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Aseptic Technique

Author: Nanci Donacki
Source: Contributed by Nanci Donacki
Date Added: Tue May 14 2002
Date Modified: Wed Apr 28 2004
Abstract: Aseptic techniques used by Cell Culture specialists in handling products from and/or mammalian cells.


To describe aseptic techniques used by Cell Culture specialists in handling products from and/or mammalian cells.


Protect eyes, mucous membranes, open cuts and wounds from contact with biohazard material.  Use gloves, goggles, mask, and protective clothing as necessary.


Laminar flow or biological safety hood as appropriate to the hazardous nature of the project.


  1. Wipes, lint-free.
  2. Disinfectant, or quaternary ammonium.
  3. Alcohol. 70% ethanol.
  4. Pipets, sterile, of appropriate size.
  5. Pipet-Aid, or equivalent.
  6. Biohazard waste container.


  1. Carry out all culturing operation is a laminar flow hood.
  2. Disinfect all surfaces prior to use with a disinfectant solution.
  3. Swab down the working surface liberally with 70% ethanol.
  4. Periodically spread a solution of 70% ethanol over the exterior of gloves to minimize contamination.  Replace them if torn.
  5. In case of any spill, spread a solution of 70% alcohol and swab immediately with non-linting wipes.
  6. Discard gloves after use and do not wear them when entering any other lab area.
  7. Bring into the work area only those items needed for a particular procedure.
  8. Leave a wide clear space in the center of the hood (not just the front edge) to work on.  Do not clutter the area to prevent blockage of proper air flow and to minimize turbulence.
  9. Swab with 70% alcohol all glassware (medium bottles, beakers, etc.) before placing them inside the hood.
  10. Arrange the work area to have easy access to all of it without having to reach over one item to get at another (especially over an open bottle or flask).
  11. Use sterile wrapped pipets and discard them after use into a biohazard waste container.
  12. Check that the wrapping of the sterile pipet is not broken or damaged.
  13. Inspect the vessels to be used:
    • T-flask - Must be free from visible contamination or breakage, or lack container identification. The plastic covering the flask must be intact.
    • Bottles - Check for cracks, expiration dates.
    • Spinner flasks - Check for cracks, expiration dates, and proper assembly.
  14. Discard any biohazard or contaminated material immediately.
  15. Never perform mouth pipetting.  Pipettor must be used.
  16. When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.
  17. Make sure not to touch the tip of the pipette to the rim of any flask or sterile bottle.
  18. Clean the work area when finished by wiping with 70% alcohol.
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