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Aerobic and Anaerobic

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LAB6_AEROBIC AND ANAEROBIC GROWTH

As humans, we are accustomed to thinking that oxygen is essential to life. Microorganisms, however, are quite adaptable and diverse and vary considerably in their oxygen requirements. Some bacteria require oxygen; some can grow with or without it and some are actually killed by oxygen. Bacteria are generally classified into three main groups with respect to oxygen:

1. Obligate aerobes: Like humans, these organisms have an absolute requirement for oxygen. Because aerobic metabolism generates the toxic byproduct hydrogen peroxide, obligate aerobes must produce the enzyme catalase which breaks down hydrogen peroxide to water and oxygen gas.

2. Obligate anaerobes: Not only do these organisms not require oxygen, they are often killed in its presence. Because anaerobic metabolism does not generate hydrogen peroxide, obligate anaerobes generally do not produce catalase. The causative agent of botulism, Clostridium botulinum is an obligate anaerobe. Since the canning process removes the air, Clostridium botulinum can grow in inadequately sterilized canned foods. This isn't normally a problem in fresh foods since they are exposed to air.

3. Facultative anaerobes: These organisms will use oxygen in their metabolism if it is available, but can also grow without oxygen. Again, since aerobic metabolism generates hydrogen peroxide, they produce catalase. E. coli, which is normal flora of the intestine, is an example of a facultative anaerobe.

In addition to these three basic categories of oxygen requirements, bacteria may also be classified as microaerophillic (requires oxygen, but can only tolerate limited amounts), aerotolerant anaerobe (grows in the presence of oxygen, but never uses aerobic metabolism), capnophilic (requires less oxygen and an increased concentration of carbon dioxide), or other variations.

In this experiment you will utilize two methods for the cultivation of obligate and aerotolerant anaerobes. Reducing media contain reagents that chemically combine with free oxygen, reducing the concentration of oxygen (e.g. Thioglycollate broth contains sodium thioglycollate). A small amount of agar is also added in order to increase the media’s viscosity, which reduces the diffusion of air into the media. The dye Resazurin, which is pink in the presence of excess oxygen and colorless when reduced is commonly used as an oxygen indicator.

Another method of cultivation is to incubate the organisms in a Brewer Anaerobic jar or bag. In this system a palladium catalyst in the form of a “Gas Pack” is added to the jar where it combines hydrogen with oxygen to form water. A methylene blue indicator tablet is placed inside the bag. Methylene blue is blue in the presence of oxygen and white when reduced.

Reference: Brown, A.E. Benson’s Microbiological Applications, Laboratory Manual in General Microbiology, Short Version, 12th Edition, McGraw Hill, 2012. LAB EXERCISE 24: Effects of Oxygen on Growth

PERIOD 1

Materials per table of 4: 8 Thioglycollate Broths (FTM) 8 TGYA Shake tubes 2 TSA plates Brewer’s Bag

Plate cultures: Your 6 stock cultures on slants: A1, A2, A3, B1, B2, B3 Additional cultures provided on plates: Enterococcus faecalis Clostridium sporogenes

Procedure:

1. Do not shake or disturb the Thioglycollate broth tubes. Why not?

2. Label the eight tubes of Thioglycollate broth, and aseptically inoculate each of the eight organisms into each of the eight tubes and incubate the tubes at 370C until next lab period.

3. With a marker, divide each TSA agar plate into eight sectors on the bottom of the plate. Label one plate “Aerobic” and the other “Anaerobic”.

4. Streak (straight line) one loopful of each of the eight organisms onto a separate quadrant of each plate. (DO NOT inoculate too much – may be misinterpreted as slight growth)

5. Incubate the “Aerobic” plate, inverted at 370 C. Place the “Anaerobic”plate, inverted, in the Brewer’s Bag. The instructor will demonstrate how to remove the oxygen from the Bag.

PERIOD 2

1. Examine the Thioglycollate broths and TGYA shake tubes and record all observations in the results section of your lab notebook for this lab. Note whether growth occurs at the surface, below the surface or throughout the agar. Classify all eight organisms according to their ability to grow in the presence or absence of oxygen. Some bacteria may produce gas as they grow. This may split the agar or raise the entire deep off the bottom of the tube as the gas displaces the solid material.

2. Discard tubes.

3. Observe the growth of the organisms on the plates which were incubated aerobically and anaerobically or using the reducing medium such as Brewer’s agar. Record all observations in the results section of your lab notebook for this lab. Compare the results and indicate the effects of oxygen on the growth of each of the organisms.

4. Record all observations and make colored drawings as appropriate in your laboratory notebook results section for this lab. Generating a table of results is a great idea to help you answer the questions that follow. Name_________________________________________________________

Section Number______________________________________

6_ AEROBIC & ANAEROBIC GROWTH Post Lab Report (5 pts)

Please answer the following questions using your lab notebook for information then attach the Results and Discussion Sections from your notebook. The results section should include a data table summarizing the growth of all 8 organisms in all the different types of media used. The data table should facilitate a discussion section that addresses the following questions:

1. Based on your results from the Thioglycollate broth (FTM tubes), what are the oxygen requirements of the different organisms tested?

2. Based on your results from the TGYA shake tubes, what are the oxygen requirements of the different organisms tested?

3. Based on your results from the TSA plates (in/out of Brewer’s bag), what are the oxygen requirements of the different organisms tested?

4. Based on your answers in #1-3, was there consistency between the results obtained using the Thioglycollate broths (FTM), the TGYA shake tubes and the TSA plate (+/- Brewer’s bag)? If not, can you hypothesize as to why there may be differences? (You can also look up known oxygen requirements of the organisms and compare to that.)

Questions:

1. What is the role of oxygen in aerobic cellular respiration?

2. What TWO types of metabolism can occur in the absence of oxygen? What is the difference between these 2?

3. Name TWO enzymes that are present in obligate aerobes but lacking in obligate anaerobes. What is the function of each enzyme? Which of the 8 microbes would have these two enzymes?

4. Why is resazurin a useful media additive for the study of anaerobes?

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