Choosing the best media for your anaerobe

By Allison Faust

Petri dishes and eyedropperUnderstanding your anaerobe’s nutritional requirements is essential for selecting the correct media. Presently, there are a number of broth and agar media that can be freshly prepared or purchased from commercial suppliers to support the growth of your cultures. Here, we will provide an overview of these types of media as well as the importance of using indicators and reducing agents.

One of the most frequently used media for anaerobic culturing is pre-reduced, anaerobically sterilized (PRAS) media. This medium is available commercially and is superior because it is autoclaved, dispensed, and packaged anaerobically in light-proof packaging. If freshly prepared in the laboratory, this type of medium should be stored in the dark and used within 7 days. Agar plates in particular should be moved into an anaerobic environment, such as a jar or chamber, within several hours of making the media.

Various types of blood agar are commonly used to propagate anaerobes. Common bases for blood agar media are brucella, which is enriched and ideal for fastidious organisms; Columbia, which is a combination of animal and plant proteins to support fastidious and non-fastidious organisms; tryptic soy agar, which supports a number of fastidious and non-fastidious strains; and brain heart infusion agar with 0.5% yeast extract, which is an enriched medium for the cultivation of most anaerobes and other fastidious strains. All of these blood agar bases are non-selective and will support growth for most anaerobes. It is important to note that some anaerobes may perform better on one base over another, so knowing your anaerobe’s nutritional requirements is essential for successful growth. If you have difficulties achieving growth on agar, choose a medium that has supplements. Selective agars are useful when trying to identify an unknown culture or to separate a mixed culture. One example is Bacteriodes Biel Esculin (BBE) agar, which selects for Bacteroides, Bilophila, and Fusobacterium; this agar is supplemented with gentamicin to inhibit aerobes, and bile to inhibit most other anaerobes.

Jars and test tubes on deskBroth can also be used to propagate anaerobes. Chopped meat broth will support the growth of the vast majority of anaerobes. Another option is reinforced clostridial broth, which is a non-selective medium commonly used for growing Clostridium. When experiencing growth problems, peptone yeast glucose (PYG) broth or supplemented tryptic soy broth can stimulate growth in anaerobes due to the addition of carbohydrates, yeast extract, hemin, and vitamin K1. For good growth in broth media, inoculations should not be too dilute and subcultures should not be made too late in the growth curve.

To help ensure that your broth and agar are free from oxygen, reducing agents can be added to most anaerobic media. Reducing agents are oxidized, effectively removing oxygen from the media while depressing the redox potential. Some reducing agents can perform quickly, but generate toxic bi-products (like sodium dithionite). Sodium sulfide precipitates essential metal cations and can be fairly toxic to some anaerobes like ruminants. Cysteine has low toxicity and does not have ancillary chemical reactions, but the rate of reduction is slow (several hours at room temperature); this is the reducing agent ATCC recommends most often. You should select the reducing agent that is used in the recommended media formulation; it is typically added at a ratio of 2 mL of reducing agent to 100 mL of media. Allow the reducing agent to react for at least 1 hour; however, 12 to 24 hours is preferable.

To help monitor the redox potential of agar and broth media, we recommend the use of indicators. Resazurin is commonly used indicator as it is non-toxic to bacteria and effective at low concentrations. Resazurin becomes colorless at a redox potential below -110 mV and remains pink above -51 mV; though, some organisms require redox potentials lower than -110 mV and may not start to grow even if medium is colorless. It is important to note that if the organism produces nitrate, which is a strong oxidant, the redox potential may rise above -51 mV; this will turn the media pink even though it has not been exposed to oxygen.

Lastly, proper media storage is important to prevent inhibitor formation. Inhibitors that typically affect the quality of the media are oxygen, light, moisture, or dehydration; oxygen causes the oxidation of organic ingredients and light creates peroxides. By storing media in the dark and using it within the recommended time frame, you can help prevent inhibitors from forming.