Cultivation of aerobic and anaerobic bacteria and their preservation

Cultivation of aerobic and anaerobic bacteria and their preservation :

Culture of Aerobic bacteria:

To grow aerobic or facultative bacteria in tubes or small flasks,incubation of the medium under normal atmospheric conditions is generally satisfactory. However, 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, which special containers are available. Aeration can also be increased by constantly shaking the inoculated liquid cultures.

Anaerobic culture:

Microorganisms vary in their need for, or tolerance of oxygen and accordingly, they are divided into several groups. Aerobes are capable of growth at full oxygen tensions (21% O₂ in air) and many can even tolerate elevated concentrations of oxygen (hydrobromic oxygen). Microaerophiles, by contrast, are aerobes that can use O₂ only when it is present at levels reduced from than in air, usually because of their limited capacity to respire or because they contain some oxygen sensitive molecule such as an oxygen-labile enzyme. Organisms that lack a respiratory system cannot use oxygen as a terminal electron acceptor.Such organisms are called anaerobes. there are two kind of anaerobes: aerotolerant anaerobes which can tolerate molecular oxygen and grow in presence even though they cannot use it, and obligate or strict anaerobes, which are killed by molecular oxygen.The lethal effect on strict anaerobes is probably due to inability to detoxify, superoxide and hydroxyl radical formed.Many obligate anaerobes are rich in flavin enzymes,which can react spontaneously with O₂ to yield these toxic products. By contrast, aerobes have enzymes that decompose toxic oxygen products, whereas anaerobes seem to lack all or some of these enzymes.

For anaerobic culture, the problem is to exclude oxygen, and because oxygen is ubiquitous in air, special methods are needed to culture aerobic microorganisms. Obligate anaerobes vary in their sensitivity to oxygen, and a number of procedures are available for reducing the O₂ content in cultures, some simple and suitable mainly for less sensitive organisms, other more complex but necessary for the growth of strict anaerobes. The isolation of anaerobic bacteria by plating methods poses special problems. Provided that the desired organisms are not rapidly killed by exposure to oxygen, plates may be prepared in the small usual manner and then incubated in closed containers, from which the oxygen is removed either by chemical adsorption or evacuation. For more oxygen sensitive anaerobes a modification of pour plate method known as the dilution shake culture is preferred. for strict anaerobes, techniques as roll tube and anaerobic glove box are employed.

Methods for cultivation of anaerobes:

• Evacuation and replacement of oxygen atmosphere in sealed jars.

1. Brewer anaerobic culture plate
2. The gas Pak system
3. Vacuum and Gas Displacement Method
4. Anaerobic chamber
5. The Candle method

• Specialized techniques were evacuated sealed jars are not used.

1. Solid medium
2. Shake culture method
3. Pyrogallic acid method
4. Roll tube method

• Liquid medium

1. Paraffin plug medium

Plate methods:

There are several techniques of growing anaerobic bacteria on an agar plate. The most common use methods are brewer’s anaerobic plate, Gas Pak jar, Vacuum Gas displacement method

Brewer’s Anaerobic culture plate:

In 1942, J.H. Brewer. Introduced a specially designed Petri dish cover to used with an anaerobic medium for the surface cultivation of anaerobes. It is a relatively simple device and gives satisfactory results. The cover is provided with a circular ridge that rests on the medium, providing very rare space above the medium. An anaerobic condition in the space results from the presence of reducing agents in the medium.

After streaking on the agar surface, the continental petri dish cover is replaced by the sterile brewer cover. The medium should be deep enough so that the Brewer cover does not rest on the edge of the bottom dish. The weight if the cover will cause the cover ridge to settle somewhat into the medium.

The Gas Pak jar:

The anaerobic system is composed of the Gas Pak generator envelope and the anaerobic indicator strip kept inside the anaerobic jar.Medium plates are inoculated and kept inside the jar. Water is added to the Gas Pak generator envelope, causing the evolution of H₂ and CO₂. The H₂ react with O₂ on the surface of the palladium catalyst, forming H₂O and establishing anaerobic environment carbon dioxide is also produced which enhances the growth of fastidious bacteria which sometimes fail to grow, or grow only poorly in its absence.

Vacuum and Gas displacement method

Ideal atmospheric conditions for strict anaerobes can be achieved by displacing the air in a closed container with a mixture of nitrogen and carbon dioxide. A vacuum pump is used to withdraw the air from the chamber containing the culture plates to be incubated. A mixture of the two gasses is then allowed to fill the chamber. The CO₂ serves the same purpose as in Gas Pak system. Many bacteria have difficulty starting growth in the absence of CO₂.

Anaerobic chamber:

This refers to anaerobic glove box which is sealed chamber in which objects are manipulated with the hand inserted into rubber gloves that are attached to the front of the unit; transparent panel allows the manipulation to be viewed from the outside. The chamber has an atmosphere of H_2,CO₂, and N₂. Culture media are placed within the chamber by mean of an air lock, which can be evacuated and refilled with N₂.

The candle method:

Although this method is an old one,it is still used for culturing some bacteria such as Neisseria gonorrhoeae. For many organisms,however, it is considered too toxic because of the amount of carbon monoxide produced by the burning candle. When a lighted candle is placed in a container that is tightly sealed, the O₂ will be used up, CO₂ will be produced, and the candle will quite burning after the O₂ has been exhausted.

References:

Debey, RC and D K Maheshwari. A textbook of Microbiology. India: s.chand and company Ltd., 1999.

MacFaddin, H W. Biochemical test for Identification of medical bacteria. the Williams and Wilkins company, n.d.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.