Overcoming the Challenges of Growing Anaerobic Bacteria

To grow anaerobically means to grow without the presence of molecular oxygen; instead, energy is produced via anaerobic respiration. In general, anaerobes are classified as facultative, aerotolerant, or strict. Facultative anaerobes can grow with or without the presence of oxygen and can metabolize energy aerobically or anaerobically. Aerotolerant anaerobes are uninhibited by oxygen but generate energy without using oxygen via fermentation. Strict anaerobes grow only in the absence of oxygen and may be inhibited or killed if it is present. There are also extremophilic anaerobes, which grow in niche environments that are acidic, excessively hot, or irregular in air pressure; these may also require a gas mixture free of carbon dioxide (CO₂). Finally, methanogens are a class of strict anaerobe that require nitrogen or ammonia sources in their media; as their name suggests, methane is produced as a biproduct of their metabolism.

Selecting the right media

Knowing your organisms’ nutritional requirements is key to establishing healthy growth. The superior choice is pre-reduced anaerobically sterilized (PRAS) commercial media, which is boiled free of molecular oxygen, autoclaved, sealed anaerobically, and stored in light-proof packaging. There are a wide variety of medium options available depending on the organisms’ requirements. Blood-based media are popular for their high density of nutrients, which include brucella, tryptic soy-based blood, and brain heart infusion (BHI) with 0.5% yeast extract. Various supplements can be added to media to meet specific metabolic requirements, including 5% sheep, horse, or rabbit blood; vitamin K1; or hemin. 

It is important to note that most anaerobic organisms prefer to grow in broth (liquid) over agar (solid) media. Common broth media are chopped meat, reinforced clostridial, and peptone yeast extract broth with glucose (PYG). Finally, an indicator compound called Resazurin can be added to some media that has been reduced or drained of oxygen. Resazurin will turn pink at higher redox potentials (more oxygen present) and will appear clear once the biologist has added a sufficient amount of reducing agent. 

Growth conditions

Propagation of anaerobic bacteria has evolved over the years to meet the growth needs of newly discovered isolates. The various media described come in specialized Hungate tubes, which are test tubes with screw caps and a butyl rubber stopper that allows gas exchange through a needle and syringe. There are also Balch tubes, which have a crimped aluminum seal for culturing extremophiles. 

Reduction of the redox potential of growth media is achieved by adding reducing agents such as co-enzyme, cysteine, or sodium sulfide. Reducing agents bind to any oxygen in the media, which prevents the oxygen from interacting with anaerobic organisms. The pre-reduced media and organism are then opened in an anaerobic chamber to ensure a constant flow of oxygen-free gas. Inoculated agar plates and broth can be incubated in special anaerobic jars. Then the jar is connected to an Anoxomat™ system to rapidly remove the oxygen from the jar and replace it with precise anaerobic gas mixtures. Agar plates can also be placed in anaerobic pouches with enclosed sachets that remove unwanted oxygen during incubation. 

Need more information? No problem!

Growing anaerobes can be challenging, but at ATCC we make use of various technologies for atmosphere control as well as specifically formulated media to provide the best possible conditions for propagation. For further information on achieving optimal anaerobic growth conditions, please refer to the following ATCC webinar: Biology of Anaerobic Bacteria and Predominant Propagation Practices. To view our growing portfolio of anaerobic bacteria, please visit the ATCC link: www.atcc.org/Anaerobes. 

Did you know?

ATCC has over 1,000 anaerobic bacteria in our collection, including strains isolated as part of the Human Microbiome Project.