittleavailablenutritionforthe growth of mushroom mycelium, and this feature also sends a signal to the mushroom culture to beginmushroomformation.

Almondmushroomsgrowingfromacasinglayer

Mostcasingcontainspeatmoss,andasimpleformulaIhaveusedforalmondmushroomssimply callsforonepartpeatmixedwithonepartgardensoil,plusahandfulofgypsum(calcium sulfate)fortwoorthreegallonsofmixture,allmoisteneduntildampbutnotclumpy.Thecasing isappliedtothetopofthemushroomculturetoadepthoftwoinchesatthemost.Becareful nottotampitdown,astheporousstructureisessentialforencouragingformationofmushroom primordia. Peatbogsareanendangeredhabitatworldwide,soweneedtofindalternativestotheuseofpeat incasing.Growersindifferentpartsoftheworldhavebeguntodevisealternatives,asonecan discover by searching the US patent database (see http://www.uspto.gov/). In some cases, soil alone may suffice as a casing, or soil plus vermiculite. Vermiculite by itself is a possible alternative(althoughitwillnotsupplymicroorganisms).AlmondmushroomsandKingOystersdo notabsolutelyrequireacasing(thecasingdoestendtoaccelerateprimordiaformationwithP. eryngii,however),somanipulationofconditionsmayleadtogoodfruitingswithoutitforthese organisms.


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Ifyoudoapplyacasing,youwillthenneedtowaitaweekortwoforthemyceliumtogrowup intothecasingbeforemushroomswillbegintoform.Withthealmondmushrooms,thisisthetime to warm up the cultures (I put my apple-box cultures of almond mushroom on an electric "cat warmer"tobecertaintheyarewarmenough).Itisalsothetimetosprinklethecasinglightly withwatereverycoupleofdaystokeepitmoist.(WithP.eryngii,warmingandsprinklingare notnecessary).Mushroomsusuallybegintoformafewdaysafterthemyceliumbeginstoreachthe surfaceofthecasing. Seasonalplanning

BacktoContents Iyouareonlygrowingafewmushrooms,andyouhaveacool,insulatedindoorspacesuchasa lightedbasement,youwillprobablybeabletogrowyourfavoritemushroomsallyear.However,if your are growing outdoors, or you are growing a lot of mushrooms indoors (so that you need ventilationfromoutdoors),youmayneedtoplanaheadtohavetheappropriatemushroomcultures readyintherightseason.Ifruitmymushroomsinabasementwithopenwindowsandafanto bringinfreshair,sothemushroomgrowinggetsharderinthehottestpartofthesummerandthe coldestpartofthewinter.Closingthewindowsisnotanoption,ascarbondioxidewillbuildup andinhibitmushroomformation.Heatingorcoolingtheincomingairiscertainlypossible,butit runsuptheenergybilltoomuchformytaste.Soallmymushroomsdobestinfallandspring. In the coldest part of the winter, both the temperature and the light levels fall. All the mushroomstakelongertofinishcolonizingthebulksubstrate.P.eryngiifruitswithdifficulty, andH.ulmariusgrowsveryslowly,producinglongerstalks(anddeformedcaps,ifthelightand temperaturelevelsaretoolow).H.erinaceusalsogrowsslowlyatthistime,butthisspecies still produces normal, but small, fruiting bodies even in very cold weather. Agaricus subrufescensneedswarmth,butparadoxicallyitmakesagoodindoormushroomforwinter,because itwillfruitinheatedroomanditdoesn'tneedasmuchairorlightastheothermushrooms. In the hot parts of summer, there is plenty of light, but it can be a problem to initiate fruitingatthehighertemperatures.Keepinghumidityupcanbedifficult,too.However,Ihave hadH.erinaceusblocksfruitonadrycompostpilein90degreeweather;evidentlythefreshair initiatedfruitinginthatcase,sincemyindoorH.erinaceusblocksrefusedtofruituntilthe temperaturecamedownsubstantially.A.subrufescenslikeswarmweatherandtendstofruitasthe peaktemperaturespass.Ganodermalucidumalsopreferswarmweather,asdoesStropharia. Outdoorgrowingvs.indoorgrowing

BacktoContents I used to grow almost all of my mushrooms indoors. This allowed me to grow them year 'round becauseofthemoremoderatetemperature,anditalsosavedmefromhavingtodealwithslugsand snails,whichlovemushroomsandgrowingreatnumbersinmyarea.(Actually,Istillgetafew slugsthatmanagetoclimbinmywindows,traveldownthecementbasementwallandacrossthe cementfloor,andeventuallyentermyfruitingcultures).Deermayalsoeatmushrooms,andfungus gnatscanbeaprobleminoutdoormushroompatches.Sointhepast,Ihavealwaysrecommended indoorgrowing.Butoutdoorgrowinghasitsadvantages,too.Foronething,mushroomsfruited outdoorshaveadistinctlysuperiorflavortomushroomsgrownindoors.Also,mushroomformation canbemorerobustinthefreshairoftheoutdoors.There'smorephysicalspaceforcrops.And thereislittleproblemwithbreathingmushroomspores.Fungusgnatscanbereducedsomewhatby coveringcultureswithafinelightfabricsuchasReemay(TM)orotherrowcovermaterials.And ifyouliveinatemperateregionontheseacoast,youmaybeabletogrowmushroomsoutdoorsall year'round.Soifyouthinkyoucankeepallthepestsaway,goaheadandfruityourcultures outside.Youwilljustneedashadedareathatcanbekeptdamp. Harvesting

BacktoContents Knowingwhentoharvestmushroomsislargelyamatterofknowinghowlargetheygrowandwhat


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changestheygothroughastheymature.WithP.eryngiiandH.ulmarius,theuncurlingofthe marginofthecapisusuallyasignthatthemushroomhasreachedmaturity,butyouwillneedto correlatethischangewiththesizeofthemushroomtobesure.WithA.subrufescens,thecap opensandthegillsbegintoturnreddishincolor.WithH.erinaceus,small"icicles"formand themushroomsoftens. Most mushrooms are said to be tastier if harvested before they start releasing many spores, althoughtheymaystillgainmoremassiflefttogrowfurther.ItiscertainlytruethatH. ulmariusistastierwhenyoung,butIhaven’tpersonallymadetastecomparisonswiththeother mushroomspeciesIgrow.

