Purpose: To observe the effects of exposure to different concentrations of ethanol on the behavior of C. elegans.

Materials:

A plate of N2 (normal wild-type) worms
2 clean, unseeded plates
Dissecting microscope
Small test tubes, 1 with 1% ethanol solution, 1 with 10% ethanol solution
Sterile transfer pipettes
1 eppendorf tube, in a rack
spreader (made from a paper clip or a glass Pasteur pipette)
Kimwipes or tissue
Test tube with S-BASAL solution
Small beaker for waste solution
Bunsen burner or alcohol lamp

Procedure:

1. Label one of the clean, unseeded plates Ethanol 1% and the other Ethanol 10%.

2. Using a transfer pipette, pipette approximately 200 microliters (3 to 4 large drops) of 1% ethanol onto the plate labeled 1%. Heat sterilize the spreader and spread the solution over the surface of the plate. Be careful not to dig into the agar use a light touch! Put the lid on the plate and let it sit so that the ethanol can soak into the agar.

3. Using a clean pipette, pipette approximately 200 microliters of the 10% ethanol solution onto the plate labeled 10%. Heat sterilize the spreader and lightly spread the solution over the surface of the plate. Put the lid on.

4. Using a new, sterile pipette, transfer approximately 500 microliters (half a pipette) of S-BASAL solution onto the plate of N2 worms. Tip the plate so that the worms are "washed" into a pool at one end. Use the pipette to "suck up" the pool of worms, and squirt them into the eppendorf tube. Close the top of the tube and place it in a rack for approximately 5 minutes so that the worms will settle to the bottom of the tube.

5. When there is an obvious "worm pellet" at the bottom of the eppendorf tube, carefully pipette most of the solution out of the tube, squirting it into a waste beaker.

6. Using the same pipette, carefully "suck up" the worm pellet and release approximately half of it onto the plate with 10% ethanol and the other half onto the plate with 1% ethanol. Pay close attention to the amount of worms in the pipette, trying to get approximately half of the worms onto each plate.

7. Twist a kimwipe or tissue into a point and carefully touch the point to any excess S-BASAL on the 1% plate, sucking it up so that all that remains on the plate is the worms. Repeat with the 10% plate. On your data table, write down the time worms were placed on each plate.


8. Observe worms on the 1% ethanol plate for approximately one minute. Write down the time of observation on your data table. While your partners observe the worms, write your observations in the space on the data table how did the worms' movement appear (normal? abnormal?) Did you notice egg-laying? Other behaviors?

4. When everyone in your group has observed the 1% ethanol plate, change to the 10% ethanol plate and repeat your observations, recording the time and writing down specific behaviors.

5. Repeat the cycle of observing and recording for each plate at least one more time, noting any changes from the first round of observations.

6. Clean up your area, being sure that all plates are covered, microscopes are turned off, and waste beakers are handled according to teacher instructions.

7. Compare your group's observations with other groups in the class. Brainstorm explanations for the behaviors observed when worms are exposed to different concentrations of alcohol. If there are variations in the behaviors observed, brainstorm reasons for these variations. Are the effects of alcohol on worms similar to the effects on humans? How might humans benefit from research on worms? (What could be happening at the cell level? Which cells are likely to be involved? Do humans have similar cells?) How could you write a hypothesis to explain the effects of alcohol on worms? How could you test your hypothesis? Write up possible answers to these questions in the form of Conclusions to this lab. NOTE: There has been much work done on worms and the effects of alcohol. Try searching C. elegans web sites for information.