Encyclopedia of Experiments: Biology
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Transcript
- To make sense of encountered conditions and respond to them accordingly, the worm C. elegans has, among others, a highly developed chemosensory system able to detect a wide variety of different chemicals.
Sensory neurons in the head, the amphid and inner labial neurons, as well as neurons in the tail, the phasmid neurons, are either directly or indirectly exposed through the cuticle to the outside conditions. These chemosensory neurons code relevant information used by the worm to produce an appropriate behavioral response: a behavior termed chemotaxis.
To test the chemotactic response to either volatile odorants or gustatory water-soluble cues, isolate worms of the desired developmental stage and expose them to the test chemical on a previously prepared experimental arena. When exposed to favorable chemicals like those produced by a bacterial food source, C. elegans displays positive chemotaxis towards the source.
Reversely, when exposed to less favorable or toxic chemicals like heavy metals, the worm displays negative chemotaxis, avoiding the chemical. Hence, olfactory or gustatory mutants fail to avoid aversive chemicals and remain in unfavorable conditions in contrast to wild type animals who avoid the aversive condition.
In the example protocol, we will see a demonstration of a chemotaxis assay, testing the worm's aversion to copper.
- After overnight incubation, remove plates from the incubator and using the marked underside of the plate as a guide, pipette 100 microliters of freshly prepared 0.5 molar copper two sulfate solution on the edge of the agar to create an outer copper barrier. Pipette 25 microliters of the copper two sulfate solution to create a midline barrier.
Ensure that the copper two sulfate solution does not contact the bacterial patch, and allow the copper solution to dry onto the plate. Check for dryness every five minutes after transfer with a laboratory tissue by lightly dabbing the solution near the edge of the plate to discern.
Immediately prior to the assay, transfer the experimental organisms to a bacteria-free agar plate and allow the nematodes to move freely for one minute to remove excess bacteria.
Next, pipette 1 milliliter of M9 onto the plate with young adults that were transferred 24 hours previously in order to wash worms into a microcentrifuge tube. Centrifuge the nematodes at 3,000 times g for one minute.
Worms should form a pellet at the bottom of the tube. Aspirate M9 solution without disrupting the worm pellet. Add 1 milliliter of M9 solution to the worm pellet. Invert tube to mix worms with the solution.
If excess bacteria were initially transferred with the worms, repeat for a total of five times. After the final wash, aspirate the supernatant until 100 microliters of M9 solution and the worm pellet remains. Immediately transfer worms from solution once the wash steps have been completed.
Pipette 20 microliters of the worm pellet from the bottom of the tube onto the bacteria-free half of the assay plate. Ensure that 10 worms are transferred to the assay plate and that no contaminating food is present. No bacteria should be transferred to the copper food race plates.
Remove excess M9 solution from the nematodes with a laboratory tissue within one minute. Ensure the M9 solution does not contact to the copper two sulfate solution, and that the worms and agar surface remain intact. Discard worms that have accidentally been removed with the laboratory tissue.
Once the M9 solution has been removed and all worms have commenced non-liquid locomotor patterns, i.e. when they have stopped thrashing, start the assay stopwatch. If extra worms were accidentally included, remove them by picking with halocarbon oil to ensure that no bacteria are added to the plate.
Check assay plates every 30 minutes. For the assay plates with bacterial patches, positively score organisms if they reached the food patch over a four-hour period. For the negative control plates, positively score organisms if they have crossed the barrier.
