Successful cell culture depends heavily on keeping the cells free from contamination by microorganisms such as bacterial, fungi, and viruses.  Nonsterile supplies, media, and reagents, airborne particles laden with microorganisms, unclean incubators, and dirty work surfaces are all sources of biological contamination.

Aseptic technique, designed to provide a barrier between the microrganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these sources.  The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.

 

Video 2: Sterile Technique

Video: Sterile Techniques

This video is focused on the steps you should take to prevent contamination of your cell culture.  All of the basic actions required to perform cell culture using best practice sterile techniques are demonstrated.

Sterile Work Area

The simplest and most economical way to reduce contamination from airborne particles and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a cell culture hood.  

  • The cell culture hood should be properly set up and be located in an area that is restricted to cell culture that is free from drafts from doors, windows, and other equipment, and with no through traffic.  
  • The work surface should be uncluttered and contain only items required for a particular procedure; it should not be used as a storage area.
  • Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely.
  • For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially after any spillage.
  • You may use ultraviolet light to sterilize the air and exposed work surfaces in the cell culture hood between uses.
  • Using a Bunsen burner for flaming is not necessary nor is it recommended in a cell culture hood.
  • Leave the cell culture hood running at all times, turning it off only when they will not be used for extended periods of time.

Good Personal Hygiene

Wash your hands before and after working with cell cultures. 

In addition to protecting you from hazardous materials, wearing personal protective equipment also reduces the probability of contamination from shed skin as well as dirt and dust from your clothes.

Sterile Reagents & Media

Commercial reagents and media undergo strict quality control to ensure their sterility, but they can become contaminated while handling.  Follow the guidelines below for sterile handling to avoid contaminating them.  Always sterilize any reagents, media, or solutions prepared in the laboratory using the appropriate sterilization procedure (e.g., autoclave, sterile filter).

Sterile Handling

  • Always wipe your hands and your work area with 70% ethanol.
  • Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood. 
  • Avoid pouring media and reagents directly from bottles or flasks.
  • Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to avoid cross contamination.  Do not unwrap sterile pipettes until they are to be used.  Keep your pipettes at your work area. 
  • Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resealable bags to prevent microorganisms and airborn contaminants from gaining entry.
  • Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and never leave it open to the environment.  Return the cover as soon as you are finished. 
  • If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down.
  • Use only sterile glassware and other equipment. 
  • Be careful not to talk, sing, or whistle when you are performing sterile procedures. 
  • Perform your experiments as rapidly as possible to minimize contamination.