Chemotaxis plates areprepared with 10cm Petri dishes containing 10ml of assay agar (2% agar, 5mM KPO4pH6, 1mM CaCl2, 1mM MgSO4). c.elegans have to be washedtwice in S-basal, once in distilled water and placed onto spot on the plate.

Mark two spots equidistant from center diameter on the bottom of petri dish,mark them as A and B. Mark the spot X on the circumference of the petri dishforming a triangle with A and B1µl of 1:100benzaldehyde:EtOH is placed at test spot A and 1µl of ethanol was applied tothe other spot B. C.Elegans are added at spot X.

For each spot, 1µl of 1 M NaN3was applied to immobilize the worms when they reached the spot. After one hour ofchemotaxis, animals were counted, and Chemotaxis Index (CI) is calculated as{number of animals at the test odorant A – number of animals at the solventB} / Total number of animals tested (A+B). Repeat the steps 2,3 with adifferent concentration of 1µl of 1:1000 benzaldehyde:EtOHFor locomotor assay, takeC.elegans are grow on NGM with OP50 in nicotine-free plates.

They are placed onfresh plates 5-6 hours before the behavioral test. C.Elegans are thentransferred to plates with appropriate nicotine concentration. Observations arerecorded.First 10-12 minutes of thetest are discarded for results because the elegans have to get acclimated tothe new environment. The results give us insight of Acute Locomotor Assay.Elegans from step 3 are thentaken to measure the Locomotor Sensitization Assay.

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Transfer worms to a clean plate containing a lawn of OP50and 100 ?M nicotine before transferring them to a second clean, nicotine-freeplate for an additional hour. Observe how the eleganscrawl on a plate covered in 40 ?L OP50 and a 10 ?M Nicotine concentration. For chemotaxis assays, weused standard 10 cm Petri dishes containing 15 mL of an optimized nematodechemotaxis medium 1.6% BBL agar; 1 mM MgSO4; 1 mM CaCl; 5 mMKPO4. On test day, plates are allowed to warm on the bench to 20ºC, beforepartitioning the outside of the dish into three sections – A,B and X forattractant, control and middle section.Transfer about 50-150 L4elegans to the chemotaxis plate by adding 10?L nicotine diluted in ethanol at Aand the control, ethanol diluent at B. We then placed the dishesupside down and allowed to sit undisturbed for one hour.

Observations are recorded atthe end of the hour. Chemotaxis index (CI) is calculated by counting the numberof elegans using the formula: (A-B)/(A+B+X) = CI.