Is Oxygen Required for Culturing? – Explained!

Some of the microbes that cause infections are normally found on the human body. Specimens sent to the lab for identification will in all likelihood contain Staphylococcus aureus, Streptococcus pyogenes coliforms (e.g., E. coli and Enterobacter), Pseudomonas spp., and Proteus spp.

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Most of these microbes are aerobic and may be grown on petri plates, agar slants, and in broth culture. However, a number of other microbes which might be found on the surface of the body or in the intestinal, respiratory, and genitourinary tracts may function anaerobically.

Microbes in this category include members of the gener” Clostridium, Bacteroides, Yersinia (facultative), Salmonella (facultative), Shigella (facultative), Peptococcus, and Peptostreptococcus.

When resistance to infection is lowered or microbes become displaced, these microbes are able to cause severe and possibly fatal infections. In order to isolate and identify anaerobic pathogens, special lab techniques and procedures must be followed.

As with any culture, successful identification begins with specimen collection. Anaerobes are suspended in infections that are foul smelling, such as gas gangrene. When a specimen is taken, it is sent to the lab in a special “gassed out” collection tube.

This protects the suspected anaerobe from the deadly effects of atmospheric oxygen. To prepare a “gassed out” tube, the atmospheric oxygen normally found in a tube, the atmospheric oxygen normally found in a tube is replaced with CO₂ and stopped before autoclaving.

Swab and body fluid specimens are quickly placed in these special tubes to prevent the loss of CO₂ and the entrance of O₂. (Since CO₂) is a heavy gas, it will stay in the tube unless it is “poured out” by tipping the tube.) If these special tubes are not available, the specimen should not be refrigerated but inoculated into the proper culture media as soon as possible.

Special media such as semisolid sodium thioglycolate may be used to create the necessary reducing conditions (thioglycolate removes oxygen from the medium), or the cultures may be placed in special containers that are kept oxygen-free.

Anaerobic containers used in the lab are the Brewer’s plate, spray plate, Brewer jar, and GasPak anaerobic jar. The simplest of thee is the Brewer’s plate. This petri plate is filled with sodium thioglycolate agar and inoculated with the anaerobic specimen.

The lid is designed with a deep lip to make contact with the surface of the agar when it is placed on the plate. This prevents the outside oxygen- containing air from making contact with the center portion of the culture.

A spray plate looks like a petridisk with an exceptionally deep base divided into two compartments. Potassium hydroxide (KOH) is placed in one side and the chemical pyrogallol is placed in the other.

The inoculated agar-containing lid is placed on top and sealed. By shaking gently, the KOH and pyrogallol are mixed in the base to produce a reaction that will remove oxygen from the atmosphere in the jar.

The anaerobic bacteria have will then are able to grow on the inverted agar surface. Plates incubated upside-down like this have an advantage in that the moisture that condenses in the plate will form on the upper surface. Since the plate is inverted, the culture will be “watered” automatically.

The Brewer jar is more complex and dangerous than either the Brewer’s plate or spray plate. After the cultures have been placed inside and lid sealed, a mixture of H₂, C0₂, and N₂ is flushed through the Brewer anaerobic jar.

This exchange of gases is repeated two or three times before the gas tube is shut off. The oxygen remaining in the jar is then removed by electrically heating a catalyst in the lid.

The catalyst is allowed to operate for about ten minutes before the entire jar is unplugged and placed in an incubator. A more practical and convenient method for culturing anaerobic is the GasPak anaerobic jar.

This is basically the same as a Brewor jar however; the anaerobic atmosphere is not generated in the same manner. A disposable envelope, which generates hydrogen and carbon dioxide, is torn open and placed inside.

The hydrogen gas produced in the jar reacts with the oxygen on the surface of a catalyst to form water. As the oxygen disappears from the atmosphere inside the jar, a mist or fog will appear on the inner wall of the jar and the lid will warm.

When the oxygen has been eliminated, a disposable anaerobic indicator inside the jar will change colour. Anaerobic conditions should be generated in about 25 minutes, and the jar may then be placed in an incubator.

Some pathogenic bacteria are not anaerobic, but do require a reduced amount of oxygen in their growth environment. In order to successfully culture microbes such as mycobacteria and Neisseria in the lab, a modified environment must be established.

These bacteria do not require anaerobic conditions but grow best when the atmosphere surrounding them has been enriched with about 5 per cent carbon dioxide.

This environment can be produced in the lab using a candle jar, biobag environmental chamber, or a carbon-dioxide incubator. Any wide-mouth jar can be used as a candle jar.

The cultures are placed inside with a lighted candle and the lid is sealed. As the candle burns, the oxygen content is reduced from about 20 per cent to 16 per cent, and the CO₂ content is increased from about 0.4 per cent to 4.0 per cent, generating a favourable growth environment.

The biobag environment chamber method is similar to the increase in carbon dioxide is the result of the rapid aerobic metabolism of another plate of bacteria cultured along with the mycobacteria. Usually only one culture plate is needed for about a dozen mycobacteria plates. The aerobe utilizes the O₂ from inside the bag for its own respiration and releases CO₂ into the bag.

Carbon dioxide incubators are usually small units that have been specially adapted to maintain a constant temperature and CO₂ level. Cultures can be placed directly inside and no special techniques are needed.

After the suspected pathogen has been isolated, cultured and identified by means of these special techniques and equipment, the technician may then be asked to perform the antibiotic susceptibility test, one of the most important tests leading to the control of the infection.