Cultivating anaerobic microorganisms, which thrive in environments devoid of oxygen, requires specific techniques and equipment to create conditions conducive to their growth. Here’s a detailed note on the cultivation of anaerobes:
1. Anaerobic Chambers
Anaerobic chambers, also known as glove boxes or anaerobic workstations, provide an oxygen-free environment for handling and cultivating anaerobes.
These chambers typically have a controlled atmosphere with low oxygen levels (less than 1 ppm).
2. Anaerobic Jars
Anaerobic jars are a simpler alternative to anaerobic chambers. They create an oxygen-free environment through chemical oxygen scavengers and gas-generating systems.
A common method involves using gas-generating packs (e.g., GasPak™) that release hydrogen and carbon dioxide, creating an anaerobic atmosphere in an airtight jar.
3. Anaerobic Culture Media
Design specialized anaerobic culture media to support the growth of anaerobic bacteria and other microorganisms.
Thioglycolate broth, reduced media, and Brewer’s anaerobic agar are media for cultivating anaerobes.
4. Inoculation Techniques
Inoculation of anaerobic media should be performed in the anaerobic environment to prevent exposure to oxygen.
Techniques include using syringes with long needles or cannulas to transfer cultures from one environment to another without exposure to air.
5. Gas Packs and Gas Mixtures
Gas packs containing chemical sachets generate anaerobic conditions by removing oxygen from the chamber and releasing gases that support anaerobic growth.
Often, use nitrogen and carbon dioxide gas mixtures to replace the oxygen in the culture environment.
6. Anaerobic Indicator Systems
Anaerobic indicator systems, such as redox indicators, help monitor the anaerobic conditions within the culture environment.
For example, resazurin is a common indicator that changes color in the presence of oxygen.
7. Use of Anaerobic Joints and Valves
In some setups, anaerobic joints and valves transfer liquids or gases between different vessels or compartments without exposing the cultures to oxygen.
8. Pre-Reduced Media
Prepare and dispense media under anaerobic conditions or pre-reduce them by boiling to remove dissolved oxygen.
Pre-reduced media are then dispensed into tubes or plates in an anaerobic chamber.
9. Temperature and pH Control
Maintain anaerobic cultures at the optimal temperature and pH for the specific anaerobe being cultured.
Use temperature-controlled incubators with anaerobic conditions to support growth.
10. Strict Aseptic Techniques
Maintaining strict aseptic techniques is crucial to prevent contamination with aerobic microorganisms during inoculation and handling.
Cultivating anaerobes requires specialized equipment, media, and techniques to create and maintain an oxygen-free environment. Researchers working with anaerobes must be well-versed in these methods to ensure successful cultivation and accurate study of these microorganisms.
