Purpose

TodescribeaseptictechniquesusedbyCellCulturespecialistsinhandlingproductsfromand/ormammaliancells.

Safety

Protecteyes,mucousmembranes,opencutsandwoundsfromcontactwithbiohazardmaterial.Usegloves,goggles,mask,andprotectiveclothingasnecessary.

Equipment

LaminarfloworBIOLOGicalsafetyhoodasappropriatetothehazardousnatureoftheproject.

Materials

1. Wipes,lint-free.
2. Disinfectant,orquaternaryammonium.
3. Alcohol.70%ethanol.
4. Pipets,sterile,ofappropriatesize.
5. Pipet-Aid,orequivalent.
6. Biohazardwastecontainer.

Procedure

1. Carryoutallculturingoperationisalaminarflowhood.
2. Disinfectallsurfacespriortousewithadisinfectantsolution.
3. Swabdowntheworkingsurfaceliberallywith70%ethanol.
4. Periodicallyspreadasolutionof70%ethanolovertheexteriorofglovestominimizecontamination.Replacethemiftorn.
5. Incaseofanyspill,spreadasolutionof70%alcoholandswabimmediatelywithnon-lintingwipes.
6. Discardglovesafteruseanddonotwearthemwhenenteringanyotherlabarea.
7. Bringintotheworkareaonlythoseitemsneededforaparticularprocedure.
8. Leaveawideclearspaceinthecenterofthehood(notjustthefrontedge)toworkon.Donotcluttertheareatopreventblockageofproperairflowandtominimizeturbulence.
9. Swabwith70%alcoholallglassware(mediumbottles,beakers,etc.)beforeplacingtheminsidethehood.
10. Arrangetheworkareatohaveeasyaccesstoallofitwithouthavingtoreachoveroneitemtogetatanother(especiallyoveranopenbottleorflask).
11. Usesterilewrappedpipetsanddiscardthemafteruseintoabiohazardwastecontainer.
12. Checkthatthewrappingofthesterilepipetisnotbrokenordamaged.
13. Inspectthevesselstobeused:
    • T-flask-MustbefreefromvisIBLecontaminationorbreakage,orlackcontaineridentification.Theplasticcoveringtheflaskmustbeintact.
    • Bottles-Checkforcracks,expirationdates.
    • Spinnerflasks-Checkforcracks,expirationdates,andproperassembly.
14. Discardanybiohazardorcontaminatedmaterialimmediately.
15. Neverperformmouthpipetting.Pipettormustbeused.
16. Whenhandlingsterilecontainerswithcapsorlids,placethecaponitssideifitmustbelaidontheworksurface.
17. MakesurenottotouchthetipofthePipettetotherimofanyflaskorsterilebottle.
18. Cleantheworkareawhenfinishedbywipingwith70%alcohol.

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