BIOS 242 Week 1 Lab 2; Isolation of Pure Cultures

Lab2:TechniquesforIsolationofPureCultures LearningObjectives: • Isolatepureculturesfromamixedpopulationofmicroorganismsusingaseptictechnique. • Learnthestreakplatemethod Introduction: Inthislaboratoryexercise,coloniesfrommixturesofEscherichiacoliandSerratiamarcescenswillbeisolatedusingthefour-waystreakmethodassummarizedbelow(Figure1). We will need this image from Materials: A24hournutrientbrothmixtureofSerratiamarsecens(S.marsecens)andEscherichiacoli(E.coli),twonutrientagarplatesper student,inoculatingloops,incinerators/burners Method:StreakPlateMethod–Day1 1. Removeallextraneousmaterialsfromthelabbenchanddisinfectitusing10%bleachsolution. 2. Labelthebottomofoneofthenutrientagarplateswiththebacteriayouwillbestreaking,thedate,and yourinitialsorgroupdesignation. 3. ObtainanutrientbrothtubecontainingS.marcescensandE.coli. 4. Loopsterilizationforthoselabswithreusablemetalloopsfollowtheinstructionsbelow(aandb). If your lab uses sterile plastic loops, use a new loop for each transfer being mindful not totouchit on any surface. Steps5-7usetheaseptictechniqueslearnedinthepreviouslaboratoryexercise. 5. Open the tube of broth culture. While holding the cap in your hand (do not put it down, as thisactionwillcompromiseitssterility)passthemouthofthetubethroughtheflameafewtimes. 6. Placethesterilizedinoculatingloopfromstep5intothemixedbacteriaculture,beingcarefulnottotouch the sidesof the tube. 7. Removetheinoculatingloopwiththebacteriasample,passthemouthofthetubethroughtheflameagain,andcapit.Youarenowfinishedwiththesample.Putitaside. 8. Whileholdingontotheinoculatingloop,liftthelidofthenutrientagarplateuntilitformsabouta45°angle. 9. Deposityoursampleontheouteredgeoftheagarplateanduseyourlooptospreadoutthesample. Spread it in a zig-zag manner along one quadrant of the plate from the edge inwardapproximately1.5cm,asshown inthe figure1 above. 10. Metalloopsonly:Flamesterilizetheloop.Allowtocoolforapproximately10seconds. 11. Dragtheloopgentlythroughyourfirstquadrant(onlyonce).Continuetostreakthesecondquadrantofyourplateinasimilarzig-zagmannerfromtheouteredgeoftheplateinward. 12. Repeatthesameprocedureforquadrants3and4,rememberingtoflamesterilizetheinoculatingloopbetweeneachquadrant ifusingmetalloop. 13. Metallooponly:Flamesterilizetheinoculatinglooppriortoputtingitaway. 14. Whenfinished,incubatetheagarplates(agarsidefacingupwards)atapproximately22°C(roomtemperature)for24 to48 hours. Method:StreakPlateMethod–Day2 PartA:IdentificationofIndividualColonies 1. Cleananddisinfectyourbenchspaceusing10%bleachsolution. 2. Removeyourplatefromtheincubator,sealtheplateswithparafilmanddonotopentheplates. 3. Makeobservationsusingsealedplatesonlyandstudythedistributionofthebacterialgrowth.Individualcoloniesshould beidentifiable. 4. InPartAoftheLabReport,recordyourobservations. 5. Youwillnowusethismixedcultureplateforthesecondpartofthislab. PartB:IsolationofPureCultures 1. Obtainanutrientagarplatefromyourinstructor. 2. Usingamarkingpen,drawalineonthebottomoftheplate,dividingitintotwoequalsections. 3. Labeltheagarplateineachsectionwiththebacteriayouwillbestreaking(S.marcescens,E.coli).Inaddition,writethedate,andyourinitialsorgroupdesignationontheplate. 4. Sterilizeaninoculatingloopintheflameofthegasburnerandallowittocool. 5. Pickasingleisolatedcolonyfromthemixedcultureplateandstreakitontheappropriatesectionof the nutrientagar plate. 6. Flamesterilizetheinoculatingloopandrepeatwithanisolatedcolonyfromtheothertypeofbacteria. 7. Incubatetheplatesatapproximately22°Cfor24to48hours. Method:StreakPlateMethod–Day3 ObserveyourplateandrecordyourfindingsintheLabReport. LabReport Purpose:Pleasedescribeincompletesentencesandinyourownwords,thepurposeofthisexperiment. Observations: PartA:Drawthedistributionandcolorofcoloniesastheyappearonyourplate. ColonyDescription: Part B: Draw your observations and describe the two sides of the plate. If you have individual coloniescomparethemtotheoriginalisolate.Ifnot,giveageneraldescription. S.marcescens: E.coli: Questions: 1. Wereyousuccessfulinisolatingpureculturesfromthemixedbroth?Ifnot,whydoyouthinkthathappened?What couldyouhavedone differently? 2. Whatwasthepurposeofstreakingthefourquadrantsonthefirstplate?Describethegrowthineachquadrantandgivethe reasonsforthispattern. 3. Iftherewerethreemicroorganismsinthemixture,wouldthestreakplatemethodworkforisolatingthedifferentbacteria?Whatmightbesomeproblems?