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Methods:Sterile Technique

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Sterile technique when working with laboratory strains of E. coli is mostly about preventing contamination of your cultures and media. It's mostly common sense, but those with years of experience watching new lab workers know that what seems like obvious common sense often isn't. Poor sterile technique can not only ruin your experiments - if your poor sterile technique leads to a labwide T1 infection, it will affect a lot more than your own experiments.

Your work area

Think of your work area on your lab bench as being like the mise en place of a professional chef. By preparing what you need ahead of time, your work will be smooth and efficient. Some layouts are shown in the figures [need some pictures]. Some of the common components of your setup will be:

  • Flame: Generally, your work area will have a bunsen burner or alcohol lamp for flaming things. This must be placed in a position where it won't set things on fire, even if you bump it (You should know where the nearest fire extinguisher is in any case). The burner is usually on all the time, creating an updraft that pushes airborne contaminants away. This isn't needed if you are working in a containment hood, but this page is about BL1 work at a normal lab bench.
  • Surface: Do not put down bench paper. See above about fires.
  • Bottles of media
  • Agar plates.
  • Pipets
  • Loops, sticks, and/or toothpicks
  • Jar of ethanol for flaming glass spreader (not needed if you use glass beads)
  • Bucket for used pipets
  • Waste container for solids
  • Your lab notebook

Liquids

Sterilizing liquids

Most labs use one of two methods for sterilizing liquids, depending on what the liquid is.

A lot of bad habits in sterile technique are found in molecular biology labs. The use of antibiotics often lets people get away with bad practices, but antibiotics should not be viewed as a way to sterilize liquids.

Measuring and Pipetting sterile liquids and liquid cultures

Most E. coli biologists use large numbers of glass serological pipets for handling liquids. Plastic disposable serological pipets are also used. When using glass pipets, it is customary to flame the pipet before putting it into anything. This is not to heat the pipet to a temperature that would kill microorganisms; the pipet should already be sterile. The reason for flaming is to create an area of convection that will prevent dust and spores from the air from settling on the pipet.

Working with serological pipets well requires learning some skills to avoid setting a pipet, a bottle cap, or a tube closure down on a surface of questionable sterility (i.e. your bench).

Pipetman and similar products: If you are using a disposable-tip pipettor, such as Rainin's pipetman or similar, for liquid handling, there is a strong risk of contamination. First, since it is impractical to sterilize the barrel of pipetman, you should not use one for anything where the barrel has to extend inside what you are inoculating. It's OK to use pipetmen for things like shallow multiwell plates, where only the sterile tip (assuming you've sterilized it) goes inside the cavity. Even so, it is important to draw the liquid into the tip slowly, to prevent aerosols from forming inside the barrel. Barrier tips can prevent this, but tend to be expensive.


Inoculating liquid cultures from colonies

There are a number of methods that are commonly used:

  • Inoculation loops. Reusable loops are available in platinum and nichrome wire. These two metals heat quickly and cool rapidly when flamed between uses. The platinum loops are much more expensive. Disposable plastic loops are also available.
  • Sterile sticks. Many E. coli biologists use sterile toothpicks or round wooden applicator sticks. Don't flame these!

Touch a colony with a stick. You do not have to pick up a visible glob of cells. Remove the closure on the flask or tube you are inoculating (don't set it down!). Flame the mouth of the flask or tube. Swish the end of the stick in the liquid you are inoculating in a flask or tube. It may help to tip the flask or tube so that the liquid is close enough to the top for the stick or loop to reach. When using sticks or toothpicks, do not drop the stick into the medium. The end you are touching is not sterile!

Inoculating liquid cultures from freezer stocks

Long term storage of E. coli is often in "freezer stocks" (also known as "frozen permanents" stored at -70°C or below. For obvious reasons, you don't want to flame these. You also don't want to thaw then, as freezing and thawing is a good way to lyse E. coli. Scrape a bit of the frozen stock onto your sterile stick or loop, and use that to inoculate your culture, or to streak out a plate for single colonies.

Plates

When working with agar plates, sterile technique is important for both making the plates and using them. See:

Waste disposal

See also

References

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