BIOS 242 Week 2 Lab 2; Simple Staining

Lab4:SimpleStaining LearningObjectives: • Tolearnaboutprincipleofstaining • Tolearntheprocessofsimplestain • Topreparewetmountslides Exercise1:SimpleStainMaterials: OvernightculturesofS.epidermidis,B.subtilis,S.cerevisiae(yeast),inoculatingloop,glassslides,incinerators,crystalvioletoranyotherstainchosenbyinstructor;tongs,stainingracks/trays,immersionoil,lenspaper,bibulouspaper,microscope Method: 1. Obtainacleanglassslide,loopandovernightcultures.Holdtheglassslidesfromtheedgestoavoidtransferringgreaseoroilfromfingerstothe slide. 2. Usingaseptictechnique,transferaloopfullofS.epidermidiscultureatthemiddleoftheglassslide.Spreaditinathinlayer. 3. Allowthesmeartoairdrycompletely.Oncethesmeariscompletelydry,holdtheglassslideusingtongsandquicklypasstheslide1-2timesthroughflame of Bunsenburnertoheatfix thebacteria. 4. Oncebacteriaisheatfixed,allowtheglassslidetocooltoroomtemperature. 5. Add1-2dropsofdilutedstainandwaitfor1minute. 6. RemovethestainandgentlywashitoffusingminimalamountofDIwater. 7. Blotdrytheslideandobserveusing10Xandthen40Xlensofacompoundmicroscope. 8. Recordyourobservationsat40XlensinLabreport. 9. Repeatthemethodusingculturesof B.subtilisandS.cerevisiae.RecordobservationsinLabreportandanswerquestions. Exercise2:Wet-mountSlidesMaterials: OvernightculturesofS.cerevisiae(yeast),inoculatingloop,sterileswabs,glassslides,coverslips,incinerators,dilutedsolutionofcrystalvioletoranyotherstainchosenbyinstructor;tongs, stainingracks/trays, immersionoil,lenspaper,microscope YeastCulture: 1. ObtainS.cerevisiae(yeast)culture,twoglassslides,twocoverslips,inoculatingloop,andstain. 2. Wearglovesbeforehandlingglassslidesandcoverslipsneededtomakewet-mountslides. 3. Handleglassslidesandcoverslipsbyholdingfromsidesratherthanbetweenyourfingerstoavoidgettingtheslidesdirty. 4. TransferasmalldropofS.cerevisiae(yeast)cultureonbothslidesusinganinoculatingloop. 5. Ononeoftheslides,addadropofstain(incaseofstainedspecimen),whiledonotusestainonthesecondslide(unstainedspecimen). 6. Putacoverslipsonbothslidesbystartingwithonesideofcoversliptouchingtotheliquidcultureonthespecimenandthendroppingthecoverslipatanangle.Thistechniqueisusefulinpreventingcatchingofairbubblesunderneaththecoverslip. 7. Usewipestocarefullyremoveexcessliquidfromtheedgesofthecoverslip. 8. Observeslidesusing10X,and40Xlenses.Recordobservationsmadeusing40XlensintheLabreport. CheekCells: 9. Obtainglassslide,coverslip,stain,sterileswab. 10. Swabinsideofyourcheekusingsterileswabandrubthesurfaceoftheglassslide. 11. Add1-2dropsofdilutedstain. 12. Putacoverslipatananglebyfirsttouchingonesideofcoversliptotheliquidontheslideandthendroppingthecoverslipatanangle.Thistechniqueisusefulin preventingcatchingofairbubblesunderthe coverslip. 13. Usewipestocarefullyremoveexcessliquidfromtheedgesofthecoverslip. 14. Observeusing10Xand40Xlenses.Recordobservationsmadeusing40XlensintheLabreport. LabReport: Purpose:Describethepurposeofthelab.Results: Exercise1:SimpleStain Drawtheobservationinsidethecirclesprovided.Labelyourimageappropriately.Writethemagnificationatwhichtheobservationwasmade. Bacteria:B.subtilis Additionalobservation/notes: Bacteria:S.epidermidis Additionalobservation/notes: Exercise2:Wet-mountSlides Drawtheobservationinsidethecirclesprovided.Labelyourimageappropriately.Writethemagnificationatwhichtheobservationwasmade. S.cerevisiae:Unstained Additionalobservation/notes: S.cerevisiae:Stained Additionalobservation/notes: CheekCells: Additionalobservation/notes: Completethefollowingtable: Questions: 1. Describedifferencesbetweenawetmountslideandsimplestaining. 2. Whyisair-dryingimportantbeforeheatfixingthespecimen? 3. Ifyouforgottoheatfixthespecimen,howwillitimpactobservationofspecimen? 4. Youobservedunstainedandstainedyeastspecimen.Whichonewaseasiertoobserve?Explainthereason?