THYROID AND IODINE AND METAMORPHOSIS 317 



EXPERIMENTAL PROCEDURE 



1. Inseminate eggs of Rana pipiens and separate them into groups of 5 to 10 eggs, and 

 place about 200 in a flat, white enamel pan measuring about 4 x 12 x 20 inches. 

 Cover the pan with a glass plate and allow the eggs to develop at about 23-25°C. At 

 the 11 mm. stage (about 14 days) begin to feed the tadpoles a uniform diet, prefer- 

 ably of fresh, washed, and boiled spinach. Change the water and add fresh food three 

 times per week. If available, separate the tadpoles so that there are no more than 

 100 per enamel pan and they will grow faster. 



2. When the hind liinb buds have attained a length of about 1 mm. , select as many as 

 are available at exactly the same stage of development. Place 5 such tadpoles in 

 each finger bowl containing 50 cc. of Standard Solution or Spring Water. (If avail- 

 able, the 12 inch crystallizing dishes may be used with 25 such tadpoles. ) In any 

 case the stage of development, volume of medium, and size of container must be 

 identical for all groups of tadpoles. 



3. Treat the experimental animals as follows (the controls receiving the spinach diet 

 only while the experimentals receive, in addition, the following). 



Thyroxin: Place tadpoles in various concentrations for 1 week . Do not suspend 

 normal feeding. 



a. Concentration of 1/1, 000, 000 



b. Concentration of 1/10, 000, 000 



c. Concentration of 1/100, 000, 000 



Doses of 1/500, 000, 000 have been known to accelerate development. 



Thyroid Tablets: Add approximately 50 mgm. of thyroid-wheat mixture per day 

 per tadpole, for a period of one week. Do not suspend normal feeding, and 

 change the culture medium daily just before adding thyroid. 



Iodine: Add concentration and normal food but add no further iodine unless the 

 medium is changed. 



a. Concentration of 1/500,000 for 7 and for 14 days. 



b. Concentration of 1/1, 000, 000 for 7 and for 14 days. 



Fresh Mammalian Thyroid: This experiment can have only qualitative significance 

 because it is difficult to control the dose of the thyroid colloid expelled from the 

 living (fresh) glands or the amount ingested by each of the tadpoles. It will be 

 significant, however, if the student can demonstrate any acceleration of amphib- 

 ian metamorphosis by the use of mammalian thyroid gland tissue or colloid. It 

 is possible simply to squeeze fresh thyroid glands directly into the experimental 

 dishes, thus liberating some of the colloid to be ingested. 



If bullfrog tadpoles are available, their thyroids may be used in the same manner. 

 Such tadpoles can also be used as test animals, providing they are second-year 

 tadpoles and have begun the development of their hind limbs. 



4. Neotony is a condition of permanent larval state, during which the forms can repro- 

 duce. Examples are Necturus and the Mexican Axolotl. Such forms can be caused 

 to complete their arrested development, and metamorphose, by treating them with 

 the thyroid hormone or iodine. If available, attempt to get rid of the otherwise 

 permanent external gills of such forms by treating them with thyroxin. Even A. 

 punctatum and A. tigrinum may be hastened through metamorphosis by thyroid 

 treatment. 



