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Oxygen has a tendency to form very reactive by-products (H₂O₂ and O^2-(superoxide)) inside a cell. These by-products create havoc by reacting with protein and DNA, thus inactivating them. Cells that are able to live in the presence of oxygen have evolved enzymes to cope with H₂O₂ and O^2- and thus are not inhibited by O₂. Also many anaerobes have oxygen labile Fe-S centers and no cellular machinery to protect them from the oxidizing power of oxygen. Organisms that cannot deal with the problems presented by oxygen cannot survive in air and are killed.

On the basis of oxygen tolerance, microorganisms can be placed into four classes. Strict aerobes cannot survive in the absence of oxygen and produce energy only by oxidative phosphorylation. Strict anaerobes, in many cases, generate energy by fermentation or by anaerobic respiration and are killed in the presence of oxygen. Aerotolerant anaerobes generate ATP only by fermentation, but have mechanisms to protect themselves from oxygen. Facultative anaerobes prefer to grow in the presence of oxygen, using oxidative phosphorylation, but can grow in an anaerobic environment using fermentation.

Oxygen utilization is a primary diagnostic tool when identifying microorganisms. Special media has been developed for the purposes of determining the oxygen relationship and method of metabolism (fermentation vs. respiration) of microorganisms. One such medium, Thioglycollate Agar is useful for determining the oxygen relationship of a microorganism. The medium contains thioglycollic acid, cystine and 0.35% agar, among other things. The thioglycollic acid and agar prevent oxygen from entering the entire medium. A dye, resazurin, is used as an indicator of the amount of oxygen in the medium. Resazurin is red in the presence of oxygen and turns colorless under anaerobic conditions. The medium is steamed just before use, which removes all oxygen from the tubes. After inoculation and incubation, oxygen is able to diffuse into the top part of the medium and support growth aerobically, while the bottom half of the medium remains devoid of oxygen.

A second medium used to investigate the general type of metabolism used by a microorganism is glucose O/F medium. This is a rich medium that contains glucose as primary carbon source. A pH indicator dye, brom thymol blue, is added and is green/blue under alkaline-Oxidative conditions or yellow under acidic-Fermentative conditions. Each test organism is inoculated into two tubes of glucose O/F medium. One tube is overlaid with mineral oil and the other is not. The mineral oil serves as a barrier to oxygen, which helps to create an anaerobic environment.

In this experiment you will first investigate the reactions of several known microorganisms having different types of metabolism. You will determine the characteristic reactions of thioglycollate medium and glucose O/F medium. You will then use this information to determine the oxygen relationships and catabolism type of your two unknown isolates.

Protocol for testing oxygen relationships

Period 1

Materials

8 tubes of Glucose O/F Medium

4 tubes of sterile mineral oil

4 tubes of Thioglycollate Agar (melted, in 50 °C water bath)

Cultures of

Pseudomonas fluorescens

Clostridium sporogenes

Enterococcus faecalis

Escherichia coli

2 plates of Brain Heart Infusion Agar

Anaerobe jar

In this exercise we will be first testing the oxygen relationships of some known organisms in Glucose O/F medium and Thioglycollate Agar. This will give you a sense of inoculating test media and allow you to observe their characteristic reactions.

1. Get four tubes of thioglycollate agar from the 50 °C water bath. The thioglycollate agar has been steamed for several minutes to drive off any oxygen. Keep the agar melted by incubating the tubes in a container containing 50 °C water. Label the tubes with the culture names.
2. Inoculate each culture into one tube of Thioglycollate agar. Mix the tubes by placing the palm of your hand over the top of the tube and moving the bottom of the tube in a circular fashion.
3. Incubate the tubes at 30 °C for 2-5 days.
4. Inoculate each culture into two tubes of glucose O/F medium by stabbing the medium the full length of the tube with the inoculating needle.
5. Overlay one tube of each culture of Glucose O/F medium with 2-3 cm of mineral oil. What is the purpose of the mineral oil? Incubate at 30 °C for 2-5 days.
6. Divide each plate into four sectors. Label one plate aerobic and the other anaerobic. Streak each culture onto one sector on each of the plates.
7. Incubate one plate aerobically at 30 °C. Place the other plate in the anaerobe jar. The air will be evacuated and replaced with H₂ + CO₂ atmosphere. The jar will also be incubated at 30 °C.

Period 2

1. Observe the thioglycollate tubes for the known cultures. Notice the growth pattern of each organism. Is growth seen throughout the tube? Is there more growth at the top of the tube? What does that mean? Did any of the cultures exhibit growth only in the bottom of the tube?
2. Make drawings of each test tube culture in your lab notebook. From looking at the tubes can you infer which organisms are strict aerobes, facultative anaerobes, aerotolerant anaerobes or strict anaerobes? Record this information in your lab notebook.
3. Observe the Glucose O/F medium. Look for growth in the tubes and production of acid (yellow color). Bacteria that grow only in the tube without mineral oil have a respiratory form of metabolism. Bacteria that grow in both tubes and produce acid under anaerobic conditions can also use fermentation. What would be the reaction of a strict aerobe in this medium? Record the results in your notebook. Do the results obtained here agree with the results from the thioglycollate experiment?
4. Observe the BHI plates. Record anaerobic and aerobic growth as + or -. Do the results here agree with that which was observed for the other media in this experiment.
5. Catalase test. Add several drops of (H₂O₂) to each area of growth on the plates incubated aerobically. Observe through the top lid of the closed plates so you don't cause an aerosol of live cells to spread from a positive reaction! A positive reaction is indicated by the constant evolution of bubbles. Figure 5-15 is a movie of the catalase test for these six microbes. However, C. butyricum cannot be tested, why?

Figure 5.15 the catalase test

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