Storing Live Frogs

·  If cooling the frogs to decrease activity before double pithing, frogs can be kept in a covered cake pan with ice and should be kept moist with amphibian Ringer’s solution.

·  Clean the frogs before beginning preparation for any experiment.  Handle frogs with care always ensuring that you wear latex gloves and moisten the gloves in water.

Frog Anatomy Reference

Cranial cavity (brain)

Spinal cord

Between the eyes, soft spot at the base of the (hard cartilage of the) skull

Extends from cranial cavity down to about the middle of the back

Pithing Procedure

Before the experiment, you need to render the frog insensitive to pain. Pithing is one procedure to accomplish this. Pithing will destroy the brain. (For some experiments, double pithing will include severing/destroying the spinal cord.) Pithing is relatively painless to the frog.

Read the entire procedure before beginning so you can perform the procedure quickly.

  1. Hold the frog facing away from your body, with the lower extremities extended.
  2. Grasp the frog with your first two fingers: first finger on the nose, second finger under the jaw. Flex the head forward (away from your body).
  3. Move probe down midline, over a bump (as a reference point) that is the occipital process until you come to the soft spot of the foramen magnum.
     
        Do not use undo pressure on the frog skin.
        The frog skull is fairly hard cartilage; soft spot is at the end of the skull.
     
  4. Insert the probe quickly into the cranial vault and sever the brain and spinal cord.
  5. Move the probe into the cranial vault and move it from side to side to destroy the brain.
     
           Without the brain, perception of any sensory phenomena is impossible.
     
  6. Test for reflexes to confirm that sensory perception has been destroyed and ensure that the spinal cord is still intact, although it is now in shock.

Corneal Reflex
(sensory perception check)

Reflex Withdrawal Response 
(spinal cord check)

In a normal frog, as soon as you touch its eyelid, it will blink. With no brain, there is no response.

Pinch the frog's leg and it should withdraw as shown below.

NOTICE: The frog may crouch, jump, or even make noise, but it is not in pain.  These actions are neural reflexes controlled via motor senses in the spinal cord, not brain functions.

  1. Keep the frog moist. The frog will still be breathing because frogs breathe primarily through the skin, not the lungs, so it is important to keep the frog moist.
  1. Sever the spinal cord: Keep the probe in the cranial canal and turn the probe around into the vertebral canal (do this without removing the probe).

When the spinal cord is severed, the frog legs become completely limp (due to flacid paralysis of skeletal muscle). The frog will never assume a crouching position again, and if you pinch the frog after the spinal cord is severed, the frog will not feel anything and there will not be a reflex withdrawal.  In other words, spinal severing makes work easier because the muscle no longer reflexes.  Local muscle twitching resulting from spinal nerve ending stimulation may still occur.

Squirt the frog with amphibian Ringers (a special saline solution for frog electrolyte concentration).

OR


When you no longer require tissue experimentation, place a moist napkin (soaked in Ringers) over the frog.

Continue to keep the frog moist so it can continue to respire through the skin (tissue remains vital) and the heart can beat.