Cultivation of anaerobic bacteria –Production of vacuum, displacement of oxygen with other gases, chemical methods, biological methods and reduction of medium.

  Cultivation of anaerobic bacteria–Production of vacuum, displacement of oxygen with other gases

Anaerobic bacteria differ in their sensitivity and requirement to oxygen. Some are aerotolerant while some are strict anaerobes. Anaerobiosis is achieved by different methods such as exclusion of oxygen, or production of vacuum, displacement of oxygen with other gases, absorption of oxygen by physical or chemical means, and reduction of oxygen.

1.     Production of vacuum

Cultivation in vacuum is done by incubating cultures in a vacuum dessicator. This method is unsatisfactory since some oxygen is always left behind.  The fluid cultures may boil over and the media may get detached from the plates in the vacuum produced. So, this method is not in much use now.

2.     Displacement of oxygen with other gases

Displacement of oxygen with other gases such as hydrogen, nitrogen, helium, or carbon dioxide is another method but this rarely results in complete anaerobiosis.

Another popular method is the use of candle jar.

Candle jar

A candle jar is a container into which a lit candle is introduced before sealing the container's airtight lid. The candle's flame burns until extinguished by oxygen deprivation, which creates a carbon dioxide-rich, oxygen-poor atmosphere in the jar. Candle jars are used to grow bacteria requiring an increased CO₂ concentration (capnophiles). Candle jars increase CO₂ concentrations but there is some amount of O₂ left and compete anaerobiosis is never achieved to grow complete anaerobes.

Microaerophiles can be easily cultivated in a candle jar in the laboratory. A microaerophile is a microorganism that requires lower levels of oxygen than are present in the atmosphere (20% concentration), to survive. Many microaerophiles are also capnophiles, as they require an elevated concentration of carbon dioxide. Candle jars are ideal for cultivating them. The candle jar provides a concentration of carbon dioxide which stimulates the growth of microaerophiles/ capnophiles. 

 

Overall efficiency of candle jars:

For Anaerobic Bacteria:

• Limited Effectiveness: Candle jars are not generally considered reliable for culturing strict anaerobes. While the burning candle consumes oxygen, it doesn't create a truly anaerobic environment. Some oxygen usually remains, which can inhibit or kill strict anaerobes.
• Microaerophilic Conditions: The candle jar creates more of a microaerophilic environment (low oxygen) than a truly anaerobic one. This might be sufficient for some microaerophiles, but not for strict anaerobes.  
• Inconsistent Results: The level of oxygen reduction in a candle jar can be variable, depending on the size of the jar, the size of the candle, and other factors. This makes it difficult to control the exact atmospheric conditions and leads to inconsistent results.

For Capnophilic Bacteria:

• Enhanced CO₂: Candle jars are more effective for growing capnophilic bacteria. The burning candle produces carbon dioxide (CO2), which these organisms require for optimal growth.  
• Suitable CO₂ Levels: The CO₂ concentration achieved in a candle jar is usually sufficient to promote the growth of most capnophiles.
• Convenience: Candle jars are a relatively simple and inexpensive method for creating a CO2-enriched atmosphere for capnophiles.  

Overall Efficiency:

• Capnophiles: The candle jar is a reasonably efficient and convenient method for cultivating capnophilic bacteria. It provides a reliable way to increase CO2 levels, promoting their growth.
• Anaerobes: The candle jar is not an efficient or reliable method for growing strict anaerobes. It does not consistently create a truly anaerobic environment, and other methods (e.g., anaerobic chambers, GasPak systems) are much more suitable. It might work for some microaerophiles which prefer reduced oxygen, but its anaerobic capability is limited.

In summary: The candle jar is a useful tool for culturing capnophiles due to its CO₂ production. However, it is not a reliable method for culturing strict anaerobes, and inconsistent for microaerophiles. More stringent methods are required for anaerobic work.

 

3.     Chemical methods for absorption of oxygen

In the chemical method, alkaline pyrogallol absorbs oxygen. First introduced by Buchner in 1888, this method has since been used with different modifications to produce anaerobiosis.

Pyrogallic acid is added to a solution of sodium hydroxide in a large test tube placed inside an airtight jar to provide anaerobiosis. A small amount of carbon dioxide is formed during the reaction, which may be inhibitory to some bacteria. The method is applied to single tube and plate cultures.

The spray anaerobic dish is a glass dish with its bottom partitioned into two halves. Pyrogallic acid and sodium hydroxide are placed in the separate halves at the bottom of the dish. The top half of petri dish contains the medium.  The inoculated top half  is inverted on top of the bottom half and sealed completely. The dish is then rocked to mix the reagents, producing complete anaerobiosis. The anerobic dish is not in much use now.

A simple modification consists of a Petri dish, between the two halves of which is inserted a metal disc of slightly larger diameter, with a hole in the centre. The metal disc is attached to the bottom half of the petridish with plasticine. Through the central hole, a few pellets of sodium hydroxide and 10 ml of a 10% solution of pyrogallic acid are added. The inoculated half of the petri dish in then inverted on the metal disc and sealed tightly.

