Main Principle: Provide Oxygen
Atmospheric condition is generally satisfactory for culture of aerobes or facultative anaerobes but for the growth of many aerobes, it is necessary to provide extensive aeration. Forced aeration of cultures is therefore frequently desirable and can be achieved either by vigorously shaking the flask or tube on a shaker or by bubbling sterilized air into the medium. When aerobic organisms are to be grown in large quantities, it is advantageous to increase the exposure of the medium to the atmosphere. This can be accomplished by dispensing the medium in shallow layers or by providing aeration by constantly shaking the inoculated liquid cultures.
B. Cultivation of Anaerobic Bacteria
- Bottles or tubes filled completely to the top with culture medium and provided with tightly fitting stopper. Suitable for organisms not too sensitive to small amounts of oxygen.
- Addition of a reducing agent that reacts with oxygen and reduces it to water e.g., Thioglycolate in thioglycolate broth. After thioglycolate reacts with oxygen throughout the tube, oxygen can penetrate only near the top of the tube where the medium contacts air.
- Obligate aerobes grow only at the top of such tubes.
- Facultative organisms grow throughout the tube but best near the top.
- Microaerophiles grow near the top but not right at the top.
- Anaerobes grow only near the bottom of the tube, where oxygen cannot penetrate.
- 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.
- Anaerobic Chambers
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 air lock which can be evacuated and refilled with N2. Any oxygen in the media is slowly removed by reaction with hydrogren, 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).
- Anaerobic Jar
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 jar and dissolved in the culture medium, is gradually used up through reaction with the hydrogen in the presence of catalyst. The air in the jar is replaced with a mixture of H2 and CO2, thus leading to anoxic conditions.