Procedure

1. Obtainacultureofmousefibroblastcellsinsuspensionculture.Thiswillbeasimpleculturewithminimalrequirements,andoneselectedforexcellentgrowthcharacteristics.Thetransferprocedurewillbesimilartothatforprokaryotes,withafewmajorchanges.First,alltransferswillbedoneinatissueculturehoodinordertomaximizeasepsis.Secondly,thecellswillbetransferredwithsiliconizedpipettesratherthanwireloops.Thesiliconepreventsadheranceofthecellstotheglasswallofthepipette.

   Atissueculturehoodisadevicethathasairmovinginlayersandunderpositivepressure.Sincetheairisfiltered,itcontainsminimalnumbersofbacteriaorfungalspores,andsinceitisunderapositivepressure,thoseparticlesthatarepresentareblownoutofthehood.Thelayeringpreventsairborneorganismsfromsettlingontheworksurfaces.

2. Arrangethematerialsinfrontofyou,easilyaccessIBLethroughtheopeningofthetissueculturehood.Ensurethatanyalcoholsandwrappingpaperarekeptclearofthebunsenburner.Pre-sterilizethehoodbeforeuse,andusedisposablesterilegloves.

3. Loosenthecapofatissuecultureflaskandthecapofastockbottleoftissueculturemedia.6

   Insertthetipofasterilepipetteintothestockbottleandremove10mlofmedia.Transferthemediatothetissuecultureflask.

4. Openthetopofthesuspensioncultureanduseasterile1.0mltransferpipettetoremovea1.0mlsampleoftheculture.Transferittothefreshmediainthecultureflask.Secureallcapsthathavebeenloosened.

5. Placethenewculturesinanincubatorat37°C.

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