FDABAMYeast and Moulds推荐文档.docx

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FDABAMYeastandMoulds推荐文档

BAM:

Yeasts,MoldsandMycotoxins

ReturntoBAMtableofcontents1

April2001

BacteriologicalAnalyticalManual

Chapter18

Yeasts,MoldsandMycotoxins

Authors:

ValerieTournas,MichaelE.Stack,PhilipB.Mislivec,HerbertA.KochandRuthBandler

Thelargeanddiversegroupofmicroscopicfoodborneyeastsandmolds（fungi）includesseveralhundredspecies.Theabilityoftheseorganismstoattackmanyfoodsisdueinlargeparttotheirrelativelyversatileenvironmentalrequirements.Althoughthemajorityofyeastsandmoldsareobligateaerobes（requirefreeoxygenforgrowth）,theiracid/alkalinerequirementforgrowthisquitebroad,rangingfrompH2toabovepH9.Theirtemperaturerange（10-35°C）isalsobroad,withafewspeciescapableofgrowthbeloworabovethisrange.Moisturerequirementsoffoodbornemoldsarerelativelylow;mostspeciescangrowatawateractivity（aw）of0.85orless,althoughyeastsgenerallyrequireahigherwateractivity. 

Bothyeastsandmoldscausevariousdegreesofdeteriorationanddecompositionoffoods.Theycaninvadeandgrowonvirtuallyanytypeoffoodatanytime;theyinvadecropssuchasgrains,nuts,beans,andfruitsinfieldsbeforeharvestingandduringstorage.Theyalsogrowonprocessedfoodsandfoodmixtures.Theirdetectabilityinoronfoodsdependsonfoodtype,organismsinvolved,anddegreeofinvasion;thecontaminatedfoodmaybeslightlyblemished,severelyblemished,orcompletelydecomposed,withtheactualgrowthmanifestedbyrotspotsofvarioussizesandcolors,unsightlyscabs,slime,whitecottonymycelium,orhighlycoloredsporulatingmold.Abnormalflavorsandodorsmayalsobeproduced.Occasionally,afoodappearsmold-freebutisfounduponmycologicalexaminationtobecontaminated.Contaminationoffoodsbyyeastsandmoldscanresultinsubstantialeconomiclossestoproducer,processor,andconsumer.

Severalfoodbornemolds,andpossiblyyeasts,mayalsobehazardoustohumanoranimalhealthbecauseoftheirabilitytoproducetoxicmetabolitesknownasmycotoxins.Mostmycotoxinsarestablecompoundsthatarenotdestroyedduringfoodprocessingorhomecooking.Eventhoughthegeneratingorganismsmaynotsurvivefoodpreparation,thepreformedtoxinmaystillbepresent.Certainfoodbornemoldsandyeastsmayalsoelicitallergicreactionsormaycauseinfections.Althoughmostfoodbornefungiarenotinfectious,somespeciescancauseinfection,especiallyinimmunocompromisedpopulations,suchastheagedanddebilitated,HIV-infectedindividuals,andpersonsreceivingchemotherapyorantibiotictreatment.

Thedilutionplatingandthedirectplatingmethodsmaybeusedtodetectfungiinfoods.Thedirectplatingmethodismoreefficientthanthedilutionplatingmethodfordetectingindividualmoldspecies,includingmostofthetoxinproducers,butitislesseffectiveindetectingyeasts.Itisalsousedtodeterminewhetherthepresenceofmoldisduetoexternalcontaminationorinternalinvasion.Methodologyfortestingtheabilityofisolatesoftoxigenicmoldspeciestoproducemycotoxinsonsterilericewatersubstrateisincludedhere.

EnumerationofYeastsandMoldsinFood--DilutionPlatingTechnique

A.Equipmentandmaterials

1.Basicequipment（andappropriatetechniques）forpreparationofsamplehomogenate,seeChapter12

2.Equipmentforplatingsamples,seeChapter33

3.Incubator,25°C

4.Arnoldsteamchest

5.pHmeter

6.Waterbath,45±1°C

B.Media4 andreagents5

 Media

1.Dichloranrosebengalchloramphenicol（DRBC）agar（M1836）

2.Dichloran18%glycerol（DG18）agar（M1847）

3.Platecountagar（PCA）,standardmethods（M1248）;add100mgchloramphenicol/literwhenthismediumisusedforyeastandmoldenumeration.Thismediumisnotefficientwhen"spreader"moldsarepresent.

4.Maltagar（MA）（M1859）

5.Maltextractagar（YeastsandMolds）（MEAYM）（M18210）

6.Potatodextroseagar（PDA）,dehydrated;commerciallyavailable（M12711）

 Antibioticsolutions

Antibioticsareaddedtomycologicalmediatoinhibitbacterialgrowth.Chloramphenicolistheantibioticofchoice,becauseitisstableunderautoclaveconditions.Therefore,mediapreparationiseasierandfasterduetotheeliminationofthefiltrationstep.Therecommendedconcentrationofthisantibioticis100mg/litermedium.Ifbacterialovergrowthisapparent,preparemediabyadding50mg/literchloramphenicolbeforeautoclavingand50mg/literfilter-sterilizedchlortetracyclinewhenthemediahavebeentempered,rightbeforepouringplates.

