16/03/2015

BacteriophageIsolation

BacteriophageIsolation

Aswediscussedinclass,bacteriophagearevirusesthatinfectbacteria(bacteriophagemeansbacteria
eater).BacteriophagethatinfectE.colisometimesarereferredtocoliphage.Generally,
bacteriophagearereferredtosimplyasphage.Asistrueforallviruses,phagecanreplicateonly
withinhostcells.Inotherwords,coliphagecanreplicateonlywithinE.coli.Phagemustattachtoa
receptoronthesurfaceofabacterialcellinordertoinitiateaninfection.Thisinteractionbetweenthe
phageandreceptorisveryspecificagivenphagetypeonlywillbindtoaspecificreceptor
molecule.Thus,allphagearenotalike.
Aswehaveseeninthelab.,notallE.coliarealike.Variabilityexistsinthegrowthcurvesand
antibioticresistanceofdifferentE.coliisolates.OnecouldhypothesizethattheseisolatesofE.coli
alsopossesssomedifferencesintheirsurfacemoleculesand,asaresult,differencesinthephage
receptorspresentontheirsurfaces(rememberthesesurfacemoleculesservesomeimportant
functionforthebacteriatheydonotexistonthecellsurfacemerelyasportalsofentryforphage!).
Toexamineif,infact,suchreceptordifferencesdoexist,wewillconductaphagetypingexperiment.
Eachpersonwillisolateaphagethatgrowsreadilyinher/hisE.coliisolate.Then,wewillinvestigate
whetherornoteachofthesephageisolatesiscapableofreplicatingwithintheotherE.coliisolates.
Attheendofthisexperiment,wewillbeabletoproduceatablethatshowsthesensitivityor
resistanceofeachperson'sE.colitoeachofthephageisolates.Eachgroupalsoisencouragedto
developandconductadditionalexperimentswiththeirphageandE.coliisolatesinanattempttolearn
moreaboutthediversityoftheseisolates.Formostoftheseexperiments,yourgroupcanworkonit's
own.Eachpersonmust,however,haveanenrichedphagepreparationbynextweek(April2or3).

BacteriophageIsolation
EachpersonwillisolateabacteriophagespecificforhisorherownisolateofE.coli.Isolatethe
phagefollowingthedirectionsprovided.Becausemostofthestepsareveryshortandrequire1to2
dayincubationsbetweenthem,wewillnotdotheprocedurestogetherasaclass.Rather,wewillgo
overtheprotocolstoday.Youcancarryoutthephageisolationaccordingtoyourownschedule.The
onlyrequirementisthatyoucompleteDay4intimeforlabonApril2or3.

Obtainingbacteriophage
Notsurprisingly,bacteriophagecanbefoundinplaceswherenumeroushostsexist.Because
coliphageuseE.coliasahostorganism,thesephagecanbefoundinplaceswhereE.coliare
plentiful.WesawearlierinthesemesterthatallofusharbornumerousE.coli(andotherbacteria)in
ourlowerintestines.Obviously,thoseE.colileaveourbodies.Thus,sewagecontainsE.colifrom
everyoneintown.And,asaresult,sewagealsocontainsnumerouscoliphage.
IhaveobtainedasewagesamplefromtheRockyRiverwastetreatmentplantinMooresville.From
thismaterial,wewillisolatedifferentstrainsofphage.
Isolationofbacteriophage
Day1:Inoculate5mLtrypticsoybrothwithyourE.coli.Aswedidbefore,touchasterileneedleto
acolonyandtransferittothetubeofbroth.Incubateovernightat37oC.
Day2:Inoculateanothertubewith4.5mLsewage,0.5mLofyourovernightE.coliculture,and
0.5mLof10xtrypticsoybroth(whatdowemeanby'10xtrypticsoybroth'?Whydoweuse10xTSB
forthisstep?).Incubate2448hoursat37oC.Duringthisincubation,phageinthesewagesample
capableofbindingtoyourE.coliwillreplicateinandeventuallylysethebacteria.Thus,thisstep
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16/03/2015

BacteriophageIsolation

servesasameansofamplifyingthephagethatcaninfectyourbacteria.
Centrifugeyourtubeinthetabletopcentrifugefor10minutesat2500rpm.Remembertobalance
thecentrifuge!Thiscentrifugationstepshouldcausethebacteriaandothercelldebristoformapellet
atthebottomofthetube.
Takeup3mLofthesupernatentintoa5ccsyringe.Attacha0.22micronfiltertothebaseofthe
syringeandfilterthesupernatentintoasteriletube.Thisfilterwillpreventanyremainingbacteria
frompassingthrough,butwillallowphagetopassthrough.Labelthefiltrate"Enrichedphageprep."
Storeintherefrigerator.
SetupanotherovernightcultureofyourE.coli.
Day3:Transfer100microlitersofanovernightE.coligrowthontothecenterofa60mmPetriplate
containingtrypticsoyagar(theseplateswillbeintherefrigerator).Withabentglassrod,spreadthe
bacteriaovertheplate.Next,place10microlitersofyourenrichedphageprepinthecenterofthe
plate.Incubateat37oCovernight.
Day4:Examineyourplate.Becauseweaddedthousands(ormaybemillions!)ofbacteriatothe
plate,wewillnotseeisolatedcolonies.Ratheraconfluentlawnofbacteriawillcovertheagar.The
phage,though,willinfectandkillthebacteriainthecenteroftheplate,creatingavisibleregionofno
bacteriaaplaque.Thissimpleexperimentdemonstratesthatyouhaveisolatedphagecapableof
infectingyourstrainofE.coli.Returntheplatetotheincubatorandincubateitforseveralmore
days.Doyounoticeanythingunusual?
Onthedaybeforenextweek'slab,inoculateatubeoftrypticsoybroth(approximately5mL)
withyourE.coliisolateandincubateovernightat37oC.

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