The Fundamentals of Anaerobic Cultivation
Anaerobic cultivation refers to the laboratory process of growing microorganisms that do not require oxygen for growth. While many of the organisms we encounter in daily life are aerobicmeaning they thrive in the presence of oxygena significant portion of the microbial world, including many bacteria and archaea, are obligate anaerobes, facultative anaerobes, or microaerophiles.
Why Anaerobic Cultivation Matters
Understanding anaerobic life is crucial for several scientific and industrial sectors. In medicine, many human pathogens, such as those responsible for tetanus, botulism, and gangrene, are anaerobic. In environmental science, anaerobic microbes are essential for waste treatment, biogas production, and the natural cycling of nutrients in oxygen-depleted environments like deep soil layers or aquatic sediments. In the food industry, anaerobic fermentation is the basis for producing products like yogurt, cheese, and sourdough.
Classifications of Anaerobic Microorganisms
To cultivate these organisms successfully, it is important to categorize them based on their relationship with oxygen:
- Obligate Anaerobes: These organisms cannot survive in the presence of oxygen and may even be killed by it.
- Facultative Anaerobes: These organisms prefer oxygen but can switch to fermentation or anaerobic respiration if oxygen is absent.
- Aerotolerant Anaerobes: These do not utilize oxygen but are not harmed by its presence.
Key Concept: The primary challenge in anaerobic cultivation is the complete removal of molecular oxygen from the growth environment, as oxygen can produce toxic reactive oxygen species (ROS) in organisms lacking protective enzymes like superoxide dismutase or catalase.
Methods of Cultivation
1. Anaerobic Jars
The most common method for small-scale cultivation is the use of an anaerobic jar. After inoculated plates are placed inside, a chemical sachet is added. When water is added to the sachet, a reaction occurs that removes oxygen and produces carbon dioxide or hydrogen. A catalyst (usually palladium pellets) is often included to facilitate the reaction between hydrogen and remaining oxygen to form water.
2. Anaerobic Chambers (Glove Boxes)
For more demanding research, anaerobic chambers are utilized. These are sealed, rigid cabinets with built-in gloves that allow researchers to manipulate cultures in a controlled, oxygen-free atmosphere. The internal environment is typically maintained using an inert gas mixture, such as nitrogen, hydrogen, and carbon dioxide.
3. Reducing Agents
In liquid media, chemical reducing agents are added to decrease the oxidation-reduction potential of the broth. Common additives include sodium thioglycollate, cysteine, or ascorbic acid. These chemicals chemically bind to any residual oxygen in the medium, ensuring an environment suitable for anaerobic growth.
Best Practices for Success
Success in anaerobic microbiology relies on precision. Researchers must ensure that all media are pre-reduced before inoculation. Furthermore, it is essential to minimize the time that samples are exposed to the ambient atmosphere during handling. When working with strict anaerobes, oxygen-free gas flushing (using nitrogen or argon) is often required during the preparation of culture tubes or flasks.
By mastering these techniques, scientists can effectively study the complex metabolisms of anaerobic organisms, leading to advancements in everything from clinical diagnostics to sustainable energy production.
