Tips for Aseptic Technique in the Laboratory -- Handling Microbial Cultures and Media (Part 2 of 4)

Cara N. Wilder, Ph.D.

When working with any microbial strain, propagation success depends heavily on the prevention of cross-contamination by other microorganisms. Sources of contamination can include non-sterile supplies, media, reagents, unclean work surfaces and incubators, airborne particles, and unclean gloves. Below, we describe some tips recommended by ATCC for handling microbial cultures and media.


Maintain a clean, uncluttered bench
Photo provider: ATCC

Maintain a Sterile Work Area

• Before and after use, disinfect all work surfaces with 70% ethanol. This is especially important after any spills.
• Maintain an uncluttered work space; all work surfaces should only contain equipment that is required for your experiment. 
• Ensure that you have all necessary supplies before beginning an experiment. Being prepared will reduce the likelihood of careless contamination.
• Work may be performed in a thoroughly sterilized biosafety cabinet. Biosafety cabinets can be sterilized via ultraviolet light in conjunction with 70% ethanol.



Use sterilized incubators and
equipment
Photo provider: ATCC

• Do not open windows or use fans that circulate outside air.  If possible, work in laboratory settings that have air vents covered with filters. This will prevent the contamination of cultures by airborne particles. 
• Frequently clean water baths used for thawing or warming media or solutions. 
• Routinely sterilize incubators used for microbial propagation. 

Handling Media

• Before and after use, sterilize the outside container of all media and reagents with 70% ethanol.  Also, do not leave containers of media open longer than necessary.
• Aliquot sterile solutions into smaller volumes whenever possible. If you are unsure of the sterility of your media, it is best to discard it immediately.
• Avoid pouring sterile liquids from one container to another, this increases the likelihood of aerosolization as well as media contamination. Rather, use filtered pipette tips for the aseptic transfer of media.  This will prevent contamination of the pipettor and any subsequent cross-contamination. 
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  Aseptically transfer media using a filtered pipette tip.
  This is best performed in a sterile environment such as a
  biosafety cabinet.
  Photo provider: ATCC

  Never mouth pipette. This poses a health risk for personnel as well as increases the risk of contamination.
• Always use sterile glass or disposable plastic pipettes to work with liquid media.  Use each pipette only once to avoid cross contamination. 
• Only unwrap sterile pipettes or open sterile media and petri dishes when you are ready to use them.
• Avoid sharing media and reagents with coworkers. 
• Always use separate bottles of media with each microbial strain, this will prevent cross contamination.

Handling Microbial Cultures

• Before working with media and microbial cultures, wipe your hands and work area with 70% ethanol.
• Ensure you are wearing appropriate protective clothing. This will protect you from the culture as well as reduce accidental culture contamination.
• Only use sterile glassware, equipment, media, and reagents. Check media for contamination by observing for turbidity.
• Handle only one microbial culture at a time. The risk of cross contamination or misidentification increases when more then one strain is handled at a time. 
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  If possible, handle microbial cultures within a biosafety cabinet
  Photo provider: ATCC

  When handling a microbial culture, work quickly and carefully in an environment that has minimal distractions.
• Avoid passaging and subculturing microbial strains too many times. Continually subculturing a strain increases both the risk of contamination as well as genetic drift.
• Inspect cultures daily for signs of contamination.