316 THYROID AND IODINE AND METAMORPHOSIS 



Iodine Solutions: Dissolve 0. 1 gram of pure crystalline iodine in 5 cc. of 95% alco- 

 hol, and then dilute to 1 liter with distilled water. This will provide a concentration 

 of 1/10,000 as a stock solution, which can be further diluted when required for ex- 

 perimental uses. 



Freshly Dissected Thyroid Glands: Three sources of fresh glands are recommended. 



a. From the frog: Since the amphibian thyroid gland is difficult to locate, use 

 the large bullfrog (Rana catesbiana) if available. Rana pipiens glands can be 

 used, however. 



Remove the lower jaw by cutting through the angles of the jaw and posteriorly 

 to the xiphisternum. Deflect the ventral skin forward and expose the under- 

 lying muscles in the vicinity of the glottis. Clip off the anterior end of the 

 xiphisternum, exposing the hypoglossal muscle which should be cut. With 

 forceps, strip these muscles forward, locate the hyoid cartilage. The thy- 

 roids will be seen posterior to the lateral hyoid processes and close to the 

 jugular veins. The preliminary dissections should be checked by micro- 

 scopic examination of the removed gland, for there are other glands in the 

 same general vicinity. 



When the dissection technique has been perfected, add a known number of 

 crushed glands to each of the experimental (finger) bowls each day. It is 

 difficult to control the amount of thyroid tissue consumed by a tadpole, but 

 if the glands are thoroughly crushed the distribution will be the more homo- 

 genous. 



b. From the rat : Experimental rats are generally available in the laboratory 

 and fresh rat thyroids can be excised, crushed (with clean sand, if neces- 

 sary) and fed to tadpoles directly. Again it is important to reduce the size 

 of the pieces of thyroid tissue to a minimum. 



c. From the slaughter house: Fresh thyroids of large mammals (sheep, pig, 

 cow) are generally available. Such thyroids may be weighed, macerated in 

 1% NaOH, and squeezed (broken up with mortar and pestle) and the mash 

 made up to a known volume (e. g. , 100 cc. ) with Standard Solution. The 

 maceration liberates the thyroid colloid into the surrounding medium and 

 the fresh and homogenous thyroid mixture may be added in known quantities 

 to the various experimental finger bowls. Such a freshly made thyroid mash 

 will remain usable for several days if frozen quickly. 



A second procedure is to dehydrate the fresh glands in acetone, freeze them 

 quickly and solidly, later to chop the pieces into small bits which can be in- 

 gested by the tadpoles. 



PREPARATION OF THE (NORMAL) CONTROL FOOD 



Anuran tadpoles can be reared to and through metamorphosis on a variety of foods. The 

 most consistently satisfactory diet is washed and par-boiled spinach or lettuce. Spinach 

 must be washed to remove arsenic powder used to destroy insects and must be boiled 

 to soften the tissues. Such spinach cannot be kept more than 24 hours at refrigerator 

 temperatures as it becomes acidified and will kill the larvae. A rough estimate of the 

 amount to provide is 1 square inch of spinach leaf per tadpole, until they are about a 

 month old when they will require more, per day. The larger bullfrog tadpoles naturally 

 require more food. The Urodele larvae (e. g. , Necturus or Amblystoma) are fed small 

 Daphnia, and later small white worms, Enchytrea. Other "normal" foods are rolled 

 oats, oatmeal and dried shrimp, liverwurst, etc. Some investigators use a mixture of 

 wheat flour with egg yolk or alfalfa. Since all foods are a source of bacterial infection 

 and growth, the food and medium should be changed daily and the culture should be kept 

 at a uniform and fairly low (18 C. ) temperature. 



