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Main Principle: reduce the O2 content of culture medium and remove any oxygen already present inside the system or in the medium .
Oxygen is ubiquitous in the air so special methods are needed to culture anaerobic microorganisms. A number of procedure are available for reducing the O2 content of cultures; some simple but suitable mainly for less sensitive organisms, others more complex but necessary for the growth of strict anaerobes.
Methods -
(1) Pre-reduced media
During preparation, the culture medium is boiled for several minutes to drive off most of the dissolved oxygen. A reducing agent e.g., cysteine, is added to further lower the oxygen content. Oxygen-free N2 is bubbled through the medium to keep it anaerobic. The medium is then dispensed into tubes which are being flushed with oxygen-free nitrogen, stoppered tightly, and sterilized by autoclaving. Such tubes are continuously flushed with oxygen-free CO2 by means of a cannula, restoppered, and incubated.
(2) Anaerobic Chamber
This refers to a plastic anaerobic glove box that contains an atmosphere of H2, CO2, and N2. Culture media are placed within the chamber by means of an airlock which can be evacuated and refilled with N2. Any oxygen in the media is slowly removed by reaction with hydrogen, forming water; this reaction is aided by a palladium catalyst. After being rendered oxygen-free, the media are inoculated within the chamber (by means of the glove ports) and incubated (also within the chamber).
(3) Anaerobic Jar
The anaerobic jar is a heavy-walled jar with a gas-tight seal within which tubes, plates, or other containers to be incubated are placed along with H2 and CO2 generating system (GasPak system). After the jar is sealed oxygen present in the atmosphere inside the jar and dissolved in the culture medium, is gradually used up through reaction with the hydrogen in the presence of a catalyst. The air in the jar is replaced with a mixture of H2 and CO2, thus leading to anoxic conditions.
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4 сен 2024
