微生物培养试验

EXERCISE2

PRIVATE

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.Petridishwith Figure2.2.Placementof

ComponentsofFahraeusslide seedlingsonFahraeusslide

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(1mmx24