Anaerobic bacteria 

• In the broadest sense, obligate anaerobes can be defined as microorganisms which are unable to utilize molecular oxygen for growth.
• Obligate anaerobes do not carry out oxidative phosphorylation.
• Obligate anaerobes are bacteria that can live only in the absence of oxygen. 
• Obligate anaerobes are destroyed when exposed to the atmosphere for as briefly as 10 minutes. 
• Some anaerobes are tolerant to small amounts of oxygen such as Facultative anaerobes are those organisms that will grow with or without oxygen. 
• The methods of obtaining specimens for anaerobic culture and the culturing procedure are performed to ensure that the organisms are protected from oxygen.
• Anaerobes are commonly found on mucous membranes and other sites such as the vagina and oral cavity. Therefore, specimens likely to be contaminated with these organisms should not be submitted for culture (throat or vaginal swab). 
• Anaerobic are killed by oxygen because they lack enzymes such as catalase [which breaks down hydrogen peroxide (H2O2 ) to water and oxygen], peroxidase [by which 1NADH + H2O2 are converted to 2NAD and O2] and superoxide dismutase [by which superoxide, O2., is converted to H2O2]. 
• These enzymes detoxify peroxide and oxygen free radicals produced during metabolism in the presence of oxygen. 
• Aerotolerant anaerobes are only slightly inhibited by significant levels of oxygen in the atmosphere.
• The other extreme is represented by strict anaerobes, which die, or immediately stop growing, upon exposure to low levels of oxygen. It is therefore important to retain anoxic conditions during all steps of handling of these microorganisms.
• Anaerobic respiration includes glycolysis and fermentation. During the latter stages of this process, NADH (generated during glycolysis) is converted back to NAD by losing hydrogen. The hydrogen is added to pyruvate and, depending on the bacterial species, a variety of metabolic end-products are produced.
• Aerobes can survive in the presence of oxygen only by virtue of an elaborate system of defenses. Without these defenses, key enzyme systems in the organisms fail to function and the organisms die. 
• While, Obligate anaerobes, which live only in the absence of oxygen, do not possess the defenses that make aerobic life possible and therefore can not survive in air. 
• The tolerance to oxygen is related to the ability of the bacterium to detoxify superoxide and Hydrogen peroxide, produced as byproduct of aerobic respiration.
• The assimilation of glucose in aerobic conditions results in the terminal generation of free radical superoxide (O2 -). The superoxide is reduced by the enzyme superoxide dismutase to oxygen gas and Hydrogen peroxide (H2O2).
• Subsequently, the toxic hydrogen peroxide generated in this reaction is converted to water and oxygen by the enzyme catalase, which is found in aerobic and facultative anaerobic bacteria, or by various peroxidases which are found in several aerotolerant anaerobes.

Characteristics of Anaerobic Bacteria

• Unable to grow in an atmosphere containing oxygen
• Use fermentative pathways in which organic compounds serve as final electron acceptors
• killed when exposed to the atmosphere for as briefly as 10 minutes
• Some anaerobes tolerant to small amounts of oxygen
• Facultative anaerobes grow with or without oxygen
• Anaerobic bacterial culture: the method used to grow anaerobes from a clinical specimen

Cultivation of Anaerobic Bacteria

Cultivation of Anaerobic Bacteria. Stringent anaerobes can be grown only by taking special precautions to exclude all atmospheric oxygen from the medium. Such an environment can be established by using one of the following methods:

Cultivation of Anaerobic Bacteria using Prereduced media

• During the preparation of Prereduced media, the culture medium is boiled for several minutes to drive off most of the dissolved oxygen. 
• Then 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 N2. stoppered tightly, and sterilized by autoclaving. 
• Such tubes can be stored for many months before being used. 
• During inoculation, the tubes are continuously flushed with oxygen-free Co, by means Oi a cannula, restoppered, and incubated.

Procedure

1. Take a tube of Prereduced media containing an atmosphere of oxygen-free N2. 
2. To inoculate, remove the stopper and insert a gas cannula to flush the tube continuously with oxygen-free CO2 and maintain anaerobic conditions. 
3. Now inoculate the medium by adding a few drops of culture with the help of a Pasteur pipette.
4. After inoculation, the tube is restoppered and incubated.

