Module 3: C. elegans Chemotaxis Assay Michael Dunn Capuchino High School

Gregory Chin, Ph.D. BABEC

Introduction Chemotaxis is the process by which an organism detects a chemical in its environment and then moves toward or away from it. An odorant is a chemical that produces a detectable odor. Organisms move toward odors they like (attractants) and away from odors they dislike (repellants). In nature, the ability to undergo chemotaxis is important for survival. Many organisms, including C. elegans, can detect the odor of their favorite foods and will chemotax toward them. Similarly, the ability to detect and move away from dangerous chemicals obviously can help an organism avoid toxic environments. As much as 1 5% of the C. elegans genome is dedicated to sensing environmental chemicals. Mutants that lack chemosensory cilia on neurons in the head or tail (such as che-2, che-3, daf-6, daf-10) are unable to detect chemicals in their environment and will not move preferentially toward a stimulus that would normally attract a wild-type (N2) worm. Other mutants are able to chemotax normally in virtually all situations except one. The mutant odr-10, for example, has a substantially reduced ability to detect the chemical attractant diacetyl but has normal sensitivity to other volatile attractants. ODR-10 is a 1 receptor protein for diacetyl. Therefore, a defect in this gene only prevents worms from detecting diacetyl., the chemical that gives synthetic butter its characteristic aroma. In contrast, odr-3 mutants exhibit weak olfaction to several different odorants due to defective responses from several different classes of neurons. In this exercise, you will study chemotaxis in C. elegans by testing the response of your worms to one or more volatile chemicals. Can you formulate a hypothesis to predict the effect of your odorant on C. elegans? What research could you perform to improve your chances of making an accurate hypothesis?

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Bargmann CI. 2006. Chemosensation. in WormBook. (ed. The C. elegans Research Community), WormBook, doi/10.1895/wormbook.1.123.1, http://www.wormbook.org/chapters/www_chemosensation/chemosensation.html Module 3: Chemotaxis Introduction (Student Guide)

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Chemotaxis Assay 1. Use an extra-fine black Sharpie marker to draw a straight line down the center of the bottom of the 100 mm NGM plate.

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2. Turn the plate 90 degrees and draw another straight line so that the plate is divided into 4 equivalent quadrants.

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3. Place a small dot in the intersection of the lines at the center of the dish (this is where the worms will be placed at the beginning of the experiment).

Worms

4. Measure 3 cm from right of the center dot in the middle of the plate and make a dash. Write “W” at the top of the quadrant.

O

W

Worms

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Water (Control)

5. Make another dash on the midline 3 cm to the left of the center dot. Write “O” (for odorant) at the top of that quadrant.

Odorant

O

W

Worms

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Water (Control)

6. Along the edge, label the bottom of the plate with your team name, date, and the strain of worms (N2).

Module 3: Chemotaxis Lab Exercise (Student Guide)

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7. Add 1.0 mL of S-basal saline solution to a plate of synchronized N2 worms (which you prepared in Module 2).

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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 solution and worms.

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9. Remove the lid from the Petri dish and tilt the plate 45 degrees.

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10. Transfer the liquid and worms to a 1.5 mL microfuge tube. Be sure that the micropipette tip is pressed against the edge of the Petri plate and is NOT touching the agar when you draw up the SBasal and worms. Otherwise the agar will clog the tip.

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11. Set a P-20 micropipette to 5 µL and place a tip on it.

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12. Finger flick the 1.5 mL microfuge tube containing worms until all the worms are evenly dispersed throughout the liquid. Note: There should be no pellet of worms at the bottom of the tube, and the tube should look uniformly cloudy.

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13. Immediately insert the micropipette tip into the middle of the liquid (between the 0.5 and 0.1 mL marks on the tube) and remove 5 µL of the worm suspension. Note: It is important to do this quickly before the worms settle at the bottom.

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14. Add 5 µL of worm suspension to the middle dot of the chemotaxis plate. Wait for the excess liquid to evaporate.

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15. Remove the lid. Using a P-20 micropipette, add 2 µL of 1 M sodium azide to the center of the lefthand dot on your Petri dish. Be careful not to gouge the agar with your pipette tip. To avoid gouging, try holding the pipette tip above the dot and carefully touch the dot with the bubble that emerges from the tip as you depress the plunger. Do not move your Petri plate at this point. Caution: Do not allow sodium azide to touch your skin or eyes. If you have an accident, inform your teacher immediately.

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16. Add 2 µL of 1 M sodium azide to the center of the right-hand dot on your Petri dish.

Module 3: Chemotaxis Lab Exercise (Student Guide)

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17. Add 2 µL of water to the control dot (W). Try not to gouge the agar with your pipette tip. Rather, hold the pipette tip above the dot labeled “W,” slowly depress the plunger to the second stop, and hold it there until a tiny droplet of water is expelled. If the drop does not fall from the tip by itself, gently touch it to the agar.

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18. Repeat steps 16-17, adding 2 µL of chemical odorant to the dot labeled “O.”

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19. Place the Petri dish on a microscope stage and focus on the worms at low magnification. Remain at the lowest magnification, so you can see as much of the plate as possible.

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20. Observe the worms. Initially, the worms will be swimming, which looks like flailing or thrashing. This will continue until all the excess liquid has evaporated. At this point, all the worms should be able to move freely and will start to move around the plate.

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21. Parafilm the plate.

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22. Allow the worms to migrate from the center of the plate for 15-30 min.

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23. Then place the plates in the refrigerator until the next day.

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24. Clean up your lab area. • All plates and used tips should be dumped into the biological waste container. • Clean your work area with disinfectant. • Be sure to turn off the microscope. • Wash your hands.

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Scoring the Assay 25. Place the N2 plate upside down on the stage of your microscope and focus on the worms.

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26. Using the red extra-fine Sharpie marker, make a dot on the Petri dish at every place you see a worm.

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27. Count the total number of worms on the “O” side of the center line—This will give you the number of worms that moved toward the chemical odorant.

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28. Count the total number of worms on the “W” side of the center line—This will give you the number of worms that preferred the water (or control) side of the plate. Module 3: Chemotaxis Lab Exercise (Student Guide)

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29. Worms that are on the center line or in the center dot should be culled (not counted).

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30. Record your results in a data table.  31. The chemotaxis index (I) is a measure of how attracted the worms are to a particular odorant. It is calculated using the following equation:

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O–W I = -------O+W O = # worms on odorant side of center line W = # worms on control/water side of center line O + W = Total # worms on Petri dish 32. Clean up your lab area. • All plates should be dumped into the biological waste container. • Clean your work area with disinfectant. • Be sure to turn off the microscope. • Wash your hands.

Module 3: Chemotaxis Lab Exercise (Student Guide)

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Module 3: Chemotaxis Lab Exercise (Student Guide)

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