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Anaerobic Jar Technique

In this anaerobic jar technique post we have briefly explained about mcintosh and filde’s anaerobic jar, anaerobic jar system procedure, catalyst, indicator, and disadvantages of anaerobic jar system.

Anaerobic Jar Technique

Anaerobic jar system provide the method of choice when an oxygen-free or anaerobic atmosphere is required for obtaining surface growths of anaerobes.

Anaerobic Jar System

Anaerobiosis obtained by McIntosh and Filde’s anaerobic jar system is the most dependable and widely used method. The jar (c. 20 × 12.5 cm) should be made of metal or robust plastic with a lid that can be clamped’ down on a gasket to make it air tight. 

Glass jars have been used in the past, but, as explosions occasionally occur, their use is not justified. The lid is furnished with two tubes with valves, one acting as gas inlet and the other as the outlet. 

The lid also has two terminals which can be connected to an electrical supply. On it’s under surface it carries a gauze sachet carrying alumina pellets coated with palladium (palladinized alumina). It acts as a room temperature catalyst for the conversion of hydrogen and oxygen into water.

Anaerobic jars

Anaerobic Jar System: McIntosh and Filde’s anaerobic jars

Procedure

Inoculated culture plates are placed inside the jar with the medium uppermost and lid downwards and the lid clamped tight. 

The outlet tube is connected to a vacuum pump and the air inside is evacuated. Approximately 6/7 of the air is evacuated (pressure reduced to 100 mm Hg, i.e. 660 mm below atmospheric) and this is monitored on a vacuum gauge. 

The outlet tap is then closed and the inlet tube connected to a hydrogen supply. Hydrogen is drawn in rapidly. As soon as this inrush of gas has ceased the inlet tap is also closed and the jar is held on the bench for 10 minutes. 

If the catalyst is normally active, a decrease in pressure of at least 20 mm Hg occurs within the 10 minutes period after the admission of hydrogen. This can be detected on the gauge when the valve is opened.

Catalyst

After the jar is filled with hydrogen, the electrical terminals are connected to a current supply to heat the catalyst and if room temperature catalyst is used, heating is not required. 

Catalysis will continue until all the oxygen in the anaerobic jar system has been used up. The catalyst will help to combine hydrogen and residual oxygen to form water. The anaerobic jar system is then incubated at 37°C.

Indicator

An indicator should be employed for verifying the anaerobic condition in the anaerobic jar system. Reduced methylene blue is generally used as indicator (mixture of NaOH, methylene blue and glucose). It becomes colorless anaerobically but regains blue color on exposure to oxygen. 

In addition to, or instead of, using a chemical indicator, some workers include in the jar a plate inoculated with a known strict anaerobe such as Clostridium tetani or Bacteroides fragilis, and of a strict aerobe, such as Pseudomonas aeruginosa. This method is quite reliable if the indicator anaerobe grows and the aerobe does not.

Disadvantages

Any anaerobic jar system has the major disadvantage that the plates have to be removed from the jar to be examined and this, of course, exposes the colonies to oxygen, which is especially hazardous to the anaerobes during their first 48 hours of growth. 

For this reason, a suitable holding system always should be used in conjunction with anaerobic jars, placed in an oxygen free holding system, removed one by one for rapid microscopic examination of colonies, and then quickly returned to the holding system. Plates never should remain in room air on the open bench.

Further Readings

Reference