微生物培养试验.docx

微生物培养试验.docx

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微生物培养试验

EXERCISE2

TOOBSERVETHEINFECTIONPROCESS

Cloverrhizobiaentertheirhost'srootsthroughtheroothairs. Infectionisprecededbyadeformationofroothairsandtheformingofaninfectionthreadwhichcanbeobserveddirectlyunderthemicroscope. RoothairdeformationsmayalsobecausedbynonnodulatingstrainsofRhizobium. Non-nodulatingstrainsusedinthischaptercausenoinfectionthreadstoform.

Keysteps/objectives

l）CulturestrainsofRhizobiuminYMbroth

2）Sterilizeandgerminatecloverseeds

3）Mountseedlingonmicroscopeslide

4）Incubatetheseedlingsininoculatedmineralmedium

5）Observeroothairdeformationandinfectionthreads

6）Compareroothairdeformationscausedbydifferentkindsofrhizobiastrains

（a）CulturingstrainsofrhizobiainYMbroth

（Keystep1）

Inoculate50mlflasksortesttubescontaining20mlofYMbrothinduplicatewiththestrainslistedbelow:

1）Rhizobiumleguminosarumbv.trifolii（TAL382）isolatedfromnodulesofTrifoliumsemipilosum

2）R.l.bv.trifoliinoninfectiveisolatedfromnodulesofTrifoliumsp.

3）R.l.bv.trifolii（TAL1185）isolatedfromnodulesofTrifoliumrepens

4）R.l.bv.phaseoli（TAL182）isolatedfromnodulesofPhaseolusvulgaris

5）R.meliloti（TAL380）isolatedfromnodulesofMedicagosativa

6）Bradyrhizobiumsp.（TAL764）isolatedfromnodulesofLupinusangustifolius

Otherstrainsofthesamespeciesofrhizobiamaybesubstituted.

Incubateat2530Cfor57daysonarotaryshaker.

（b）Germinatingseeds

（Keystep2）

Chooseasmallseededlegume. Clover,especiallyTrifoliumrepensorT.glomeratum,ismostsuitableforthisexercise.SurfacesterilizeseedsaccordingtotheprocedureoutlinedinAppendix10. Somecloverspeciesmayneedscarificationwithsulfuricacid. Others,likeNolan'swhitecloverandstrawberryclover（Trifoliumfragiferum）germinateeasilywithoutscarification. Washseedswithatleasteightchangesofsteriledistilledwater. Asepticallyplacetheseedsontowater-agarplatesforgermination. Incubatetheplatesinvertedfor48hormoreuntilrootsare68mmlong.

（c）PreparingaFahraeusslide

（Keystep3）

Prepare10mlofFahraeuscarbonandnitrogenfreemedium（Appendix3）containing0.6%agarina15mltube. Cooltheliquidagarmediumto48Cinawaterbath. 

ForeachstrainofRhizobiumused,preparetwosterile50mlboiling-tubescontaining25mlFahraeuscarbonandnitrogen-freemediumwithoutagar. Setuptwoadditionaltubesforuninoculatedcontrols.Coverwith50mlbeakers.

Transferapproximately0.2mlofagarmediumtoasterilemicroscopeslideusingaPasteurpipettefittedwitharubberbulb. Leaveone-halfoftheslideempty. ThisisbestdonebylininguptheslideandalongcoverslipsidebysideinasterilePetridish（Figure2.1）. Placetheagarinfiveorsixdropsontothebottomhalfoftheslide. Immediately,transferawellformedseedlingtotheslidewithasterileinoculationloop. Placetheseedlingontotheslideinsuchawaythattheroottipisimmersedintheagarandthecotyledonsareintheemptyhalfoftheslide. Withsterileforceps,carefullyplacethelongcoverglassovertheagarandtheroottips. Iftheseedcoatadherestothecotyledonsontheseedling,carefullyremoveitwithsterilefinetippedforceps.

Figure2.1.PetridishwithFigure2.2.Placementof

ComponentsofFahraeusslideseedlingsonFahraeusslide

TransfertheslidemountedseedlingstothetubescontainingtheFahraeusmineralmedium.

