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Some Like It Hot, Some Like It Cold
Microbial Life in Hot Springs and Antarctic Lakes

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"Imagine diving into a refreshingly cool swimming pool. Now think instead of plowing into water that is boiling or near freezing." These images, provided by Dr. Michael Madigan and Dr. Barry Marrs in the arcticle "Extremophiles" published in 1997 in Scientific American, has me asking why would any organism plunge into freezing or boiling waters? And how could they survive? Are there organisms that live in these kinds of environments? I worked for six weeks in the Dry Valleys in Antarctica, studying a bacterium that survived in a permanently ice-covered lake, as well as, assisted in the isolation of an organism living in a hot spring in Yellowstone National Park! What are the known temperature limitations for living things on our planet?

Microbial life in the permanently ice-covered lakes of the Dry Valleys in Antarctica and in the hot springs of Yellowstone National Park can be used to show that temperature affects microbial populations. 'Extremophile' is a realitively new term used to describe organisms like bacteria, that live in environments that are very hot, cold, acidic, deep, and salty. They can be classified as thermophilic (heat-loving), psychrophilic (cold-loving), halophilic(salt-loving) or by other chemical or physical requirement extremes.

What are the temperature ranges for the growth of common bacteria, such as Eshcerichia coli and Pseudomonas aeruginosa? Do either of these organisms withstand extremes of heat or cold? What kinds of bacteria are found in these unfriendly environments? In this activity, you will have the opportunity to witness the fragile nature of these organisms, with regard to temperature. You will understand the role of temperature in determining what kinds of organisms are found in these unforgiving environments.

Per Group of Two Students

  • four to five nutrient broth tubes
  • two nutrient agar plates
  • several inoculating loops
  • official letter
  • cultures
  • oil-immersion microscope, oil, lens paper, bibulous paper
  • gram staining chemicals (crystal violet, iodine, acetone-alcohol, safranin) and trays (per group of four students)
  • 2 microscope slides
  • water bottles
  • disinfectant for cleaning lab areas
  • alcohol burner and matches
  • forceps

    Day 1

    1. Read the letter from the laboratory and obtain your culture.

    2. Discuss with your lab partner, an appropriate experimental design to solve this problem. Write this in your lab notebook! What is your hypothesis?

    3. Present your proposal to the class. Finalize your experimental design, including your negative control, after the class discussion. Why do we include a control in our experimental design?

    4. Label the agar side of your plates with your name, date, course section, and sample number.

    5. Each student should streak one nutrient agar plate to obtain isolated colonies. This is essential in determining if our culture is pure. What is a pure culture?

    6. Incubate at room temperature (overnight at 30oC if you are not on a block schedule).

    Day 2

    1. Observe the colony morphology on your petri plate. Document observations in your lab notebook. Will the morphology of the colonies help in proving your hypothesis?

    2. Using a single colony, prepare a gram stain following these steps:

  • place a drop (only a drop!) of sterile water on your slide

  • aseptically (using a sterile loop) transfer a small amount of a single colony from your petri plate to the water drop on the slide, spreading the liquid to thin it

  • allow the water with the culture to air dry

  • heat fix the bacteria by passing it through your alcohol burner flame several times, holding the slide with forceps (it will become quite hot)
  • Place the slide in the Gram Stain tray

  • flood the slide with crystal violet; wait 1 minute

  • rinse with water

  • flood the slide with iodine; wait 1 minute

  • rinse with water

  • holding the slide with forceps and at an angle, rinse the slide with acetone-alcohol until no more blue is seen dripping from the slide (10 - 30 seconds)

  • flood the slide with safranin; wait 1 minute

  • blot the slide dry with bibulous paper

  • observe the cell shape, arrangement of cells, and gram stain reaction and record
  • What does the gram stain tell us? Can we NOW prove our hypothesis?

    3. Label all nutrient agar tubes with initials, date, and sample number. Why is it necessary to put so much information on our test tubes?

    4. Inoculate each tube with a single colony using a sterile inoculating loop. Why do we use only a single colony?

    5. Document the temperatures of your chosen incubation sites in your lab notebook. What scale do scientists use for measuring temperature?

    6. Incubate each tube in its appropriate place.

    7. Clean your lab area.

    Day 3

    1. Observe growth (or lack of) in your culture tubes.

    2. Describe the growth as

    'NDG' (no detectable growth; water-like)

    'Light' (you can still see through the tube but, it is slightly cloudy),

    'Medium' (the tube is cloudy but, not completely opaque)

    'Heavy' (the broth tube is completely opaque, that is, light does not pass through; milk-like).

    3. Prepare a graphical display of your results.

    4. Dispose of your cultures in the biohazard can. Why can't we throw the cultures into the regular waste can?

    5. Disinfect your lab area.

    6. Present your data to the class.

    7. Copy all class data into your lab notebook.

    8. Begin your final report and letter.

    Day 4

    1. Complete your final report and letter to the laboratory and hand in.


    Bacterial Repository, Inc.
    2000 Agar Avenue
    Ooze, OH 45202

    Dear Students,

    Recently, bacterial cultures in our repository have not been growing as they should. We have analyzed our growth media and have eliminated that as the cause of our problems. Our scientists have suggested that technicians might have mislabeled our storage tubes and the cultures, because of this mislabeling, are being grown at incorrect temperatures.

    We need you to help us. Would you please test the temperature tolerances of the enclosed bacterial samples and suggest possible labeling schemes? That is, we would like the samples to be identified according to the optimal temperature at which they grow. Your assistance would be greatly apprecitated.

    Bacterial Repository, Inc. is aware of your expertise in this area and trust that accurate and proficient analyses will be performed. Thank you in advance for your recommendations.


    Janet Bjierinkia

    Discussions Questions/Extensions ......
    Answer these questions in your lab notebook.

    Are there organisms that live in these kinds of extreme environments? (Absolutely! Read "Extremophiles" ">http://www.sciam.com/0497issue/0497marrs.html"> http://www.sciam.com/0497issue/0497marrs.html .

    What are the known temperature limitations for living things on our planet?

    What are the temperature ranges for the growth of common bacteria, such as Eshcerichia coli and Pseudomonas aeruginosa and Streptococcus sp.?

    Do any of these organisms withstand extremes of heat or cold? (No).

    What kinds of bacteria are found in these unfriendly environments?

    List several examples (using the correct format for writing Genus species names).

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