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Living Without Oxygen
Oxygen Tolerance in Bacteria

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· lab notebooks · disposable pipettes · screw capped tubes filled with water

· screw capped tubes with molten media · ice bath · disposable 1 ml pipettes · bacterial cultures · incubator · lab notebook

· cultures from the incubator · lab notebook · ruler

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. Listen to the lecture on the nitrogen cycle. Think about these questions….Is oxygen involved in the nitrogen cycle? Bacteria carry out the processes presented to you. Are all bacteria capable of nitrogen fixation and denitrification? If not, are there different tolerances to the presence of oxygen as it affects these processes? 3. In order to prepare for the actual lab, we must practice aseptic technique so that we do not contaminate our cultures and make our experimental results invalid. 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 loop to touch ANYTHING! DO NOT set the loop 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 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. · 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, loosen the cap to allow oxygen to seep into the tube, and incubate at 32oC 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.

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. 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 ......

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