Table of Contents
A. Aerobic Bacteria
Main Principle: Provide Oxygen
Atmospheric condition is generally satisfactory for the culture of aerobes or facultative anaerobes. However, for the growth of many aerobes, it is necessary to provide extensive aeration. Forced aeration of cultures is therefore frequently desirable. It is achieved either by vigorously shaking the flask/tube on a shaker or by bubbling sterilized air into the medium. When aerobic organisms are to be grown in large quantities, it is advantageous to increase the exposure of the medium to the atmosphere. This is accomplished by dispensing the medium in shallow layers or by providing aeration by constantly shaking the inoculated liquid cultures.
B. Cultivation of Anaerobic Bacteria
Main Principle: reduce the O2 content of the culture medium and remove any oxygen already present inside the system or in the medium.
Oxygen is ubiquitous in the air so culture of anaerobic microorganisms require special methods. A number of procedures are available for reducing the O2 content of cultures. Some are simple but suitable mainly for less sensitive organisms. Whereas others are more complex but necessary for the growth of strict anaerobes.
- Bottles or tubes filled completely to the top with culture medium and provided with a tightly fitting stopper. Suitable for organisms not too sensitive to small amounts of oxygen.
- Addition of a reducing agent that reacts with oxygen and reduces it to water e.g., Thioglycolate in thioglycolate broth. After thioglycolate reacts with oxygen throughout the tube, oxygen can penetrate only near the top of the tube where the medium contacts air.
- Obligate aerobes grow only at the top of such tubes.
- Facultative organisms grow throughout the tube but best near the top.
- Microaerophiles grow near the top but not right at the top.
- Anaerobes grow only near the bottom of the tube, where oxygen cannot penetrate.
A redox indicator dye called resazurin is added to the medium because the dye changes color in the presence of oxygen. Thereby indicating the degree of penetration of oxygen into the medium.
Strict anaerobes, such as methanogenic bacteria can be killed by even brief exposure to O2. In these cases, a culture medium is first boiled to render it oxygen- free. Then a reducing agent such as H2S is added and the mixture is sealed under an oxygen-free gas. All manipulations occurs under a tiny jet of oxygen-free hydrogen or nitrogen gas, directed into the culture vessel when it is open. Thus driving out any O2 that might enter. For extensive research on anaerobes, special boxes fitted with gloves, anaerobic glove boxes, permit work with open cultures in completely anoxic atmospheres.
Methods of cultivating 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:
- Pre-reduced media
During preparation, the culture medium is boiled for several minutes to drive off most of the dissolved oxygen. 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 flushed with oxygen-free nitrogen, stoppered tightly, and sterilized by autoclaving. Such tubes are continuously flushed with oxygen-free CO2 by means of a cannula, restoppered, and incubated. - Anaerobic Chambers
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. Any oxygen in the media is slowly removed by reaction with hydrogen, 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).
- Anaerobic Jar
Anaerobic Jar: GasPak system 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). After the jar is sealed oxygen present in the atmosphere inside the jar and dissolved in the culture medium, is gradually used up through reaction with the hydrogen in the presence of a catalyst. The air in the jar is replaced with a mixture of H2 and CO2, thus leading to anoxic conditions.
References
- La Scola, B., Khelaifia, S., Lagier, J. C., & Raoult, D. (2014). Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology, 33(10), 1781–1783. https://doi.org/10.1007/s10096-014-2137-4
- Stieglmeier, M., Wirth, R., Kminek, G., & Moissl-Eichinger, C. (2009). Cultivation of anaerobic and facultatively anaerobic bacteria from spacecraft-associated clean rooms. Applied and environmental microbiology, 75(11), 3484–3491. https://doi.org/10.1128/AEM.02565-08
- Burt, R., & Phillips, K. D. (1977). A new anaerobic jar. Journal of clinical pathology, 30(11), 1082–1084. https://doi.org/10.1136/jcp.30.11.1082
- Ayers S. H. (1910). A New Form of Anaerobic Jar. American journal of public hygiene, 20(4), 844–846.
I have a question…In my micro class we discussed the different classifications of bacteria based on oxygen preferences.
we were told that if we inoculate an anaerobe and an aerobe into separate tubes of nutrient broth, the aerobe grows well, but the anaerobe wont be able to grow unless anaerobic conditions are established. This made sense to me completely, however we were then told that if the two microorganisms were inoculated together, they would both grow. I do not understand how that is possible. Are you able to explain that concept further?
Alison
The nutrient broth contains oxygen, so the aerobe grows well but the presence of oxygen (oxygen toxicity) kills the anaerobes. In the second case, if you inoculate both aerobe and anaerobe together. First aerobe will utilize the available oxygen in the system and may generate anaerobic condition. After that anaerobes can grow. Hopefully this makes sense to you.
I want to know about the Gaspak system in detail.I mean chemicals used in the process and their roles.Please!
I need to check if culture media full of facultative an aerobe it will not allow to grow strict anaerobes.any method that only refer strict anaerobe
Tankeshwar Acharya Sir,
If I need to check out the content claimed in the commercially marketed probiotics whats the procedure to be followed to identify their number of CFU and Bacterial species.
Sir,
what are the procedures to grow anaerobic bacteria?
what is the importance and significance of cultivation of aerobic and anaerobic bacteria??