306 



REGENERATION 



2. Into each finger bowl place an equal number of tadpoles, a minimum of 5. 



3. Mark the 6 finger bowls containing the same medium as follows: 



a. Vertical cut. 



b. Cut angled toward the dorsal body wall. 



c. Cut angled toward the belly. 



d. V-shaped cut, apex toward body. 



e. V-shaped cut, apex away from body. 



f. Control - no cut. 



4. Consult the accompanying diagram of a frog tadpole (stage #25) to determine the 

 angles of the various cuts prescribed. All but "a" and "f" (the control) are illus- 

 trated. 



FROG TADPOLE (STAGE =25) SHOWING LEVELS 

 OF CUTS FOR TAIL-FIN REGENERATION STUDIES 



A - V-shaped cut with apex 



away from the body. 

 B - Transverse cut angled 



toward dorsal body. 

 C - Transverse cut angled 



toward belly. 

 D - V-shaped cut with apex 



toward the body. 



5. Prepare an operating Syracuse dish with Permoplast base. Fill the dish with 

 one of the above (3) solutions, beginning with the 10% Standard. Transfer all (5) 

 tadpoles successively from each of the finger bowls containing the 10% Standard 



to the operating dish and cut the tail fin in the manner indicated on the previously- 

 marked finger bowl. That is, there will be finally 18 finger bowls, containing 

 six (6) different solutions, and representing five (5) different cuts, and a set of 

 controls. The cuts should be made with a sharp scalpel while the tadpoles are 

 immobilized with 1/10, 000 MS 222 (made up in the same medium). If the cuts 

 are made on the group from a single finger bowl, while in the same Syracuse 

 dish, it will be somewhat easier to insure the cuts being similar. 



6. Immediately return the tadpoles to the finger bowl with the appropriate medium, 

 and properly marked. It is best to make the cut in the medium to be tested. 

 (Do not save the tail tips unless for incidental chromosome counts - see "Tail 

 Tip Technique". ) 



7. Place all 18 finger bowls under identical environmental conditions of light and 

 temperature, and minimize evaporation by keeping them covered. 



The tail fins should be examined under the dissection microscope daily for about ten days, 

 and the record consists of daily sketches beginning with a sketch immediately after cutting. 

 Note the color of the regenerating (blastema) tissue and the angle of the regenerate. 



THE DEVELOPMENT OF ORGAN ANLAGEN IN REGENERATING BLASTEMAS 



This experiment involves regeneration, transplantation, and possibly some induction. It 

 is based upon the assumption that the regenerating tissue (the blastema) is essentially 

 embryonic in nature and will either supplement an implanted organ anlage or will, under 

 the influence of such an anlage, be induced to form certain organs. 



