Strict anaerobes such as anaerobic streptococci, Bacteroides species and Clostridium. These species are very sensitive to oxygen and that is why a proper anaerobic atmosphere is required for their growth. The choice of anaerobic technique is influenced by the cultures to be performed, the associated cost and space limitation. Anaerobic techniques in clinical microbiology include the use of anaerobic jars, plastic anaerobic bags the pre-reduced anaerobically Sterilized method (PRAS) and anaerobic chambers.
Chambers are used in laboratories that specialize in anaerobic culture work. They are large plastic tents with an incubator and equipment for culturing the anaerobes. The atmosphere inside the chamber is filled with a mixture of carbon dioxide and nitrogen gas. The chamber has an airlock that can be emptied or refilled with nitrogen or oxygen-free gas. It is through this airlock that you can place the culture media inside or remove it from the chamber. The oxygen remaining in the chamber is removed by its reaction with hydrogen in the presence of a palladium catalyst. You work inside the chamber by extending your arms into specialized gloves attached to the chamber walls; this is why it is also called a glove box. Anaerobic chambers can be very economical if properly constructed because the cost of gases for operating the system is minimal and it allows the use of conventional plating media.
You can use anaerobic jars when you want an oxygen-free atmosphere to obtain surface growth for anaerobes. Anaerobic jars are airtight containers with a removed or reduced oxygen level. Each container can hold about 10 to 20 Petri dishes. This type of jar is convenient when you want to culture a small number of anaerobic samples. The jar contains two things: an envelope containing chemicals that generate carbon dioxide and hydrogen, and inoculated media. The oxygen inside the jar is removed by a palladium-catalytic reaction once the jar is closed. There are two types of anaerobic jars: anaerobic jars with vents and jars without vents. Note that this technique is inadequate for cultivating strict anaerobes but can be used for cultivating micro-organisms that require minimal oxygen.
Anaerobic Bag or Pouch
An anaerobic bag or pouch is convenient when you want to process a few cultures at one time and if you want to return the Petri dishes to an anaerobic environment quickly. The pouches are made of clear plastic bags and vary in size. They can hold about one to three Petri-dishes that measure 100 millimeters in diameter. Because the bags are clear, you can visually inspect the plates incubated inside the pouches without opening the anaerobic pouch.
Pre-reduced Anaerobically Sterilized Method of Hungate
The concept of using rubber stoppered culture tubes as individual anaerobic chambers was first introduced by Hungate in 1950. It is a basic technique that provides an effective means of successfully growing the most fastidious anaerobes. The success of this technique depends on your ability to remove air from the tube using oxygen-free gas when the tube is opened. The process involves adding a media constituent using a syringe, boiling off the dissolved air and replacing the remaining air in the tube with oxygen-free gas. This technique is cheap, easy to use and does not require specialized equipment.
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