The overall goal of this amputation procedure is to selectively and completely remove the pharynx from planarians without damaging other organs. This method can help answer key questions in the regeneration field. Such as how stem cells recognize missing organs and initiate their regeneration.
The main advantage of this technique is that it is very precise and that other organs are not damaged during the process. I first had the idea for this method when I was anesthetizing planarians with sodium azide. I noticed that exposure to sodium azide caused the pharynx to pop out.
Visual demonstration of this method is critical as the amputation requires the proper selection of animals as well as a careful observation of animal movement and morphology. Demonstrating the procedure will be Divya Shiroor, graduate student from my laboratory. Before beginning the procedure, use a plastic transfer pipette to select worms that were fed five to seven days previously into a clean petri dish.
Place a flat ruler under the petri dish to confirm that each planarian is at least 6 millimeters in length as they crawl across the dish. Then transfer up to 20 selected worms into 35 millimeter petri dish and use a transfer pipette to remove all planarian water from the dish. Place the dish under a stereo microscope and adjust magnification such that multiple animals can be viewed.
Add five milliliters of freshly prepared 100 millimolar sodium azide solution to the dish. Monitor the animals without moving the dish for three to four minutes to observe the extrusion of the pharynx. When at least half of the animals in the dish have fully extended a pharynx use a plastic transfer pipette to aspirate and forcibly dispense the worms around the dish a few times.
If any pharynges were fully extended, this vigorous pipetting will cause them to detach. Alternatively, use a pair of fine forceps to grasp the pharynx while lifting the planarian upwards toward the meniscus. As the animal is raised, the surface tension will cause the pharynx to detach.
While you're moving the pharynx be careful not to cause accidental injury to the animals. Planarians are very soft bodied and easily damaged, especially when in sodium azide. Use a transfer pipette to move about 15 amputated animals into a new dish containing fresh planarian water and rinse the worms with fresh planarian water three times.
Make sure to completely wash out the sodium azide. Even traces of the chemical could cause animals to lyze. Then incubate the worms at 20 degree Celsius for seven days.
The day after the amputation, place the worms back under the microscope to determine whether a dark spot is visible on the dorsal side of the worm. To feed the worms, transfer an appropriate volume of liver paste into a microcentrifuge tube and centrifuge the tube briefly to remove any air bubbles. After estimating the volume of the paste pellet, add planarian water to one fifth of its volume.
Based on the total volume in the tube, add two percent red food coloring. Mix the food sample thoroughly until the dye is evenly distributed throughout the paste and briefly centrifuge the tube again. Transfer the amputated animals into a new petri dish and place in the dark for approximately one hour.
At the end of the incubation, trim approximately one half centimeter from the end of a P200 pipette tip and use the modified tip to transfer 25 microliters of red liver paste into the dish. Allow the animals to feed for 30 minutes. Then place the worms on a white background to score the number of animals that appear red.
After approximately 6 minutes in sodium azide solution, the white tip of the pharynx can be seen. A few minutes later the animals actively contract and forcefully eject the pharynx out of their bodies. After approximately 11 minutes of azide exposure, the animals relax at which point the characteristic bell shape of the pharynx becomes clearly visible.
It is now ready for amputation. Removing this large unpigmented mass of tissue results in the appearance of a dark spot on the dorsal side of the amputated animals. To monitor pharynx regeneration more precisely, the animals can be stained with fluorescently conjugated streptavidin overnight.
By combining food coloring with the liver paste, the animals that eat can be distinguished from the animals that do not eat. The number of red animals can then be quantified to assess the percentage of animals with a functional pharynx. Once mastered, this technique can be completed in 20 or 30 minutes.
When attempting this procedure remember to carefully observe planarian movement to pinpoint when amputation should begin. Selective amputations can help address questions such as how stem cells initiate the appropriate lineage decisions to accomplish regeneration. This video should provide a good understanding of how to perform selective removal of the pharynx using sodium azide.
Working with sodium azide can be extremely hazardous. Remember to always handle it with gloves, discard waste properly and clean your workspace after the experiment.
