Cultivation Of Anaerobes

The cultivation of anaerobes is a critical aspect of microbiology, particularly in fields such as medical research, environmental science, and industrial processes. Anaerobic microorganisms, which thrive in environments devoid of oxygen, play essential roles in various biological and chemical processes. These organisms are found in diverse habitats, including deep soil layers, sediments, the gastrointestinal tracts of animals, and anaerobic digesters used in waste treatment.

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Understanding the cultivation of anaerobes is vital for several reasons. In medical research, anaerobes are studied to understand their role in the human microbiome and their involvement in infections. In environmental science, they are crucial for nutrient cycling and bioremediation. Industrially, anaerobes are harnessed for processes such as fermentation and biogas production.

Cultivating anaerobes in the laboratory presents unique challenges due to their sensitivity to oxygen. Unlike aerobic microorganisms, anaerobes require specialized techniques and equipment to create and maintain an oxygen-free environment. This article will delve into the various methods and media used for the cultivation of anaerobes, highlighting the importance of maintaining anaerobic conditions to ensure successful growth and study of these microorganisms.

Anaerobic Environment

To cultivate anaerobes, it is essential to create an environment that mimics their natural, oxygen-free habitats. This can be achieved using several methods:

Anaerobic Chambers

Anaerobic chambers, also known as anaerobic glove boxes, are sealed containers filled with an oxygen-free gas mixture, typically nitrogen, hydrogen, and carbon dioxide. These chambers allow researchers to manipulate cultures without exposing them to oxygen. Key features include:

Gas Mixture: The chamber is filled with an anoxic gas mixture to displace oxygen.
Gloves: Built-in gloves allow for handling of cultures and equipment inside the chamber.
Catalysts: Palladium catalysts are often used to remove any residual oxygen by converting it to water in the presence of hydrogen.

GasPak Systems

GasPak systems are simpler and more cost-effective than anaerobic chambers. They consist of jars containing chemical sachets that react to remove oxygen and generate carbon dioxide. The process involves:

Chemical Reaction: The sachets contain chemicals like sodium borohydride and citric acid, which react to produce hydrogen and carbon dioxide.
Oxygen Removal: Hydrogen reacts with oxygen in the presence of a palladium catalyst to form water, effectively removing oxygen from the jar.
Indicator Strips: Methylene blue or resazurin strips are used to confirm the absence of oxygen, changing color when oxygen is present.

Reducing Agents

Reducing agents are chemicals added to culture media to lower the oxidation-reduction potential, creating a more favorable environment for anaerobes. Common reducing agents include:

Thioglycolate: Often used in thioglycolate broth, it helps maintain a low oxidation-reduction potential.
Cysteine: Another reducing agent that can be added to media to create anaerobic conditions.
Ascorbic Acid: Sometimes used in combination with other reducing agents to enhance the anaerobic environment.

Cultivation Techniques

Roll Tube Method

Developed by Robert E. Hungate, this technique involves rolling tubes of agar medium under cold water to create a thin layer. The medium is pre-reduced by bubbling anoxic gas through it, ensuring an oxygen-free environment throughout the process.

Preparation: Tubes of agar medium are boiled and then cooled under anoxic conditions.
Gas Bubbling: An anoxic gas mixture is bubbled through the medium to remove any residual oxygen.
Rolling: The tubes are rolled under cold water to create a thin layer of solid agar, ensuring anaerobic conditions throughout the process1.

Anaerobic Jars and Bags

Anaerobic jars and bags are used for smaller-scale cultivation. These methods include:

Anaerobic Jars: Similar to GasPak systems, these jars use chemical reactions to create an anaerobic environment. They are often used for culturing clinical samples.
Anaerobic Bags: These are flexible, sealable bags that can be filled with an anoxic gas mixture. They are useful for transporting anaerobic cultures.

