Cultivation of Anaerobes

Cultivation of Anaerobes

Principle - Remove any existing oxygen in the system or the medium and reduce the oxygen content of the culture medium. As oxygen is ubiquitous in the air, anaerobic microorganisms require special culture methods. In terms of reducing the oxygen content of cultures, there are several options available; some simple, others complicated, but all necessary for the growth of strict anaerobes.

• Fill culture medium to the top of bottles or tubes and provide a tightly fitting stopper. Useful for organisms that don't require a lot of oxygen.
• In thioglycolate broth, a reducing agent, such as thioglycolate, is used to reduce the oxygen. After thioglycolate reacts with oxygen throughout the tube, oxygen can only penetrate the tube near the top of the tube.

1. These tubes only support aerobes growing at the top.
2. Growing organisms are found throughout the tube, but near the top are the most productive.
3. Despite being near the top, microaerophiles are not growing there.
4. In anaerobic environments, where no oxygen can penetrate the tube, anaerobes grow only near the bottom.

The medium is stained with resazurin, a redox indicator dye that changes color when exposed to oxygen and shows how much oxygen is being absorbed by the medium. O2 can kill methanogenic bacteria, even after a brief exposure to the gas. This process involves first boiling a culture medium to make it oxygen-free, adding a reducing agent, such as H2S, and sealing the mixture under an oxygen-free gas. Manipulation is carried out under pressure while an oxygen-free jet of hydrogen or nitrogen gas is used. When a vessel is open, the jet is directed into the vessel to drive out any oxygen. Anaerobic glove boxes, which are equipped with gloves, are used for extensive studies of anaerobes on open cultures in anoxic environments. In order to grow stringent anaerobes, special precautions have to be taken to exclude all atmospheric oxygen from the medium. The following methods can be used to create such an environment:

Pre-reduced Media

A few minutes of boiling removes most of the dissolved oxygen from the culture medium during preparation. It is possible to further lower the oxygen content of the solution by adding a reducing agent, e.g., cysteine. Anaerobic conditions are maintained by bubbles of oxygen-free N2. After dispensed into tubes, the medium is flushed with oxygen-free nitrogen, capped tightly, and autoclaved to sterilize. Incubation is carried out in these tubes by continuously flushing them with oxygen free CO2 through a cannula and re-stopping them.

Anaerobic Chambers

We describe an anaerobic glove box made of plastic that contains hydrogen, carbon dioxide, and nitrogen as an atmospheric composition. It is possible to rehydrate N2 to fill the chamber after removing culture media. The chamber contains an air lock through which media are placed. Hydrogen is slowly reacted with oxygen in the media to form water, and a palladium catalyst aids this process. By inoculating the media inside the glove ports, the media are rendered oxygen-free and then incubated (also within the glove ports).

Anaerobic Jars

Anaerobic jars are sealed containers with heavy-walled walls that contain tubes, plates, or other incubating containers, along with a system for generating H2 and CO2. The oxygen present in the atmosphere of the jar and dissolved in the culture medium are gradually consumed by hydrogen in presence of the catalyst following the sealing of the jar. An oxygen-depleted environment is caused by the replacement of air with a mixture of H2 and CO2.

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