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Zero Tolerance: A Perfectionist’s Guide to Aseptic Technique

Posted in: Cells and Model Organisms
Zero Tolerance: A Perfectionist's Guide to Aseptic Technique

Arguably, molecular biology is impossible without microbiology – even if you work exclusively with transgenic mice, you may one day need to amplify a vector in E. coli. And microbiology is definitely impossible without good aseptic technique.

The main principle of good microbiological practice is a zero tolerance approach: it’s good to be a little paranoid about maintaining sterile conditions. If in doubt whether something is sterile, assume that it is not. For example, tips that have touched anything, even a supposedly sterile part of the outside of a bottle, or have just been on the pipette for too long while you carry it around, should be discarded. Here are a few pointers for maintaining good sterile technique:

1) Labcoat

  • Sleeves should be cuffed, to prevent them from getting dirty and sweeping your sterile area.
  • Separate coats for the culture room and everything else are a must.

2) Work area

  • Don’t assume that the last person who worked in the biological safety cabinet sterilised the surface: do it yourself, and make sure that everything you bring into the sterile area is treated with ethanol on the spot.
  • Do clean up after yourself, because accumulated dirt is a wonderful place to breed bacteria and spores.
  • Keep the surface of your bench reasonably tidy, so that getting your pipette from the bottle to the flask doesn’t mean completing an obstacle course.
  • While working with bacteria or yeast, keep a Bunsen burner lit: this creates an airflow similar to that in a biological safety cabinet, which keeps potential contaminants flowing away from you.
  • Briefly pass through the Bunsen burner flame the necks of glassware such as bottles and flasks, but not any plastic items such as bottle lids, stoppers, single use pipettes, etc.

3) Media

  • Label open media bottles with the opening date. Never return an open bottle to the unopened media storage area. If possible, keep the bottle for your personal use only – this is probably the only justified instance of lab selfishness.
  • Always gently swirl the bottle of medium before using it – the naked eye can see 106 cells. You will develop a six sense for contamination after awhile.
  • If you suspect contamination, your can store “rich” media such as LB at room temperature for several days to make sure that it remains clear, but ‘sterile’ water and buffers should be binned if you think they are contaminated.
  • It’s a bad idea to re-sterilise a suspected medium, because even when you kill the offending bug, you don’t know what nasty stuff it has secreted into your medium.
  • Never put anything back into a sterile container, even if you are 100% sure that it is still sterile. This is especially important for solutions – if you taken more than necessary, discard it.

What are your tips for good aseptic technique?


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  1. PLD on September 4, 2012 at 9:32 pm

    Unless you’re made of money or time, you can’t frog algae without a flame. (though even a pilot light will do)

    But if you can’t keep from smoking your hood, well…..


  2. PLD on August 30, 2012 at 5:29 pm

    Have to disagree on the Bunsen burner part. If you run a burner on your bench, you’re creating a draft and turbulence, which draws non-sterile air and dust over your plate. Once through the flame, everything’s sterile, but you’re not working above the flame.

    Here’s how to keep your plates free of contamination when on the bench:

    Don’t work under or near a air vent. (drafts)

    Spray the bench with 70% EtOH (70% works best for various physiological reasons)
    Work quickly but safely.
    EtOH spray and wipe the bottle tops prior to opening.
    Keep things covered when not drawing fluids (things only come out of the bottle, never back in)
    Keep your plates covered when not dispensing onto them.

    Finally: The bench is for BSL1 yeast and bacteria. The hood (where the flame is fine) is for all else.

    • bgarrett on September 4, 2012 at 2:44 pm

      I would argue that if you are using proper Aseptic Technique that a flame is NOT required in a “hood”. In fact, the CDC and the National Sanitation Foundation (in Standard 49: Biosafety Cabinetry: Design, Construction, Performance and Field Certification) ‘recommend’ not using open flames in a BSC. This is echoed by BSC manufacturers.

      Like you mention in your comment, flames create updrafts that can cause airflow disruptions in the BSC or laminar flow Clean Bench.

      Also, when not used properly, flames from Bunsen Burners have been known to melt the potting agent (typically polyurethane) of the HEPA filters, rendering them useless.

      This is one of the reasons why the use flammable gasses in BSCs are recommended against.

      If an open flame is absolutely necessary in your operations and needed inside a BSC, I would recommend a low profile on-demand flame system with a automatic valving and a pilot light. These days, they can be motion sensitive or touch command operated.

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