Transfer C. elegans to OP50- seeded NGM-lite Plate
• To feed worm strains it is easier to transfer a chunk of worm-filled agar from a well-grown plate to a new plate rather than pick individual worms.
1. Use a permanent marker to label the bottom of your OP50-seeded plate with the date and “WT” (wild-type).
2. Examine the plate you will be transferring the worms to under the dissecting microscope to verify the plate is free of bacterial or mold contamination. Obtain a new plate if your plate is contaminated.
3. Sterilize a metal spatula. Dip the end into ethanol, and briefly pass it through a Bunsen burner flame to ignite the alcohol. Allow alcohol to burn off away from the Bunsen flame and ethanol; the spatula will become too hot if left in the flame.
Caution: be extremely careful not to ignite the ethanol in the beaker. Do not panic if the ethanol is accidentally ignited. Cover the beaker with a Petri dish lid or other non-flammable cover to cut off oxygen and rapidly extinguish the fire.
4. Use the sterilized spatula to cut a 1-cm square chunk of agar with worms from the wild-type starter plate.
5. Transfer the chunk to your OP50-seeded plate, and place it face down in the agar. (Placing the chunk face down allows the worms to crawl quickly into the bacteria on the new plate.) Allow the worms to crawl off the chunk and on to the plate for a minimum of 30 minutes before counting them. You can answer some questions in Parts B &C while you are waiting for them to move from the chunk.
6. Observe the worms on this OP50-seeded plate using a dissecting scope. Become familiar with the various stages of development (see Figure 7.4 in the Lab Manual) and try to determine which stages are present on your plate.
7. Determine the number of worms in various stages present on your plate (questions 1-5 in the Lab Report.
8. Use this image (also in the Lab report) to answer questions 6-9) in Part A of the Lab Report.
1. Use a permanent marker to label the bottom of the OP50-seeded plate with your group name, and WT (wild-type). This is your control plate.
2. Use a permanent marker to label the bottom of each of your RNAi feeding plates (bli-1 and dpy-11) with group name and either bli-1 or dpy-11. These are your 2 experimental plates.
3. Using the rounded end of a sterile toothpick, pick a small blob of bacteria from the edge of the bacterial lawn of an unused OP50-seeded plate. 4.
While observing the worms with a dissecting microscope, transfer five adult worms from your plate of wild-type worms to the new OP50-seeded plate labeled “WT”. Use the blob of E. coli on the end of your toothpick as glue to pick up the worms. If you have trouble identifying adults, pick larger worms from your wild-type plate. You may have to pick the worms one at a time.
5. Confirm the transferred worms were not injured or killed during the picking process.
6. Identify any eggs or young larvae that may have been accidentally transferred. Carefully pick them off the plate with a sterile toothpick and dispose of them appropriately.
7. Use the same method, using new sterile toothpicks for each plate, to transfer 5 adult worms to each of the plates seeded with RNAi feeding strains (bli-1 and dpy 11). When you are done you will have carefully picked 5 adult worms (making sure you have not transferred any larvae or eggs) to 3 different plate (OP50), bli-1 (RNAi),and dpy-11 (RNAi)
8. Incubate the plates lid side down at 20 C. Choose a place where the plates will not be disturbed or exposed to direct sunlight.
9. Go to Wormbase.org to find out the normal function of the bli-1 and dpy-11 gene products. There is a search option in the upper righthand corner (you do not need to login).
10. Answer questions in Part B of the Lab Report.
• Answer questions in Part C of the Lab Report.