Method of Anaerobic Jar

Introduction

McIntosh and Fildes’ anaerobic jar is an instrument used in Microbiology laboratory, for the generation of anaerobic condition (anaerobiosis) to culture obligates anaerobes such as Clostridium spp.

Anaerobiosis obtained by McIntosh and Fildes’ anaerobic jar is one of the excellent and most widely used method for anaerobiosis. All O2 must be excluded while cultivating anaerobic organisms. This is accomplished in several ways. 

• Anaerobic media containing reducing agents such as thioglycollate or cysteine may be used. The medium is boiled during preparation to dissolve its components and drive off oxygen.
• The reducing agents eliminate  any residual dissolved O₂ in the medium so that anaerobes can grow beneath its surface.
• Oxygen also may be eliminated from an enclosed work area, often called an anaerobic chamber or anaerobic workstation.

Principle

anaerobic jar works on the principle of evacuation and replacement, where the air inside the chamber is evacuated and replaced with  mixture of gases (consisting of 5%CO₂, 10%H₂ and 85%N₂) . Anaerobic conditions can be achieved by various means.

Anaerobic media containing reducing agents such as thioglycollate or cysteine may be used. The medium is boiled during preparation to dissolve its components and drive off oxygen.

The reducing agents eliminate any residual dissolved O₂ in the medium so that anaerobes can grow beneath its surface.

Oxygen also may be eliminated from an enclosed work area, often called an anaerobic chamber or anaerobic  workstation. Most of the air is removed with a vacuum pump followed by purges with nitrogen gas.A gas mix containing hydrogen is then introduced into the workstation.

In the presence of a palladium catalyst, the hydrogen and last remaining molecules of O₂ react to form water, creating an anoxic environment. Often CO₂ is added to the chamber because many anaerobes require a small amount of CO₂ for best growth.

A method for culturing small numbers of anaerobes is the GasPak system, which also uses hydrogen and a palladium catalyst to remove O₂.

A similar approach uses plastic bags or pouches containing calcium carbonate and a catalyst, which produce an anoxic, carbon dioxide–rich atmo-sphere.

Increasingly in clinical laboratories, the GasPak system is being replaced by a bacterial enzyme that when added to a broth removes oxygen from the broth and the headspace of the container.

Construction

McIntosh and Fildes’ jar consists of a jar of stout glass or metal with a tight fitting metal lid. McIntosh Fildes Anaerobic JarThe lid can be clamped airtight with a screw and is fitted with two tubes with taps, one for introduction of gas inside (inlet) and the other as outlet for vacuum valve.

The lid also contains two terminals that can be connected to an electric supply. A capsule containing alumina pellets coated with palladium is suspended under the lid by stout wires which are connected with the terminals to heat the catalyst for its activity.

Nowadays, catalyst active at room temperature is also available.

Working

• Keep the inoculated culture plates inside the jar along with an indicator.
• Screw tight the lid
• Close the inlet tube and connect outlet tube to a vacuum pump ( at least three quarters of the air of the jar can be removed).
• Note the pressure on a vacuum gauze and when the pressure is reduced to 100 mm Hg (i.e., 600 mm below atmospheric), tightly close the outlet tap.
• Connect the inlet tap is to a hydrogen supply and then open it. Hydrogen is passed through a small wash bottle.
• Bring the reduced pressure up to 760 mm Hg (i.e., atmospheric) by monitoring on the vacuum gauze as 0.
• Switch on the electric terminals for heating the palladinised crystal (When room temperature catalyst is used heating is not required).
• The catalyst helps the combination of hydrogen and residual oxygen to form water. This process is allowed to continue for 20 minutes.
• Incubate the McIntosh and Fildes’ jar in an incubator at 37°C for 48 hours.

Monitoring efficacy of anaerobiosis

Reduced methylene blue indicator is used to check the efficacy of anaerobiasis.

A tube containing reduced methylene blue solution had to kept inside the jar along with the culture plates. Methylene blue is colorless in reduced conditions and turns blue when oxidized.