Life without oxygen – getting started with anaerobic microorganisms

For most of us, culturing bacteria is usually quick and easy. With pre-poured plates, ready-made media, purpose built flasks and shakers that stay within a fraction of a degree of the temperature you set them to, culturing cells on agar or in liquid is almost second nature to microbiologists. There is a group of microorganisms, however, that are a little more involved when being grown in the lab – the anaerobes!

In the past decade there has been a resurgence in research into anaerobic microorganisms for their application in industrial settings. One of the biggest hurdles in scaling up a biological process is finding a method to get enough oxygen into the medium for bacteria to grow optimally. With anaerobes, whether they be facultative or obligate, we can completely circumvent this hurdle for ‘easy’ industrialisation.

These microbes are also interesting from a clinical standpoint: possibly the most famous anaerobe that affects humans is Clostridium difficile – a bacterium that alongside MRSA has been one of the key examples of the dangers of overuse of antibiotics. Alongside C. tetani and C. botulinum (the strains behind tetanus and botulism) these three are incredibly important agents of anaerobic infections and much work is done into studying them, as well as other anaerobic pathogens.


Clostridium difficile is a well known anaerobic pathogen

When it comes to actually growing anaerobes in the lab, there are several things that need to be taken into consideration. ‘Anaerobe’ is a broad moniker covering several different groups of microorganisms. Generally, this is split in two to give us ‘facultative’ and ‘obligate’ anaerobes – those that can survive by using oxygen if it is available, and those that cannot use oxygen at all. These two ‘categories’ can be split down even further, which leads to some complications. Many obligate anaerobes find oxygen toxic, and even low levels will prevent growth, but some are oxygen tolerant – meaning they will grow in oxygen but not use it, making them practically very different to the oxygen-sensitive microorganisms. There are also ‘microaerophiles’ which are not truly anaerobic, but are poisoned by atmospheric concentrations of oxygen so require a lowered oxygen environment.

Clearly, the first step to ‘growing your anaerobe’ is to ‘get to know your anaerobe’ – will it be able to survive in oxygen, how much can it tolerate, and if it is a facultative anaerobe, do you want it to be growing aerobically or anaerobically for this part of your experiment. After a good literature search or a chat to with someone who’s worked with your organism before, the next step is to have the right medium for the job.

There is a vast variety of different media available for growing microorganisms, I won’t attempt to give a comprehensive list here but there are some features of media for anaerobes that make them stand out as different. First and foremost is the present of a reducing agent; cystine is common but many others appear in different media for different organisms. There may also be a redox indicator in the medium which will let you know whether you’ve truly achieved anaerobic conditions. As I touched on in the first paragraph, many anaerobic media are available ready-made so if it’s a common organism that you’re working on the chances are you’ll be able to buy your medium or plates off the shelf.

Now that you have your organism and something to grow it in, you’ll need a way to make and keep the environment anaerobic. The more sensitive your organism is to oxygen, the more of a problem this will be. There are a few methods of removing oxygen from the environment, which tend to boil down to:

  • Removing the oxygen chemically, via reducing agent or chemical catalyst.
  • Displacement of oxygen by other gases.
  • Creation of a vacuum.

If your organism can tolerate brief exposure to oxygen, you may be able to transfer plates to a small chamber known as an anaerobic jar. After streaking your plates in aerobic conditions, you can place them into the chamber, which is connected to a pump that will insert nitrogen gas or carbon dioxide (or some other gas or gas mixture), pushing the oxygen out to achieve anaerobic conditions. Variations on this that rely on gas-producing chemical reactions inside the chamber to displace oxygen are also widely chemically available.

anaerobic jar

An anaerobic jar – inside is a bag with chemicals that produce gas when mixed

If the exposure to oxygen whilst placing plates inside the anaerobic jar will be toxic to your organism, then a larger anaerobic environment will need to be used to prepare your plates or medium. Larger anaerobic chambers such as anaerobic glove boxes are available and allow you to prepare your media anaerobically to remove oxygen at all stages of the experiment. These tend to rely on gas displacement, with a vacuum air lock to allow you to take things in and out of the chamber.

An important thing to note when your microorganism is particularly sensitive to oxygen is that most commercially available gases will contain too much trace oxygen to be usable for your organisms. There are two ways to get around this. There are purer, oxygen free gases available for purchase, but these are much more expensive, so if you are running a large amount of experiments in anaerobic environments you might want to consider the second alternative. This involves removing the extra oxygen yourself, the most common method of which is by passing the gas over copper turnings heated to 350 Celsius. This promotes the reaction of the trace oxygen with the copper to form copper oxide which will be left behind when your gas emerges at the other end of the copper-filled tubes. The advantage of this is that copper oxide can be reduced back to copper by the addition of hydrogen, meaning you aren’t expending the copper in the process.

So now that you have your organism, your medium and plates, and your anaerobic environment, you’re ready to start your research into anaerobes. Of course, there are a lot of things that I haven’t touched upon in this post, particularly the vast array of techniques specific to working with anaerobes, for that I recommend looking at the literature for your organism as we could fill textbooks with the different methods used in the field. The most important thing to remember (apart from ‘don’t open the door to the airlock when it isn’t ready’) is that like aerobes, all anaerobes are different and grow optimally in different conditions – so pick the best tools for the job.

Robert G Millar (University of Warwick)

Society for Applied Microbiology

Categories: Early Career Scientists

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