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Embryology with Rhabditis

Carolina Labsheets™

In this lab students observe cleavage of fertilized eggs of female Rhabditis. Two or even three cleavages may be observed during a 1-hour lab session. The material is inexpensive and easily obtained from the common earthworm, Lumbricus terrestris. Rhabditis species are nematodes whose larval stages live within earthworms. When the earthworm host dies, the nematodes mature and reproduce, making the collection of fertilized eggs and early stage embryos an easy task


Student Lab Sheet


Needed Materials*

Earthworms, Lumbricus terrestris (141626)

petri dishes

anesthetizing solution (ethanol, etc.)

sharp dissecting scissors or razor knife

2% non-nutrient agar

teasing needles

microscope slides

coverslips

compound microscopes

stereomicroscopes


Safety

Ensure that students understand and adhere to safe laboratory practices when performing any activity in the classroom or lab. Demonstrate the protocol for correctly using the instruments and materials necessary to complete the activities, and emphasize the importance of proper usage. Use personal protective equipment such as safety glasses or goggles, gloves, and aprons when appropriate. Model proper laboratory safety practices for your students and require them to adhere to all laboratory safety rules. Have students wash hands before and after this lab. Although these nematodes are not known to be parasites of humans, they should be handled with appropriate precautions to keep them away from the face and mouth.


Procedures

Prepare the non-nutrient agar by boiling 2 g agar (796200) in 100 mL water with stirring until dissolved. Allow to cool to about 45°C before pouring into petri dishes. Sterility is not required. If you would prefer not to prepare your own agar, non-nutrient agar is available as poured plates (821182) and in prepared media bottles (156395). One bottle will pour four or five plates, enough to provide plenty of material for a lab of up to 30 students.

Allow about 30 minutes for anesthetizing an earthworm. Place the earthworm in an empty petri dish (no agar) and add enough water to cover the bottom. Add ethanol or other anesthetizing solution drop-by-drop until the earthworm shows no response and becomes limp.

When the earthworm no longer responds to stimuli, remove it from the dish and use sharp scissors or a razor knife to cut it into cross sections about 4 mm thick. Place the sections in the poured petri dishes with the cut surfaces in contact with the agar. Replace the lids and maintain the dishes at 18–20°C. (Few, if any, worms or eggs will be produced at temperatures above 22°C. Use ice if necessary to keep the temperature down.) In 2 or 3 days, you will have an abundant supply of adult Rhabditis.

As the earthworm sections decay, liquid will collect around each section and you will begin to see white worms in the liquid. These are the mature Rhabditis. Due to the odor produced by the decaying earthworm, you will want to keep the plates under a ventilated hood or in an enclosure.

Check for the presence of the worms with a dissecting microscope. You can leave the cover on the dish while searching for Rhabditis. When you detect worms in the cultures, use the procedure given in the Student LabSheet to check for eggs. Perform this lab exercise the same day that worms with eggs and early embryo stages appear in the cultures. If this is not possible, keep the cultures cool or refrigerated until the lab exercise can be performed; otherwise, the eggs will rapidly complete development into worms, and few will be left for student observation. At room temperature, this development may be completed in 3 or 4 hours.

On the day of the lab, divide the class into four groups, and give each group a plate of Rhabditis and a teasing needle. Indicate where the students are to obtain other materials.


Student Lab Sheet



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