Troubleshooting BacktoContents Despite my use of hydrogen peroxide to protect my mushroom cultures, there have been many occasionswhenthingsdidnotgoasIhadplannedandcontaminationappeared.Eachtime,Ihadto trackdowntheproblemandcorrectmyprocedure,andeachtimedIwasrelievedtolearnthatthe useofperoxideitselfwasnotflawed.TheproceduresIhavedescribedheretothebestofmy knowledgeincorporateeverythingIhavelearnedfrommymistakesandshouldcoverthekeypoints required to produce contamination-free mushrooms with peroxide-based culture. Nevertheless, troubleshootingisanunavoidablepartofmushroomculture,andyouwillhavetodoitsooneror later. Ialwaysfinditdiscouragingtoreadthroughlistsofthingsthatcangowrong,soinstead,I havecreatedalistofquestionsthatdrawattentiontodifferentaspectsofthecultureprocess forthepurposesoftroubleshootingcontaminationproblems. If you are adding peroxide, and you still suffer significant contamination, you might ask yourselfsomeofthefollowingquestions: Istheconcentrationofperoxideinyourstocksolutionwhatitshouldbe?Hasitbeenovera monthsinceyoumeasuredit? Isyourpressure-cookingequipmentfunctioningproperly? Issteamabletoenteryourjarsandequilibrate(arethelidslooseenough?Ifpressurecooking, doyouallowfiveminutesforsteamtoequilibratebeforeputtingonthepressureregulator?) Isyourstoveelement(ifelectric)heatingconsistently? Areyoucookingatahighenoughtemperatureandforalongenoughtimetoeliminateresident contaminantsand,ifnecessary,peroxide-decomposingenzymes? Isyoursubstratemoistenoughforsteamtopenetrate? Hasyoursubstrateorsupplementspoiledbeforeuse? Isyourperoxidegettingdistributedevenlythroughoutthemedium? IsyourpHreadingaccurate?(PeroxideisapparentlymoststablearoundneutralpH). Ifyourfruitingsubstrateisgettingcontaminated,isyourspawnclean? Ifyourspawnisgettingcontaminated,isyourinoculumclean? If you are free-pouring diluted peroxide into your cultures, are there drips that run over unsterilizedsurfacesbeforefallingintotheculture?


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Areyourpetridishesfreeoftracesofoldmedium? Ifyouragarplatesaregettingcontaminated,isthecontaminationonthesurfaceorwithinthe agar?Ifitisonthesurface,lookforasourceofcontaminationexternaltotheagarmedium;if itisintheagar,contaminationisgettinginbeforeorduringpouring. Areyoulettingyourmediumorsubstratecoolsufficientlybeforeaddingperoxide? Isyourwatercleanandfreeofparticulates? Haveyouoverlookedsomesourceofunsterilizedorunpasteurizedmaterialthatcangetintoyour cultures? Areyoumushroomsgettingenoughlight(butnotdirectsunlight),freshair,andhumiditytogrow toagoodsize?

Conclusion BacktoContents AsIreachtheendofthismanuscript,Iamforcedtopauseforamomentofselfexamination.I calledthisvolumeGrowingMushroomstheEasyWay, and now I have to ask myself whether I wasn't indulginginjustabitofself-servingexaggerationwhenIchosethatname.Afterall,thereare stillfarmorewaysforthingstogowronginmushroomcultivationthanforthemtogoright.And Isometimesthinkitisawonderindeedthatweevergetanyoftheseorganismstorespondtoour coaxingandproducetheirdelectablefruitingbodies. Well,itisawonder.Andevenwithperoxidemaintenanceofmushroomcultures,theprocessisfar fromfool-proof.ButIfeelgratifiedthattheproceduresdescribedheredomakeitpossiblefor hobbyistswithatleastaminimaldegreeofcomfortwithsteriletechniquetoperformallthe stepsofgourmetmushroomgrowingandmushroomcultureinanordinaryhousehold,moreeasilythan everbefore,withnomorespecialequipmentneededforcontaminantcontrolthanasteamingpot andameasuringpipette.Andwiththestresstakenoffofbattlingcontaminants,homegrowers shouldbefreedatlasttofocusonthethingthatattractsusalltomushroomgrowinginthe firstplace,thequestforevermoreofthosebeautifulanddeliciousfungi.

AbouttheAuthor

BacktoContents RushWayneholdsaMastersdegreeinBiochemistryandMolecularBiologyfromHarvardUniversity andaPh.D.inBiochemistryfromtheUniversityofCaliforniaatBerkeley.Hewasfirstexposed totheelementsofmushroomgrowingduringhisgraduateworkinthe1970’sbutdidnotbegin growingmushroomsinearnestuntilhebegantoimplementtheinnovationscontainedinthismanual in1993.Instructionsforhisperoxidemethodofgrowingmushroomsarenowinthehandsof mushroomgrowersinover75countriesaroundtheworld.


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[Psilocybin]Home Mushroom Cultivation With Hydrogen Peroxide

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