Another common method, is the use of a disc of filter paper with the same diameter as a petri dish. It is placed on the top of one half of the dish and a mixture of pyrogallol and sodium carbonate, in dry powder form is spread on it. The inoculated petri plate is inverted over the filter paper and sealed tight with molten wax. The dry pyrogallol mixture is activated by the moisture within the closed system and complete anaerobiosis develops within about two hours.

Instead of alkalline pyrogallol, anaerobiosis is achieved by the use of a mixture of chromium and sulphuric acid (Rosenthal method) or with yellow phosphorous.

1.     Gas pack

Gas packs which can generate CO₂ are generally used in place of candle jars. Gaspak is commercially available as a disposable envelope, containing chemicals which generate hydrogen and carbon dioxide on the addition of water. The inoculated plates are kept in the anaerobic jar, the Gaspak envelope with water added is placed inside and the lid screwed tight. Hydrogen and carbon dioxide are liberated and in the presence of a catalyst such as palladium in the envelope, hydrogen and oxygen combine thus resulting in an anerobic environment.  The Gaspak is simple and effective. Gaspak reduces the oxygen concentration to about 5% and provides CO₂ concentration of about 10%.

An indicator such as reduced methylene blue can be used to verify the anaerobic condition in the jars. It remains colorless anaerobically but turns blue on exposure to oxygen.

2.     Anaerobic jar

The most widely used and reliable anaerobic method is McIntosh-Filde’s anaerobic jar. It consists of a stout glass/metal jar with a metal lid which can be clamped air tight with a screw. The lid has two tubes with taps, one acting as gas inlet and the other as gas outlet. The lid has two terminals which can be connected to an electric supply. On the underside of the lid is a small catalyst chamber containing a layer of palladinised asbestos. Palladium acts as catalyst to combine O₂ in the jar with H+ thus removing O₂.

Inoculated culture plates are placed inside the jar, and the lid clamped tight. The outlet tube is connected to the vacuum pump and the air inside is evacuated. The outlet tube is then closed an dthe inlet tube connected to a hydrogen supply. After the jar is filled with hydrogen, electric terminals are connected to a current supply and palladinised asbestos heated. This acts as a catalyst for the combination of hydrogen with any remaining oxygen present in the jar. . This method ensures complete anaerobiosis. 

There is a risk of explosion which can occur rarely. This can be avoided by modifying the catalyst. Alumina pellets coated with palladium suspended from the lid of the jar act as a catalyst at room temperature, as long as the sachet is kept dry.

      

 3.     Biological methods

Biological methods to establish anaerobic conditions rely on the activity of other microorganisms or biological processes to consume oxygen within a closed environment. Biological method can be used to establish anaerobic conditions.

1. Co-cultivation with Facultative Anaerobes: A facultative anaerobe is a microorganism that can grow in both aerobic (with oxygen) and anaerobic (without oxygen) conditions.

A strict anaerobe when grown with a facultative anaerobe in a sealed container, the facultative anaerobe will use up any available oxygen in the container as it grows. Once the oxygen is depleted, the environment becomes anaerobic, allowing the strict anaerobe to grow. The facultative anaerobe acts as an "oxygen sink."   One half of the solid medium in the Petri´s dish is inoculated with the tested sample, the second half is inoculated with facultative anaerobe which is able to produce anaerobic environment by the consumption of oxygen. Petri dish is sealed with the wax or parafin and cultured.

2. Use of Anaerobic Indicator Organisms: Some microorganisms are very sensitive to oxygen and can serve as indicators of anaerobic conditions. Such an indicator organism, if included along with target anaerobe, its growth (or lack thereof) can tell if truly anaerobic conditions have been achieved. A classic example is Clostridium species, which are strict anaerobes. Their vigorous growth would suggest a suitably anaerobic environment.  
3. Metabolic Activity of the Culture: In some cases, the anaerobic culture itself can create its own anaerobic environment. As the anaerobes grow, they consume available nutrients and produce metabolic byproducts, some of which can further reduce the oxygen levels or create a reducing environment. This is particularly true in deep, unshaken liquid cultures where oxygen diffusion into the lower parts of the culture is limited.
4. Use of Oxygen-Reducing Enzymes: While not strictly a "biological method" in the sense of using whole organisms, some research or industrial applications might use oxygen-reducing enzymes (like oxido-reductases) derived from biological sources. These enzymes can be incorporated into the media to chemically remove oxygen. This is more of a biochemical approach, but it leverages biological molecules to achieve the desired result.

While these biological methods can be useful in certain situations, they are often less precise and reliable than physical or chemical methods (like GasPaks or anaerobic chambers) for establishing strict anaerobiosis. They are more likely to be employed in situations where those more sophisticated tools are not available.

 

4.     Reduction of oxygen in the medium is achieved by using various reducing agents such as 1% glucose, 0.1% thioglycolate, 0.1% ascorbic acid and 0.05% cysteine.

 

5.     Anaerobic chamber

For fastidious anaerobes, pre-reduced media and an anaerobic chamber (glove box) may be used. The anerobic chamber is an air-tight, glass fronted cabinet filled with inert gas with an entry slot for the introduction and removal of materials and gloves for the hands.