（d）Inoculatingtheseedlings

（Keystep4）

Usingthebrothcultureswhichhavebeensetupforthisexperimentin（a）,inoculatetwoseedlingswitheachofthesixstrainsofRhizobiumbyaddingfivedropsofthecellsuspensionstoindividualtubescontainingthemineralmediumandtheFahraeusslides.

Alternatively,theseedlingsmaybeinoculatedbyincorporatingacellsuspensionintotheFahraeusagarmediumbeforetheseedlingisplacedontotheslide. Thisspeedsuptheinfectionprocess.Addfivedropsofsterilebrothmediumtothecontrols.

Incubateat2530Cinawell-lightedenvironment.

（e）Observingtheroothairsunderthemicroscope

（Keystep5）

After24hremoveFahraeusslidefromthenutrienttubeandexamineitunderthemicroscope. Removetheexcesssolutionwithabsorbentfilterpaper. Observewithphasecontrastorordinarybrightfieldmicroscopeunderlowandhighpowermagnifications. Searchforroothairdeformationsand/orcurlingandinfectionthreads.Markthepositionofyourslideonthemicroscopestagesothatthesamespotmaybefoundinlaterobservationsofthesameroothairinfections. Makeobservationsinintervalsof1224h. Periodicobservationmaybemadeatshorterintervalsifinoculationwasdonebyincludingthecellsuspensionintotheagarmedium.Returntheslidetoitstubebetweenobservations.

Takeprecautionsagainstunduecontaminationwhenreturningtheslidetothemineralmedium. Asepticconditionscannotbemaintainedbeyondthefirstobservation. However,contaminationusuallydoesnotinterfereprovidedtheroothairschosenforobservationsarenotlocatedattheedgesofthemicroscopeslide.

（f）Comparingroothairdeformations

（Keystep6）

Photographordrawtheroothairdeformationsorcurlingcausedbyeachstrain. Distinguishfullcurlingfromslightcurlingandroothairbranching.Notetheeffectsofnoninvasivestrainsontheroothairs.Comparethedeformationscausedbythevariousstrainsused.

Typicalroothairdeformations,liketheshepherd'scrook,areshowninFigure2.3.

Figure2.3.DeformedwhitecloverroothairinfectedwithR.trifolii0403.Notethesheperd’scrookandtheinfectionthread.（PhotocourtesyofF.Dazzo）

Figure2.4.SelectiveproliferationandcolonizationofRhizobiumtrifoliionaroothairofitshostlegumecloverinaFahraeusslidesystem.（PhotocontributedbyB.B.Bohlool）

Figure2.5.Rhizobiumtrifoliiinsideinfectionthreadintheroothairofitshostclover（Trifoliumrepens）.（Photo

contributedbyB.B.Bohlool）

Requirements

（a）CulturingR.leguminosarumbv.trifoliistrainsinYMbroth

Rotaryshaker

Twelve50mlflasks（ortubes）containing20mlculturebrotheach

Inoculationloop,flame

SlantculturesofcloverrhizobiastrainsTAL382,TAL1185,TAL182,TAL380,TAL386,noninfectivestrainofcloverrhizobia

（b）Germinatingseeds

Incubator

Materialsandtoolsforsterilizingseeds（Appendix10）

Platesofwateragar（7.5gagarperliterdistilledwater）

Seedsofclover（Trifoliumrepens,T.glomoratumorother）

（c）PreparingaFahraeusslide

Waterbath

Sterilemicroscopeslides（1mmx24mmx40mm）

Coverslips（keptinsterilePetridishes）

Pasteurpipettes（sterile）;rubberbulbs

Inoculationloop,forceps,flame

FahraeusCandNfreemedium

Fahraeusmediumplus0.6%agarin15mltube

Seedlingsofclover

Fahraeusmedium（25ml）intubes（39mmx150mm）withcovering50mmbeakers

（d）Inoculatingtheseedlings

Growthchamber（orwelllightedenvironment）at2530C

Pasteurpipettes（sterile）;rubbernipples

Tubeswithseedlingsfrom（c）

（e）Observingtheroothairsunderthemicroscope

Microscopewithphaseorbrightfieldcondenser

Forceps

Filterpaper（sterileandabsorbent）

SeedlingsininoculatedFahraeussolutionfrom（d）

（f）Comparingroothairdeformations

Microscopeasin（e）withcameraattachment