This protocol is a straightforward and feasible way for undergraduate students to conduct authentic research that could lead to publishable results. The main advantage is that students can conduct original research on questions that they decide on their own. To begin, make a solution spreader by carefully bending a nine milliliter glass Pasteur pipette in a Bunsen burner flame and close the opening of the pipette.
Then ethanol sterilize the spreader before each spreading on the Petri plate. Using a micropipetter, place 100 microliters of the dilution on the center of the agar and spread gently across the surface with the sterilized spreader. Then set aside the Petri dishes covered and let the solution soak into the surface until dry.
For exposure periods longer than 12 hours, add 50 microliters of an overnight culture of Escherichia coli to the plates prior to adding the nematodes, whereas for acute experiments of 12 hours or less, there is no need to have E.coli on the plates. Using a micropipette, place one 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 settled by gravity, carefully remove at least 500 microliters of the water and discard. Then resuspend the nematodes, and using a cutoff 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 micropipetter. 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 this slide with the agar side up on the benchtop and allow it to dry for one to two minutes before using. To add nematodes of the prepared slide with the agarose pad, add a five microliter drop of water containing one molar sodium azide to the agar pad, which will anesthetize the worms and mobilize them. Then transfer at 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 cover slip 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 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 an 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.
The effects of manganese containing pesticide on dopamine neuron morphology using a strain in which dopamine neurons expressed GFP was studied showing a bright signal. However, the fluorescent signal bleaches if the neurons are exposed to light for long periods of time. This procedure can be part of a multi-week student-driven project that can also include behavioral or other data.
