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A Breath of Fresh Air!
Oxygen Tolerance in Bacteria

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What do a bulging can of pork and beans, gangrene, and denitrification have in common? All are the result of anaerobic bacterial action.

Many important chemical processes (including food spoilage, tissue deterioration, and refueling our air with nitrogen) are carried out by bacteria and often the efficacy of these processes is determined by the amount of oxygen present in the environment in which the bacteria live. Much of the time, these processes are carried out by anaerobic bacteria in regions of lakes, oceans, sediments, and leaf litter where there is little or no oxygen available.

Do all bacteria share the same tolerance for oxygen? How can differences in oxygen tolerance be tested?


  • lab notebooks
  • screw capped tubes filled with water
  • screw capped tubes with molten nutrient agar and tryptic soy agar
  • ice bath
  • disposable 1 ml pipettes
  • bacterial cultures
  • incubator
  • ruler
  • disinfectant

    Day 1

    1. Brainstorm with your partner about the sources of nitrogen in our atmosphere. How does it get there? Does it always comprise approximately 79% of natural air? What would happen if the nitrogen concentration in air decreased? Is the nitrogen cycle related to any other element cyles?

    2. In order to prepare for the actual lab, we must practice aseptic technique so that we do not contaminate our cultures and invalidate our experimental results. Follow these instructions and practice the mechanical movements. It may seem silly at first, but good aseptic technique is essential when working with pure culures.

  • Carefully remove the disposable pipette from the wrapping being sure to touch only the blunt end of the pipette NOT the pointed end. DO NOT allow the pipette to touch ANYTHING! DO NOT set the pipette on the lab bench!
  • Using the same hand, hold the test tube by the cap between your little finger and your hand.
  • With your other hand, gently twist the tube to remove it from the cap.
  • Carefully insert the pipette into the open tube. Place your finger over the blunt end of the pipette to keep the liquid that has flowed into the pipette from dripping. You can practice this at home with a straw!
  • Recap the test tube by twisting it back onto the cap. Place the test tube in the rack and pick up another, holding it the same way. KEEP YOUR FINGER OVER THE BLUNT END OF THE PIPETTE CONTINUOUSLY!
  • Remove the cap of this test tube and carefully place the pipette into this tube.
  • Release the liquid in the pipette by removing your finger from the blunt end of the pipette.
  • Remove the pipette, recap the test tube, and discard the pipette into the biohazard bucket.
  • Disinfect the lab area and wash your hands.

    Day 2

    1. Label your test tube of molten media with your initials, the date, and information about your bacterial culture. Record this information in your lab notebook.

    2. Inoculate molten media as you practiced in the last class.

    3. Immediately recap and invert the tube several times to distribute the bacteria throughout the media.

    4. Place the inoculated tube into the ice bath to solidify the agar.

    5. Remove the tube when the agar is solid (a few minutes), loosen the cap to allow oxygen to seep into the tube, and incubate at 30oC until the next class period. Record the time in your lab notebook.

    6. Discard all materials into the biohazard can.

    7. Disinfect the lab area and wash your hands.

    Day 3

    1. Retrieve your cultures from the incubator.

    2. Make general observations about growth patterns, color, etc, into your lab notebook.

    3. Using your ruler, measure (in mm) the distance from the top of the agar to the top of the bacterial growth. Record this in your lab notebook. Measure also, the width of the bacterial band. Record in your notebook.

    4. Discard materials in the biohazard bucket.

    5. Disinfect the lab area and wash your hands.

    6. Make a table of your results. Can you display this information in a graph? Show me!

    7. Answer the discussion questions.

    8. Wash your hands again!

    Discussions Questions/Extensions ......
    After completing the laboratory, answer these questions in your lab notebook:

    1. Which organisms were the most oxygen tolerant? Which were the least oxygen tolerant?

    3. Create a scale with which you may rate the oxygen tolerance of the bacteria (eg. on a scale of 1 to 10). Make a drawing of each test tube illustrating the bacterial growth.

    4. Could oxygen possibly be a factor in the process of denitrification in Lake Bonney?

    5. What general conclusions can be drawn about the denitrifiers in Lake Bonney and their tolerance for oxygen? Can any conclusions be drawn?

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