Studying the Effect of Pesticides on Caenorhabditis elegans Neurons

Begin with an agar plate containing transgenic Caenorhabditis elegans expressing green fluorescent proteins or GFPs in their neurons.

Add sterile water to the plate, swirl it to lift the worms, and transfer them to a tube.

Allow the worms to settle, then remove the excess water and resuspend them.

Transfer the resuspended worms to a pesticide-coated plate and a control plate without pesticide, then incubate.

Based on their actions, the pesticide specifically damages certain neurons and causes neuronal swelling, decreasing GFP expression.

Transfer the worms onto an agarose pad that contains an anesthetic agent, then place a coverslip.

Mount the slide on a fluorescent microscope. First, visualize the worms using phase-contrast mode, then switch to fluorescence mode.

In pesticide-treated worms, neurons exhibit reduced fluorescence, swollen somas, and gaps in neuronal processes, indicating neurodegenerative effects.

While in control, healthy neurons show intact soma with bright fluorescence.

Using a micropipette place, 1 milliliter of sterile water onto the plate of nematodes. Next, swish the water around to lift up the nematodes. Then, remove the liquid with the nematodes to a 1.5-milliliter microfuge tube. After 10 minutes, as the nematodes settle by gravity, carefully remove at least 500 microliters of the water and discard.

Then, resuspend the nematodes, and using a cut-off yellow pipette tip, remove 50 to 100 microliters of suspended worms to each treatment plate, ensuring that each plate has at least 10 adult worms. Prepare two slides by placing a piece of label tape on only one side of the slide to form a pad of uniform thickness.

Then, position a clean, unused slide in between them on the benchtop. Place a 10-microliter drop of melted agarose onto the center of the slide using a micropipettor. Then, flatten the drop by placing another clean slide on top perpendicular to the slide with the drop and apply pressure so the agarose drop forms the thickness of the labeling tape.

Afterward, apply steady pressure to separate the two slides. Then, rest the slide with the agar side up on the benchtop, and allow it to dry for one to two minutes before using. To add nematodes to the prepared slide with the agarose pad, add a 5-microliter drop of water containing 1 molar of sodium azide to the agar pad, which will anesthetize the worms and mobilize them.

Then, transfer at least 10 nematodes per treatment slide to the droplet using a worm pick, which should be sterilized before and after transferring the nematodes. Next, add a coverslip using forceps by placing it at an angle and slowly lowering it.

Afterward, place the prepared wet mount on a fluorescent microscope to observe the worms, first under 10x magnification and phase contrast, then under 40x magnification with phase contrast and fluorescent illumination.

Place one prepared wet mount at a time on the microscope stage and secure it in place using the microscope stage clips. Then, begin viewing the wet mounts with the lowest magnification objective, which is typically 10x. and use a bright field or phase contrast to visualize and focus on the nematodes.

Once a nematode is located in the field of view, focus on it using the fine focus knob on the microscope. Then, switch the illumination to fluorescence by turning the dial and direct the light to the camera to capture the digital images.

Next, adjust the illumination using the imaging software so that the neurons are brightly illuminated, but not oversaturated. At some point, obtain a separate image of a ruler at the same magnification as the cell images to provide a magnification scale for their figure.