Cultivation of Anaerobes 

Cultivation of Anaerobes: In an aerobic system, oxygen acts as an electron acceptor. Hence, it is very essential for metabolic activity and respiration.  Molecular oxygen can directly oxidize different essential reduced groups (thiol, – SH group) or enzymes. During oxidation, oxygen gives out a toxic superoxide radical (O2).  Superoxide radicals can inactivate cell components and produce more toxic substances such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH). These radicals are strong oxidizing agents and interfere with metabolism and destroy cellular constituents very rapidly. Aerobic and facultative anaerobic bacteria possess two enzymes, superoxide dismutase and catalase which destroy toxic radicals. Superoxide dismutase converts superoxide radicals to oxygen and hydrogen peroxide while hydrogen peroxide (H2O2) is converted to water and oxygen by the catalase enzyme.

Cultivation of Anaerobes 

Superoxide dismutase and catalase enzymes are not present in anaerobic bacteria and hence it does not show growth in the presence of oxygen. 

Strict or stringent anaerobes can be grown only in the absence of oxygen. Such an environment can be prepared by using one of the following methods. 

(a) Displacement of oxygen with gases such as hydrogen, nitrogen, helium, or carbon dioxide is employed. A popular method is the candle jar. All inoculated plates are placed inside a large air-tight container and a lighted candle is kept before the lid is sealed. The burning candle is expected to use all the oxygen but some oxygen is always left behind. 

(b) Reduction of oxygen in the medium is achieved by the use of various reducing agents e.g. 1% glucose, 0.1%, thioglycollate, 0.1% ascorbic acid, etc. 

(c) Cultivation in a vacuum was attempted by incubating cultures in a vacuum desiccator. In this method, some oxygen is always left behind and fluid culture may boil over and the media may get detached from the plates.

(d) In the chemical or biological method, alkaline pyrogallol absorbs oxygen.  Pyrogallic acid added to a solution of sodium hydroxide in a large test tube placed inside an air-tight jar provides anaerobiosis. Anaerobiosis has also been produced within jars with a mixture of chromium and sulphuric acid. 

McIntosh and Fildes’s anaerobic jar is the most reliable and widely used anaerobic method. Inoculated culture plates are placed inside the jar, with the medium in the bottom half of the plates and a metal lid that can be clamped air-tight with a screw. The outlet tube is connected to a vacuum pump and the air inside is evacuated. The jar is filled with hydrogen and electric terminals are connected to a current supply so that the platinized asbestos is heated. This acts as a catalyst for the combination of hydrogen with the residual oxygen present in the jar.

Anaerobic jar Gas Pak system
Fig.1: Anaerobic jar: Gas Pak system 

The Gaspak is the method of choice for preparing anaerobic jars (Fig.1). The Gaspak is commercially available as a disposable envelope, containing chemicals that generate hydrogen and carbon dioxide with the addition of water. The presence of a cold catalyst in the envelope permits the combination of hydrogen and oxygen to produce an anaerobic environment. Reduced methylene blue is generally used for verifying the anaerobic condition in the jars. It remains colorless anaerobically but turns blue on exposure to oxygen.

Make sure you also check our other amazing Article on : Types of Culture Media
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