BIOS 242 Week 3 Lab 1; Differential Staining

Lab5:DifferentialStaining LearningObjectives: • Explaintheprincipleofdifferentialstaining • Applydifferentialstainingtoidentifybacteria Differentialstainusestwoormorestainstospecificallystaincertainstructuresorcellularcomponentswhichcannotbeeasilyobservedusingsimplestains.Differential staining principles are based upon the specific chemical nature and composition of cellular components and therefore, different structures areobservedusingdifferentstainsandstainingprocedures.Differentialstainingoftenbecomesthebasisofidentificationofthebacteriainclinicallabs. Inthislab,wewilllearnaboutfourseparatestainingprocedures. • GramStaining • EndosporeStaining • Acid-fastStaining • CapsuleStaining(SpecialStaining) Exercise1:GramStaining Materials: BrothculturesofS.epidermidis,E.coli,amixedculturecontainingoneGrampositiveandoneGramnegativebacteria;sterileloop,Gramstainingkit,glassslides,Incinerator,DIwater,marker,immersionoil,lenspaper,bibulouspaper,microscope Method: 1. Obtainglassslidesandpurecultures.Writethenameofthebacteriaonthesideoftheglassslide. 2. Usingaseptictechniquemakeathinsmear.Allowittoairdryandthenheatfixit. 3. Addadroportwoofcrystalvioletandallowstainingfor1minute. 4. GentlywashoffcrystalvioletusingafewdropsofDIwater. 5. Addafewdropsofiodinesolutiontotheslideandwaitfor1minute. 6. Washiodinesolutionusingafewdropsofwater. 7. Rinsetheslidewithdecolorizeruntilnomorecolorwashesoff. 8. Rinsetheslidegentlywithafewdropsofwater. 9. Addafewdropsofsafraninandallowtostainfor1minute. 10. Rinsetheslidegentlywithafewdropsofwater. 11. Blottheslidedry. 12. Observeusingthe10X,and40Xobjectivelenses.Recordtheobservationsmadeusingthe40Xobjectiveinthelabreport. 13. Repeatthestepsforallassignedcultures. Exercise2:DifferentialStaining-Observationofpreparedslides Materials: Preparedslidesofendospore,capsuleandacid-faststaining;immersionoil,lenspaper,microscope Method: 1. Obtainthepreparedslides. 2. Observeslidesusingthe10X,and40Xobjectivelenses. 3. Recordobservationsmadeusing40Xobjectivelensinthelabreport. 4. Completethelabreport. LabReport: Purpose:Describethepurposeofthelab. Exercise1:GramStaining Drawtheobservationinsidethecirclesprovided.Labelyourimageappropriately.Writethemagnificationatwhichtheobservationwasmade. S.epidermidis: Additionalobservation/notes: E.coli: Additionalobservation/notes: Mixedculture: Additionalobservation/notes: Questions: Completethefollowingtablebypredictingcolorsofbacteriawith-andthincellwallsastheyareprocessedthroughthestepsofGramstaining. 1. AfellowstudentshowedyouaGramstainedslidewherecellscontainingthickcellwallswerestainedpink.Whatwouldyoutellheraboutthestainingprocedure?Why? 2. AfellowstudentshowedyouaGramstainedslidewherecellscontainingLPSandathinpeptidoglycanlayerwerestainedpurple.Whatwouldyoutellherabout the staining procedure?Why? Exercise2:DifferentialStaining-Observationofpreparedslides Drawtheobservationinsidethecirclesprovided.Labelyourimageappropriately.Writethemagnificationatwhichtheobservationwasmade. Endosporestaining: Acid-FastStaining: Additionalobservation/notes: Capsulestaining: Questions: 1. Describethedifferencesbetweensimplestaininganddifferentialstainingprinciples. 2. Astudentforgottouseheatduringtheendosporestainingprocedure.Howwillitimpacttheobservation? 3. Whyisthecapsulestainingcalledanegativestainingmethod? 4. Whatismeantbytheterm“acid-fast”?