Anaerobic Glove Box

An anaerobic glove box is an advanced setup where all manipulations are done inside a sealed box filled with an anoxic gas. This method allows for more complex procedures without exposing cultures to oxygen2. Features include:

Sealed Environment: The glove box is completely sealed to prevent oxygen entry.
Gas Purging: An anoxic gas mixture is continuously purged through the box to maintain anaerobic conditions.
Built-in Equipment: Some glove boxes come with built-in incubators and other equipment for convenience.

Media Preparation for Cultivation of Anaerobes

Selection of Media Components

The media used for anaerobes must provide all necessary nutrients while maintaining a low oxidation-reduction potential. Common components include:

Carbon Sources: Glucose, peptone, or other organic compounds.
Nitrogen Sources: Peptone, yeast extract, or ammonium salts.
Minerals: Essential minerals like magnesium, calcium, and iron.
Vitamins and Growth Factors: Some anaerobes require specific vitamins or growth factors.

Preparation of Culture Media

Solid Media: Agar-based media such as Schaedler Agar or Blood Agar.
Liquid Media: Broths like Thioglycolate Broth or Brain Heart Infusion Broth.

Deoxygenation of Media

To remove dissolved oxygen from the media, several techniques are employed:

Boiling: The media is boiled for several minutes to drive off most of the dissolved oxygen.
Reducing Agents: Adding reducing agents like thioglycolate, cysteine, or ascorbic acid helps maintain a low oxidation-reduction potential1.
Gas Bubbling: Bubbling an oxygen-free gas (e.g., nitrogen) through the media further reduces oxygen levels.

Sterilization

Autoclaving: The media is sterilized by autoclaving at 121°C for 15-20 minutes. This process must be done in sealed containers to prevent reintroduction of oxygen.

Addition of Reducing Agents

After sterilization, additional reducing agents may be added to ensure the media remains anaerobic:

Thioglycolate: Commonly used in broths to maintain anaerobic conditions.
Cysteine: Often added to solid media to lower the oxidation-reduction potential.

Dispensing Media

Solid Media: Poured into Petri dishes or tubes under anoxic conditions.
Liquid Media: Dispensed into sealed tubes or bottles, ensuring minimal exposure to air.

Storage and Handling

Storage: Media should be stored in airtight containers to prevent oxygen infiltration.
Handling: All manipulations should be done in an anaerobic chamber or using anaerobic techniques to maintain the oxygen-free environment.

Common Media for Anaerobes

Thioglycolate Broth: Contains thioglycolate as a reducing agent and is used for cultivating a wide range of anaerobes.
Schaedler Agar: Enriched with nutrients and reducing agents, suitable for fastidious anaerobes.
Blood Agar: Often used for clinical samples, enriched with blood to support the growth of pathogenic anaerobes.

Challenges and Solutions

Cultivating anaerobes can be challenging due to their sensitivity to oxygen. Here are some common challenges and solutions:

Oxygen Contamination: Ensure all equipment and media are pre-reduced and handled in an anaerobic environment.
Slow Growth: Some anaerobes grow slowly, requiring extended incubation periods. Patience and careful monitoring are essential.

Applications

Anaerobic bacteria are used in various applications, including:

Medical Research: Studying the human gut microbiome and its impact on health.
Industrial Processes: Waste treatment and biogas production.
Food Industry: Fermentation processes for products like yogurt and sauerkraut.

Conclusion

The cultivation of anaerobes is a fundamental aspect of microbiology, essential for advancing research in medical, environmental, and industrial fields. By mastering the techniques to create and maintain an oxygen-free environment, researchers can successfully grow and study these microorganisms. Methods such as using anaerobic chambers, GasPak systems, reducing agents, and specialized media preparation are crucial for achieving optimal conditions for anaerobic growth. Understanding and applying these methods not only enhance our knowledge of anaerobes but also contribute to significant advancements in health, industry, and environmental sustainability. The cultivation of anaerobes, therefore, remains a vital skill for microbiologists aiming to explore the diverse and impactful roles of these microorganisms.