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Advice for Working with Anaerobic Chambers

Posted in: Equipment Mastery and Hacks
anaerobic chambers

Working with anaerobic chambers is a unique skill set to have. It is only necessary if you are working with oxygen sensitive compounds. For example, some metallo-proteins require an oxygen free environment to stay in a reduced state, while others are sensitive and even reactive to oxygen. Sometimes working in anaerobic chambers requires a long workday to finish an experiment and is a bit more tedious than working in the oxygenated environment. It takes a long time to prepare and ensure your sample and environment are deoxygenated and working through a pair of gloves slows everything down. Below is some advice in case you find yourself working in anaerobic chambers.  Hopefully this makes them less daunting than they first appear.

Here are my tips, but remember, also get some training from someone that works with the anaerobic chamber. Each one is a little different.

Anaerobic Chambers Are Like Big Glove Boxes

  • Anaerobic chambers are either rigid boxes or a flexible polyvinyl bag.
  • Chambers have either one or two sets of gloves for single or tandem use.
  • They have an airlock transfer chamber to bring items in without affecting the inside environment.
  • The anaerobic chamber contains a gas mix of 5% hydrogen and 95% nitrogen.
  • A palladium catalyst scrubs the environment of any oxygen, down to 0-5 ppm.

Recommendations Before Entering the Transfer Chamber

  • Make a list of the things you require to limit the amount of times you go into and out of the chamber. Too many times wastes gas and time and leads to oxygen contamination.
  • Before bringing your samples or equipment into the anaerobic chambers, purge them of oxygen by bringing them into the transfer chamber. Then cycle the transfer chamber between vacuum and gas mix (95%N2/ 5% H2) no less than three times before you open the inner chamber.  Make sure the chamber isn’t under the vacuum cycle when you open the door. Remember: Always have the inner door closed and sealed!
  • Bring any required equipment into the chamber at least a night before you plan to begin an experiment.
  • Purge or degas buffers/solutions for 30 minutes to an hour with an inert gas such as argon or nitrogen before you bring them in to the intake chamber.
  • Sometimes the catalyst ‘stops working’ and the oxygen detector starts to sense oxygen. Don’t worry, just bake and renew your catalyst as recommended.
  • If you are working with a stable enough protein requiring disulfide reduction, leave the protein to incubate anaerobically with the reductant. Then dialyze the reductant away in the chamber overnight. In place of dialysis, use a desalting spin column to quickly remove any residual reductant.
  • If the protein is not stable enough for overnight dialysis in the chamber, briefly purge it with an inert gas, and then equilibrate in the chamber 30 minutes to an hour prior to disulfide reduction.

While You Are Working in the Chamber

  • Remember to bring out all your trash.
  • Don’t bring ice into the anaerobic chamber – it gives off oxygen. Instead save ice packs and use them in a Styrofoam box.
  • You can flash freeze samples inside the chamber with liquid nitrogen for storage, however, take note of these special considerations:
    • When bringing liquid nitrogen inside the chamber, remove a lot of the gas via vacuum (about half) because liquid nitrogen gives off nitrogen gas while in the chamber filling the chamber up rather quickly. This can be very dangerous due to increased pressure.
    • Always use the proper precautions for working with liquid nitrogen, regardless of where you are working.
  • Stock your chamber, or at least set it up for the next person.   Bring in some of the items listed below:
    • Tips for all sizes of pipets (stock)
    • Beakers for waste (dump and stock each time)
    • Tubes: micro centrifuge tubes, 15 mL conical tubes, and 50 mL conical tubes (stock)
    • Pipets (if possible keep inside at all times)
    • Tabletop centrifuge (if possible keep inside at all times)

Not all Chambers Are Anaerobic

You may see other chambers around the lab, but they aren’t all for anaerobic use.  Don’t confuse the following with anaerobic chambers:

  • Dry boxes
  • Full humidity control
  • Oxygen tissue culture glove boxes
  • Oxygen animal study glove boxes

Anaerobic chambers are pretty intimidating if you have never seen or worked in one. They can be tricky and there is a huge learning curve when working with gasses, regulators, seals, vacuums, redox states, and proteins precipitating or even sometimes evaporating. However, if you take good care of the anaerobic chambers they can last through several PhDs and lead to some interesting science.

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