e Techniques, Tissue Culture and Cell Counting

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1、COCBiotechnologyProgramSterileTechniques,TissueCultureandCellCountingVersion12-7-03•Tissuecultureisatechniqueusedbyscientiststogrowcellsfromvarioustissuesinalaboratorysetting.•Inthisexperimentyouwillpropagateyourowncelllineforthreeweeks,conductingbi-weeklypropagationaswellasquantifyingandcharac

2、terizingyourcellculture.•Thetypeofcellsyouwillbeworkingwithareinsectcells—theyareeasytoobtainandcangrowunderavarietyofconditions(unliketheirmammaliancounterparts).•Cellcultivationrequiresstringentprotocolsandsteriletechnique.Therefore,inadditiontothepropagationofyourcelllineyouwillbecomefamilia

3、rwiththesetechniquesandprotocols.•Youwillalsorecordandgraphthehealthofyourculturebyusinganinstrumentcalledahemocytometertocountbothlivinganddeadcellsinyourcellline.•Finally,inconjunctionwiththeadditionallabsyouwilldeterminetheaverageproteinconcentrationofthecellsinyourculture.Formoreinformation

4、ontheCollegeoftheCanyonsBiotechnologyprogramcontactJimWolf,AssociateProfessorofBiology/Biotechnologyat(661)362-3092oremail:jim.wolf@canyons.eduI.Objectives:1.Tobecomeproficientatsteriletechnique.2.Tounderstandtheimportanceoftissuecultureinthefieldofmolecularbiology.3.Tosuccessfullypropagateyour

5、owninsectcelllineforthreeweeks.4.Tobecomefamiliarwithahemocytometerandbeabletouseittoquantifylivinganddeadcellsinyourcellline.II.Background:Overthenextfewweekswewillbelearningtheart(oops,Imeantscience)oftissueculture.Manyofthemostsignificantadvancesinthefieldofmolecularbiologyhavebeenmadeviatis

6、sueculturetechniques.Isayartoftissueculturebecausealotofwhathasbeenlearnedisthroughtrialanderrorandserendipity.Eachorganismhasuniquetissues,andsothetissueculturetechniquesarebothorganismandtissuespecific.Onceatissuecultureprotocolhasbeenworkedoutitisrarelychanged.Sometissueculturetechniquesanda

7、ctualcelllinesdatebackoverfiftyyears!Therearethreebroadcategoriesoftissueculture:plant,mammalianandother.Mammaliantissuesarearguablythemostdifficult,forthesecellsrequireaconstantstreamofCO2gasinadditiontothenormalcellrequirements.