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A Breath of Fresh Air!
Oxygen Tolerance in Bacteria

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Author Contact Information

Sharon Harris
Mother of Mercy High School
3036 Werk Road
Cincinnati, OH 45211
(513) 661-2740

In this laboratory activity, students determine the oxygen tolerance of several kinds of bacteria. Students should have an elementary knowledge of the nitrogen cycle. They should grasp the importance of nitrogen in protein synthesis and understand that nitrogen, though not directly involved in primary production (photosynthesis), is part of many connected biochemical pathways.

Students will

  • practice aseptic technique
  • monitor the growth of bacterial cultures
  • display their results graphically
  • propose environmental problems associated with the oxygen tolerance of nitrogen fixers and denitrifiers

    Grade Level/Discipline
    I use this activity in my 10th grade biology class. It could also be used in an environmental science course during a discussion of elemental cycling.

    National Standards
    Teaching Standard A

  • inquiry-based science program

    Teaching Standard B

  • discussion about scientific ideas
  • modelling skills of scientific inquiry

    Teaching Standard D

  • extended investigation
  • supportive of science inquiry
  • create a safe science working environment
  • use technology and science tools and materials

    Teaching Standard E

  • collaborative studies
  • model the skills and values of scientific inquiry

    Pre-activity set-up

  • subculture bacterial samples as necessary
  • prepare nutrient agar and tryptic soy agar(according to package directions)
  • stabilize temperature of incubator (30oC


  • bacterial cultures (Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, Rhodospirillum rubrum, Bacillus subtilis can be obtained from Carolina Biological)
  • nutrient agar and tryptic soy agar in screw capped tubes
  • incubator (set at 30oC)
  • disposable 1 ml pipettes
  • biohazard bags
  • Clorox or a steam sterilizer
  • disinfectant
  • ruler
  • ice bath
  • screw capped tubes filled with water

    Time Frame
    Three to four one hour classes are necessary to complete this activity. Times may be adjusted to fit block schedules. Student pages and the following teaching sequence have been written for 135 minute block classes.


    One day for introducing aseptic technique, vocabulary, and the importance of oxygen in bacterial metabolism.

    One day for inoculating agar tubes with cultures.

    One day for observing results and writing laboratory reports.

    Engagement and Exploration (Student Inquiry Activity
    Begin by describing the action of Clostridium botulinum on food spoilage and Clostridium perfringens on gangrene development to illustrate the necessity for these organisms to live in an anaerobic environment.

    Ask the students to define denitrification. (The conversion of nitrate and nitrite to atmospheric nitrogen gas). Most often they will get the idea that it involves the removal of nitrogen from somewhere. Ask students where the nitrogen in our atmosphere originates. How does it get there? (Primarily from microbial activity through the process of denitrification). Does it always comprise approximately 79% of natural air? (Yes, in present day). What would happen if the nitrogen concentration in air decreased? (Accept all reasonable answers). Is the nitrogen cycle related to any other element cyles? (Yes! Some bacteria require anaerobic conditions to 'fix' nitrogen, that is, convert atmospheric nitrogen to ammonia compounds).

    Explanation (Discussing)
    Introduce the vocabulary terms aerobic (oxic; in air), anaerobic (suboxic and anoxic; without air), obligate (required), facultative (preferred but, not required), nitrogen fixation (conversion of atmospheric nitrogen to ammonia products), denitrification (conversion of nitrates and nitrites to atmospheric nitrogen).

    Discuss the various bacteria that live under anaerobic conditions. Discuss the processes of nitrogen fixation and denitrification both of which occur under anoxic or suboxic conditions. These processes can give the concept of living without oxygen a defined purpose.

    Are all bacteria equally tolerant of oxygen? (No). Can all bacteria fix nitrogen? (No). Do all bacteria parcticipate in the process of denitrification? (No).

    Elaboration (Polar Applications)
    Explain that the bacteria that live in the suboxic region of Lake Bonney in the Dry Valleys of Antarctica, are capable of denitrification. Briefly describe the lake and known factors affecting this process (See "Background"). Read the journal entries (listed in "Resources") describing my research.

    Describe photosynthetic nitrogen fixation by the purple non-sulfur and the green bacteria. (See "Explanation"). Ask students to read the procedure and write these into their lab notebooks. Writing the procedures will ensure that the students have read the procedures and know what to expect. Explain to them that it is necessary to work quickly once the media has been inoculated with the bacteria. Inoculated cultures (that have been inverted several times for mixing) should be placed in an ice bath immediately for rapid solidification of the agar.

    Have students practice the aseptic techniques.

    Inoculate and observe cultures that have been incubated under microaerobic conditions to determine the tolerance for oxygen of certain bacteria. Cultures are grown in loosely capped tubes in an agarose medium. The depth at which a culture band is seen can be used as a measure of the oxygen tolerance of the cultures. If the organisms grow on the top of the agar, they are very tolerant of the presence of oxygen. Cultures appearing as bands below the surface of the agar are less tolerant (i.e. the farther below the surface, the less tolerant).

    Exchange (Students Draw Conclusions)
    After completing their lab analyses, students will answer the following questions in their lab notebooks:

    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).

    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?

    Evaluation (Assessing Student Performance)
    Each student is required to keep a lab notebook. This should be graded for completeness and accuracy (eg. depth of the bacterial band).

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