BIOS 242 Week 1 Lab 1; Culture Transfer Techniques

Lab1:CultureTransferTechniques LearningObjectives: • Identifytheimportanceofaseptictechniqueinthefieldofmicrobiology • Applytheconceptofaseptictechniqueanditsimportanceinthefieldofmicrobiology. • Identifydifferentformsofbasicgrowthmedia • Transferapurebacterialculturefromonegrowthmediatoanother,aprocesscalledsub-culturing. Introduction: Materials: Nutrientbroth,Nutrientagarslants,Nutrientagarstabs,liquidandslantculturesofSerratiamarcescens, inoculatingloop,inoculatingneedle,incinerators NotetoStudents: 1. Youmayusemetalloops/needlesordisposableplasticloops/needles.Theinstructionsmayvarybased on the type. Please check with your instructor regarding usage and disposal of loops andneedles. 2. Youmayuseincineratororburnerwithflametosterilizemetalloopsandneedles.Pleasecheckwithyourinstructorregardinghowtosafelyuseincineratorsorburnerstosterilize. Method: 1. Removeextraneousmaterialsfromthelabbenchanddisinfectitwith10%bleachsolution. 2. Obtain24hourculturesofSerratiamarcescens(brothandslant). 3. Labelthesteriletubes(broth,slant,andstab)withthenameoftheorganismandyourgroupdesignation. 4. Loop sterilization for those labs with reusable metal loops following the instructions below (aandb).Ifyourlabusessterileplasticloops,useanewloopforeachtransferbeingmindfulnottotouchit on any surface. 5. Transferfromabrothculturetoanewbrothculturebyfollowingthestepsoutlinedina-h. 6. Transferfromabrothculturetoaslantculture 7. Transferfromabrothculturetoaslabculture 8. Transferfromaslantculturetoabroth,slant,orstabculture 9. Whenfinished,incubatethetubesatapproximately25oCfor24to48hours. Nextlabclass: 10. Examine the cultures for appearance of growth. Broth cultures should appear turbid (cloudy).Slantandstabculturesshouldhaveorange-redgrowthonthesurfaceoftheslantandalongthelineof inoculation in theslantculture. 11. RecordyourfindingsintheLabReport. LabReport Purpose: Pleasedescribeincompletesentencesandinyourownwords,thepurposeofthisexperiment.Allowsus topracticethesteriletechnique Observations: BrothStockCulture:EC SlantStockCulture:SM Questions: 1. Whyisproperaseptictechniqueimportantinmicrobiology? 2. Whatistheimportanceofflamingtheinoculatinglooporneedlebeforeandaftereachinoculation? 3. Ifyoudonotwait10–20secondsafterflamesterilizingtheinoculatinginstrumentsbeforeobtainingthe sample,what mightbe theconsequences? 4. Whyisitimportanttoflameneckofthetubesimmediatelyafteruncappingandbeforerecappingthe tubes? 5. Thestabtubewasinoculatedwithaneedle.Whywasthisusedinsteadoftheinoculatingloop? 6. The Serratia marcescens cultures were accidently incubated at 37o C instead of 25o C. Youobserved growth but the slant and stab cultures were white, not orange-red. Does this meanyoursample wascontaminated?