Generation and Long-term Maintenance of Nerve-free Hydra

The overall goal of this procedure is to generate and efficiently maintain nerve-free hydra by manual force feeding and burping. This method can help answer key questions in the hydra field about the role of the nerve and various behaviors and biological processes. The main advantage of this technique is that it is simpler, safer and more time efficient than previously published protocols for feeding and burping.

Using forceps to manipulate the hydra on the microscope can be difficult and frustrating at first, but it quickly gets easier with practice. Begin by adding Artemia cysts to a tall, narrow glass container that tapers at the bottom. Fill the container with Artemia water, taking care not to exceed one gram of cysts per one liter of water for a higher hatch yield.

Cover the top of the container with parafilm and insert a 10 milliliter serological pipette through the parafilm, into the container. Taking care that the pipette tip reaches the bottom of the container. Attach a piece of tubing to an aquarium air pump, then fit the tubing around the pipette to provide air ration to the container.

When the Artemia have hatched use an Artemia net to strain the shrimp from the culture and wash the animals for about 20 seconds in deionized water. At the end of the wash, transfer the Artemia into a dish with hydra medium. Under a stereo microscope, use a pair of fine forceps to euthanize the shrimp with light squeezing.

Placing the freshly sacrificed Artemia close to the hydra to facilitate a fast feeding. Now grasp the first hydra by the foot with one pair of forceps and pinch the body of the hydra with a second pair of forceps to make the hydra contract. Tap the center of the hypostome to make the hydra open its mouth.

If the mouth does not open, puncture the center of the hypostome with the forceps and slightly release the pressure on the tips to stretch out the mouth opening made by the puncture. Quickly insert as many Artemia as possible into the hydra's gastric cavity, until the cavity is full and no more Artemia can be inserted without damaging the hydra, accidentally pulling out any Artemia from inside the cavity or preventing the mouth from closing. If the mouth begins to close, stretch the mouth open with the forceps as just demonstrated, taking care that no Artemia already inside the animal, come out.

If the hydra is too small for a whole Artemia, cut the shrimp with the scalpel and fit the hydra smaller pieces. If the Artemia does not stay inside the hydra, it maybe necessary to press the forceps against the Artemia, to hold the Artemia inside until the mouth closes. When all of the hydra have been fed, use a glass pasteur pipette to transfer the hydra into a dish with fresh hydra medium, supplemented with rifampicin, taking care to avoid accidental burping.

Between eight and 20 hours after the feeding, use P320 or similar grid sandpaper, to smooth down the point of a 30 gauge hypothermic needle until the tip is flat. Attach the needle to a disposable one milliliter plastic syringe and fill the syringe with fresh rifampicin supplemented hydra medium. Place the hydra culture dish under the microscope and use fine forceps to hold the foot of the hydra.

Tap at the center of the hypostome with the syringe needle. If the mouth doesn't open, insert forceps into the hypostome to open the mouth as just demonstrated. Next, use the syringe to very gently flush the medium into the gastric cavity, taking care not to blow away the hydra until all of the debris has been expelled.

Then, use a glass pasteur pipette to transfer the burped hydra into a dish with fresh rifampicin supplemented hydra medium. Immediately following an initial eight hour culture treatment, all hydra survived. Some animals will have only tentacle stubs remaining, while others will have completely lost their tentacles.

About one week following treatment, the hydra will demonstrate signs of small, stub-like tentacle regrowth before their eventual full regeneration, over the next seven days. After a second treatment, about 10%of the original number of animals that have undergone both treatments recover and grow to a size suitable for experimentation. Dual-treated hydra exhibit tentacles that appear thinner than those of untreated animals, have tissue that is more transparent and appear bloated due to the inability to open their mouths to relieve osmotic pressure.

The nerve nets in the heads of untreated hydra display distinct fibers that radiate outward from the mouth, as well as obvious cell bodies. While the same fibers are absent in nerve-free hydra after double treatment with culturezine. Further co staining with DAPI reveals a reduced number of nuclei in nerve-free hydra compared to untreated controls, due to the loss of interstitial cell lineage cells induced by dual culture zine treatment.

Once mastered, a single hydra feeding can be completed in under one minute if it is performed properly. After watching this video, you should have a good understanding of how to force feed and burp nerve-free hydra.