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Renin Angiotensin Mechanism.

Cultivation of Anaerobes.

 

Introduction:

  • Anaerobes are the bacterias that don't require presence of atmospheric oxygen for their growth they are further divided as,

    • Obligate anaerobes: Strict anaerobes (Sensitive to oxygen).

    • Facultative anaerobes: Less sensitive to Oxygen.

  • Oxygen is ubiquitous in the air so special methods are needed to culture anaerobic microorganisms.

  • Main Principle that works here is to reduce the O2 content of the culture medium and remove any oxygen already present inside the system or in the medium .

  • The environment necessary for growth of anaerobic bacteria can be achieved by one of the following methods;

  1. Displacement of Oxygen with other gases viz. Nitrogen, hydrogen, Carbon dioxide etc.:

  • Famous example is Candle Jar, a jar in which a lit candle is kept and the jar is closed, it is expected that all oxygen will be consumed by burning candle but practically this doesn't happen, some oxygen always remains behind.

  1. Use of Chemicals:

  • Pyrogallic acid is added in a test tube containing Sodium Hydroxide and is kept in an airtight jar the mixture in the test tube removes oxygen in the jar by a chemical reaction.

  • Anaerobiosis can also be induced by jars containing mixture of chromium and sulfuric acid which also removes oxygen from the environment.

  1. Cultivation in Vacuum:

  • This is attempted by incubating cultures in a dessicator jar by applying vacuum but some oxygen is always left behind.

  • Moreover the application of vacuum causes detachment of media from plates and fluid cultures to come out of the tube due to the application of vacuum.

  1. Addition of a reducing agent:

  • 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.

  • 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.


  • All above methods are not useful for obligate anaerobe for them. A more strict environment is needed and specific instruments like “Anaerobic Chamber, Special Medias, Anaerobic jar are used”.

Anaerobic Chamber:

  • Consist of 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 hydrogen, forming water; this reaction is aided by a palladium catalyst. 

  • After being made oxygen free, the media are inoculated within the chamber (by means of the glove ports) and incubated (also within the chamber).

Anaerobic Jar:

  • Also called “McIntosh and Fildes Anaerobic Jar”.

  • It 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 systems (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 catalyst (Palladium). 

  • The air in the jar is replaced with a mixture of H2 and CO2, thus leading to anoxic conditions.

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.
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