Friday, July 30, 2010

Cultivation of Aerobic and Anaerobic Bacteria

A. Aerobic Bacteria
Main Principle: Provide Oxygen
Normally, atmospheric conditions 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
Main Principle: Exclude Oxygen (reduce the O2 content of cultures).

Oxygen is ubiquitous in the air so special methods are needed to culture anaerobic microorganisms. Obligate anaerobes vary in their sensitive to oxygen, and a number of procedure are available for reducing the O2 content of cultures- some simple and suitable mainly for less sensitive organisms, others more complex but necessary for growth of strict anaerobes.
  • 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. Thioglycoalte 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.
ü  Obligate aerobes grow only at the top of such tubes.
ü   Facultative organisms grow throughout the tube but best near the top.
ü  Microaerophiles grow near the top but not right at the top.
ü  Anaerobes grow only near the bottom of the tube, where oxygen cannot penetrate.

A redox indicator dye called resazurin is added to the medium because the dye changes color in the presence of oxygen and thereby indicates the degree of penetration of oxygen into the medium.
To remove all traces of oxygen for the culture of anaerobes, it is possible to place an O2 consuming gas in the jar holding the tubes or plates. One of the simplest devices for this is an anaerobic jar, a heavy- walled jar with a gastight seal within which tubes, plates, or other containers to be incubated are placed. The air in the jar is replaced with a mixture of H2 and CO2, and in the presence of a chemical catalyst the traces of O2 left in the vessel or culture medium are consumed, thus leading to anoxic conditions.
For Strict anaerobes, such as methanogenic bacteria can be killed by even a brief exposure to O2. In these cases, a culture medium is first boiled to render it oxygen free, and then a reducing agent such as H2S is added and the mixture is sealed under an oxygen- free gas. All manipulations are carried out under a tiny jet of oxygen free hydrogen or nitrogen gas that is directed into the culture vessel when it is open, thus driving out any O2 that might enter. For extensive research on anaerobes, special boxes fitted with gloves, called anaerobic glove boxes, permit work with open cultures in completely anoxic atmospheres.
Stringent anaerobes can be grown only by taking special precautions to exclude all atmospheric oxygen from the medium. Such an environment can be established by using one of the following 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 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. From the air lock the media are placed within the main chamber. Any O2 in the media is slowly removed by reaction with H2, 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).
Non stringent anaerobes can be cultivated within an anaerobic jar. Inoculated media are placed in the jar along with H2 and CO2 generating system. After the jar is sealed, the oxygen present in the atmosphere within the jar, as well as the dissolved in the culture medium, is gradually used up through reaction with the hydrogen in the presence of catalyst. 

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