Module 4: Effect of Alcohol on Worms

Transcription

1 Module 4: Effect of Alcohol on Worms Michael Dunn Capuchino High School Gregory Chin, Ph.D. BABEC Introduction Alcohol is a drug that affects the nervous system of many animals. The type of alcohol that is found in alcoholic beverages is called ethyl alcohol, or ethanol. In humans, ethanol impairs coordination and reaction time. In high enough doses, it is deadly. Scientists often use model organisms to learn more about the physiological effects of a drug. In this experiment, you will study the effect of alcohol on the model organism C. elegans. Studying the effect of alcohol on C. elegans has revealed a genetic component. Mutations in the C. elegans slo-1 gene results in resistance to the effects of ethanol on locomotion and egg-laying. The slo-1 gene encodes a homolog of the BK potassium channel protein found in humans. 1 Researchers have demonstrated that electrical current at the SLO-1 channel increases in response to doses of ethanol that alter normal C. elegans biology and behavior. Failure of slo-1 mutants to produce this current demonstrated that the response is SLO-1-dependent. Under the influence of intoxicating levels of ethanol, a similar increase in BK channel opening has also been observed in mammals. 2 Naturally occurring tolerance to alcohol has also been discovered in C. elegans. Wild-type worms discovered in Hawaii (strain CB4856) develop ethanol tolerance more quickly than N2 wild-type worms. 2 This is believed to be due to genetic variation between the strains. Some studies have indicated that a similar tolerance gene may play a role in the ethanol tolerance of rodents as well. In this exercise, you will take one-minute digital videos of untreated and ethanol-treated C. elegans worms. Using software to track the movement of the worms, you will then calculate the average velocity of both data sets and determine the effect of ethanol on C. elegans. How do you believe the alcohol will affect the worms? 1 Wang ZW, Salfee O, Nonet ML, and Salkoff L SLO-1 potassium channels control quantal content of neurotransmitter release at the C. elegans neuromuscular junction. Neuron, 32: McIntire SL. In press. Ethanol. in WormBook. (ed. The C. elegans Research Community), doi/ /wormbook , Introduction (Student Guide) Page 7-1S

2 1. Obtain 2 Petri dishes and label the bottom of the plate along the edge with your team name, and today s date. Also, label one plate Control and the other Alcohol. 2. Using a P-1000, pipette 150 µl of ethanol onto the center of the agar of the plate labeled Alcohol. Gently swirl the plate to help spread the ethanol evenly across the surface of the agar. Alcohol s Effect on Worms 3. Wrap parafilm around the edge of the plate so that it is airtight. The control plate can be left as it is. 4. Leave the Petri plates right side up at room temperature overnight. 5. Remove the parafilm from the alcohol plate and carefully place a filter paper ring in the center of both the alcohol and control plate. 6. Use a P-20 micropipette to apply 15 µl of copper chloride to the left side of the filter paper. Repeat this process for the other 3 sides. The paper should become saturated with copper chloride. Note: Don t get any copper chloride on the agar inside the filter paper or your results will be compromised. 7. Use a P-1000 to add 1.0 ml of S-basal to a synchronized plate of N2 worms. 8. Replace the lid to the Petri dish and gently swirl the plate for 10 seconds to wash the worms off the plate. Note: be careful not to spill the S-basal and worm solution. 9. Remove the lid from the Petri dish and tilt the plate at a 45 degree angle. 10. Using a P-1000, transfer the liquid and worms to a 1.5 ml microfuge tube. Note: The micropipette tip should rest against the edge of Petri plate. DO NOT allow it to touch the agar. 11. Finger flick the bottom of the 1.5 ml microfuge tube until all the worms are evenly dispersed. No pellet should be visible at the bottom of the tube, and the Lab Exercise--Mac (Student Guide) Page 7-2S

3 tube should look uniformly cloudy. 12. Using a P-20 set for 5 µl, lower the pipette tip into the worm suspension in the tube till it rests between the 0.1 and 0.5 ml marks on the side of the tube. Remove 5 µl of the worm suspension. Note: Removing suspension from higher or lower than this zone can lead to too many or too few worms for the experiment. 13. Add 5 µl of worm suspension to the middle of the Alcohol plate. Allow the alcohol plate to sit for 20 minutes right-side up with the lid on. This will give time for the worms to break free from the liquid and for the ethanol to take effect. 14. Repeat steps for the Control plate. 15. After about 5 minutes, the droplet on the control plate should be completely gone, and the worms should be crawling freely. Note: The alcohol plate will take about minutes to be ready. 16. Plug the Digital Blue microscope into a laptop or desktop computer via a USB port. 17. Place the microscope on top of a light box, centered above the light source. Note: The positioning of the light cord may need to be adjusted to obtain optimal lighting. Tape the light cord to the lab bench to avoid further movement once you have find the optimal light angle. 18. Using the dial on the front of the microscope, set the magnification to 10x. Note: Make sure the dial clicks into place. 19. Turn on the computer. Double-click on the mixscope alias folder and then double-click on the mixscope icon within this folder. 20. Click on the mixscope tab on the tool bar at the top of the screen and then click preferences. Click on the camera tab, and verify that the microscope is set to record images in jpeg format. lose this window. 21. Locate the drop-down menu in the lower right corner of the screen and choose Device. 22. Set the saturation to approximately 60-70%. 23. Set the brightness to nearly 0. Lab Exercise--Mac (Student Guide) Page 7-3S

4 24. Change the drop-down menu in the lower right corner of the screen back to Movies. 25. Change the Movie Type to TimeLapse. 26. Make the Frame Capture Interval 1.00 sec. 27. Make the Playback Frame Rate: frames/sec. 28. Verify the Create an image sequence box is checked. 29. Make sure all other boxes are unchecked. 30. Make sure both microscope lamps are OFF (icons are located at the bottom of the screen). 31. Remove the cover and place your control worm Petri dish upside down on the stage of the Digital Blue microscope. 32. Turn on the light box. 33. Use the adjust knob on the microscope to bring the worms into focus. 34. Center the plate so that the entire portion inside the filter paper ring (where the worms are located) fills the microscope s field of view. 35. Record a 60 second video of your control worms by clicking Start Recording. 36. After 60 seconds, click Stop Recording. 37. A window will open that tells you the name of the file and where it is saved. Record this information in your notes, so you can find your file later. 38. The video will automatically be saved in the Pictures folder as a stack of images with today s date in a folder titled ImageSeq. Click OK and close mixscope. 39. After you have completed your control video, check your alcohol plate with the dissecting microscope to verify the worms are no longer clumped. When they are all moving freely, repeat steps 20 to 38 with your alcohol plate. 40. Clean up your lab area. All plates and used tips should be dumped into the biological waste container. Clean your work area with disinfectant. Lab Exercise--Mac (Student Guide) Page 7-4S

5 Be sure to turn off the microscope. Wash your hands. Lab Exercise--Mac (Student Guide) Page 7-5S