Cultivation of Anaerobic Bacteria using Anaerobic chamber

• This refers to 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 airlock which can be evacuated and refilled with N2. 
• From the airlock the media are placed within the main chamber. Any O2 in the media is slowly removed by reaction with the H2, forming water; this reaction is aided by a palladium catalyst. 
• After being rendered oxygen-free, the media are inoculated within the chamber (by means of the glove ports) and incubated (also within the chamber).
• It has Glove ports and rubber gloves that allow the operator to perform manipulations within the chamber. 

Procedure

1. Airlock with inner and outer doors. 
2. Media are placed within the airlock with the inner door remaining sealed.
3. Air is removed by a vacuum pump connection and replaced with N2 through. 
4. The inner door is opened and the media are placed within the main chamber, which contains an atmosphere of H2 + CO2 + N2. 
5. A circulator circulates the gas atmosphere through pellets of a palladium catalyst, causing any residual oxygen in the media to be used up by reaction with H2. 
6. After media have become completely anaerobic they can be inoculated and placed in an incubator located within the chamber.

Cultivation of Anaerobic Bacteria using Gas pack

• Gas packs can generate CO2 also and are generally used in place of candle jars. 
• The packet consists of a bag containing a Petri plate and CO2 gas generator. 
• The gas generator is crushed to mix the chemicals it contains and start the reaction that produces CO2. This gas reduces the oxygen concentration in the bag to about 5% and provides CO2 concentration of about 10%.

Procedure

1. Media are inoculated and then placed in the jar. 
2. Water is added to the GasPak generator envelope, causing the evolution of H2 and CO2.
3. The H2 reacts with O2 on the surface of the palladium catalyst, forming water and establishing anaerobic conditions. 
4. The CO2 aids growth of fastidious anaerobes which sometimes fail to grow, or grow only poorly, in its absence. 
5. An anaerobic indicator strip (a pad saturated with methylene blue solution) changes from blue to colorless in the absence of oxygen.

Cultivation of Anaerobic Bacteria using Anaerobic Jar

• Anaerobic jar 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 system (GasPak system).
• Nonstringent anaerobes can be cultured within an anaerobic jar.
• Inoculated media are placed in the jar along with an H2 + CO2 generating system. 
• After the jar is sealed, the oxygen present in the atmosphere within the jar, as well as that dissolved in the culture medium, is gradually used up through reaction with the hydrogen in the presence of a catalyst.
• An Anaerobic Jar consists of the following components; The GasPak Anaerobic System with inoculated Petri dishes, the GasPak generator envelope, and the anaerobic indicator strip.
• Sodium bicarbonate and sodium borohydride are mixed with a small amount of water to produce CO2 and H2 within the Anaerobic Jar.
• A palladium catalyst is used in the jar which combines with the O2 in the jar and the H2 to remove O2.

Cultivation of Anaerobic Bacteria using Candle jar

• Many microphiles are also capnophiles (as they require an elevated concentration of carbon dioxide), they can be easily cultivated in the laboratory by using a 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. 
• Many labs also have access directly to carbon dioxide and can add the desired carbon dioxide levels directly to incubators where they want to grow microaerophiles. 
• Candle jars are used to grow bacteria requiring an increased CO2 concentration (capnophiles). 
• Candle jars increase CO2 concentrations and still leave some O2 for aerobic capnophiles.

Cultivation of Anaerobic Bacteria Biological method

Anaerobic conditions can be established by following this Biological method

• One half of the solid medium in the Petri´s dish is inoculated with the tested sample.
• The second half is inoculated with Serratia marcescens – aerobic bacteria able to produce an anaerobic environment by the consumption of oxygen. 
• Petri dish is sealed with the wax or parafin and cultured in aerobic environment.

References

• https://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/englAnaerob.pdf
• http://www.surendranathcollege.org/new/upload/AMAR_CHANDRA_DAS_GHOSHCultivation%20of%20Anaerobic%20Bacteria2018-11-11Cultivation%20of%20Anaerobic%20Bacteria.pdf
• http://www.ndvsu.org/images/StudyMaterials/Micro/Bacterial-Cultivation.pdf
• https://www.ramauniversity.ac.in/online-study-material/pharmacy/bpharma/iiisemester/pharmaceuticalmicrobiology/lecture-3.pdf
• https://www.basu.org.in/wp-content/uploads/2020/12/Anaerobic_bacteria_culture__Practical_.pdf
• https://www.biologydiscussion.com/microorganisms/culture-media-for-cultivation-of-anaerobic-bacteria-4-types/55049