THYROID AND IODINE AND METAMORPHOSIS 523 



FRESHLY DISSECTED THYBOID GLAMS: Thiee sources of fresh glands are reconnnended. 



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

 use the large hullfrog ( Eana catesblana) if available. Eana pipiens 

 glands can be used, however. 



Eemove the lower Jaw by cutting through the angles of the Jaw and 

 posteriorly to the xiphi sternum. Deflect the ventral akin forward and 

 expose the underlying muscles in the vicinity of the glottis. Clip off 

 the anterior end of the xiphistemum, exposing the hypoglossal muscle 

 which should be cut. With forceps, strip these muscles forward, locate 

 the hyoid cartilage. The thyroids will be seen posterior to the lateral 

 hyoid processes and close to the Jugular veins. The preliminary dissec- 

 tions should be checked by microscopic 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 homogenous. 



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 iii5)ortant 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 ingested 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 2h 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 dally and the culture should be kept at a 

 uniform and fairly low ( ISOQ. ) temperature. 



EXPERIMENTAL PROCEDURE 



1. Inseminate eggs of Bana pipiens and separate them Into groups of 5 to 10 eggs, and 

 place about 200 in a flat, white enamel pan measuring about U 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 ll+ days) begin to feed the tadpoles a uniform diet, 

 preferably of fresh, washed, and boiled spinach. Change the water and add fresh 