Preparestocksolutionbydissolving0.1gchloramphenicolin40mldistilledwater;addthissolutionto960mlmediummixturebeforeautoclaving.Whenbothchloramphenicolandchlortetracyclineareused,add20mloftheabovechloramphenicolstocksolutionto970mlmediumbeforeautoclaving.Then,preparechlortetracyclinestocksolutionbydissolving0.5gantibioticin100mldistilledwaterandfiltersterilize.Use10mlofthissolutionforeach990mlofautoclavedandtemperedmedium.Refrigerateinthedarkandre-useremainingstocksolutionsforuptoamonth.Stocksolutionsshouldbebroughttoroomtemperaturebeforeaddingtotemperedmedium.

C.Procedures

 Samplepreparation

Analyze25-50gfromeachsubsample;generally,largersamplesizesincreasereproducibilityandlowervariancecomparedwithsmallsamples.TestindividualsubsamplesorcompositeaccordingtorespectiveComplianceProgramforthefoodunderanalysis.Addappropriateamountof0.1%peptonewatertotheweighedsampletoachieve10-1dilution,thenhomogenizeinastomacherfor2min.Alternatively,blendingfor30-60seccanbeusedbutislesseffective.Makeappropriate1:

10（1+9）dilutionsin0.1%peptonewater.Dilutionsof10-6shouldsuffice.

 Platingandincubationofsample

Spread-platemethod.Asepticallypipet0.1mlofeachdilutiononpre-poured,solidifiedDRBCagarplatesandspreadinoculumwithasterile,bentglassrod.DG18ispreferredwhenthewateractivityoftheanalyzedsampleislessthan0.95.Plateeachdilutionintriplicate.

Pour-platemethod.Usesterilecotton-pluggedpipettoplace1.0mlportionsofsampledilutionintoprelabeled15x100mmPetriplates（plasticorglass）,andimmediatelyadd20-25mltemperedDG18agar.Mixcontentsbygentlyswirlingplatesclockwise,thencounterclockwise,takingcaretoavoidspillageondishlid.Afteraddingsampledilution,addagarwithin1-2min;otherwise,dilutionmaybegintoadheretodishbottom（especiallyifsampleishighinstarchcontentanddishesareplastic）andmaynotmixuniformly.Plateeachdilutionintriplicate.

Frompreparationoffirstsampledilutiontopouringorsurface-platingoffinalplate,nomorethan20min（preferably10min）shouldelapse. Note:

Spreadplatingofdilutedsampleisconsideredbetterthanthepourplatemethod.Whenthepourplatetechniqueisused,fungalcoloniesonthesurfacegrowfasterandoftenobscurethoseunderneaththesurface,resultinginlessaccurateenumeration.Surfaceplatinggivesamoreuniformgrowthandmakescolonyisolationeasier.DRBCagarshouldbeusedforspreadplatesonly.

Incubateplatesinthedarkat25°C.Donotstackplateshigherthan3anddonotinvert.Note:

Letplatesremainundisturbeduntilcounting.

 Countingofplates

Countplatesafter5daysofincubation.Ifthereisnogrowthat5days,re-incubateforanother48h.Donotcountcoloniesbeforetheendoftheincubationperiodbecausehandlingofplatescouldresultinsecondarygrowthfromdislodgedspores,makingfinalcountsinvalid.Countplatescontaining10-150colonies.Ifmainlyyeastsarepresent,plateswith150coloniesareusuallycountable.However,ifsubstantialamountsofmoldarepresent,dependingonthetypeofmold,theuppercountablelimitmayhavetobeloweredatthediscretionoftheanalyst.Reportresultsincolonyformingunits（CFU）/gorCFU/mlbasedonaveragecountoftriplicateset.Roundoffcountstotwosignificantfigures.Ifthirddigitis6orabove,roundofftodigitabove（e.g.,456=460）;if4orbelow,roundofftodigitbelow（e.g.,454=450）.Ifthirddigitis5,roundofftodigitbelowiffirst2digitsareanevennumber（e.g.,445=440）;roundofftodigitaboveiffirst2digitsareanoddnumber（e.g.,455=460）.Whenplatesfromalldilutionshavenocolonies,reportmoldandyeastcounts（MYC）aslessthan1timesthelowestdilutionused.

IsolateindividualcoloniesonPDAorMA,iffurtheranalysisandspeciesidentificationisnecessary.

EnumerationofMoldsinFoods--DirectPlatingTechnique 

forFoodsThatCanBeHandledwithForceps 

（DriedBeans,Nuts,WholeSpices,CoffeeandCocoaBeans,etc.）

A.Equipmentandmaterials

1.Freezer,-20°C

2.Beakers,sterile,300ml

3.Forceps,sterile

4.Arnoldsteamchest

5.Waterbath,45±1°C

6.Incubator,25°C 

B.Mediaandreagents

1.Dichloranrosebengalchloramphenicol（DRBC）agar（M18312）

2.Dichloran18%glycerol（DG18）agar（M18413）

3.Antibioticsolutions（seeprevioussection）

4.NaOCl（commercialbleach）solution,10%

5.Steriledistilledwater 

C.Analysisofnon-surface-disinfected（NSD）foods

 Sampleandmediapreparation

Beforeplating,holdsampleat-20°Cfor72htokillmitesandinsectsthatmightinterferewithanalysis.

PrepareDRBCagarasdescribedintheappendix.IfDRBCisnotavailable,orthewateractivityoftheanalyzedsampleislessthan0.95,useDG18agar.Mediashouldbepreparednomorethan24hpriortouse.

Platingandincubationofsample

Fromeachsample,transferabout50gintoasterile300mlbeaker.Using95%ethanol-flamedforcepsplaceintactfooditemsonsurfaceofsolidifiedagar,5-10itemsperplate（de