Please answer questions at the end.
In this lab, you will add different volumes of âcell-free supernatantâ (CFS) to fresh cultures of V. harveyi and observe bioluminescence. The CFS is prepared by culturing V. harveyi in liquid medium overnight, then separating the cells from the supernatant by centrifugation.
MATERIALS ⢠overnight culture of V. harveyi in AB medium ⢠fresh AB medium ⢠1 microcentrifuge tube ⢠3 glass culture tubes
PROCEDURE
1. This experiment will be performed with your lab group. Label the microcentrifuge tube and 3 glass cultures tubes with your group number. Also, label the 3 glass culture tubes as follows: âMedia only,â âCells+fresh media,â and âCells+CFS.â
2. Using aseptic technique, pipet different volumes of fresh AB medium into the glass tubes as follows: 1 mL in the âMedia onlyâ tube, 800 ul in the âCells+fresh mediaâ tube, and 500 ul in the âCells+CFSâ tube.
3. Pipet 500 ul of the overnight culture into a microcentrifuge tube and centrifuge at maximum speed (>13,000 x g) for 5 min to pellet the cells.
4. After centrifugation is complete, transfer 300 ul of the cell free supernatant (CFS) from the microfuge tube to the glass tube labeled âCells+CFSâ. Be careful to avoid the cell pellet.
5. Remove the remaining supernatant from the microfuge tube using a micropipette and discard as culture waste.
6. Add 500 ul of fresh AB medium to the cell pellet in the microfuge tube and pipet up and down to resuspend until no clumps are visible.
7. Add 200 ul of the cell suspension to the âCells+fresh mediaâ tube and 200 ul of the cell suspension to the âCells+CFSâ tube. Note the time.
8. Place the 2 glass tubes containing cells in the 30 degree incubator/shaker.
9. After 1 hour and 2 hours, observe your tubes next to the âMedia onlyâ tube in a dark room. You will need to let your eyes adjust for at least 30 seconds (count to 30 slowly in the dark) before you can evaluate whether the cultures are luminescent or not. Also, make sure you try swirling the tubes in the darkroom and observe the tubes for a minute after you have stopped swirling. After the observation at the 1 hour time point, put your tubes back in the incubator shaker until the 2 hour time point.
10. Note your observations. At the end of the lab, place your tubes in the tube waste.
Questions
1. What were the expected results? Explain the rationale of the experiment. If you didnât obtain the expected results, hypothesize why not and describe how the experiment could be improved.
2. What are differences between luminescence and fluorescence?
3. Why do V. harveyi cultures glow more brightly when swirled vigorously? Be specific.
4. Imagine you have a mutant V. harveyi strain that canât synthesize autoinducer and another mutant V. harveyi strain that makes a defective luciferase. If you streaked both strains near (but not touching) each other on the same plate, would you expect to see light produced? If so, by which strain, where, and why?
5. You have two identical tubes of wild-type V. harveyi (with the same cell concentration in each tube) in fresh media. To one tube, you add CFS from a light-producing V. fischeri culture, and to the other tube you add an equal volume of fresh media. If both tubes of V. harveyi have the same light production, what can you conclude? Explain.
6. Many biomedical research labs study quorum sensing in V. fischeri or V. harveyi. How is studying light production in marine bacteria relevant to human health? List at least 3 different examples.
7. Why do you think it is important for bacteria to be able to control the expression of some genes based on the number of individuals present?
