EGG ALBUMEN AS CULTURE MEDIUM FOR CHICK TISSUE. 49 



these processes disappeared, were retracted apparently, in the 

 course of fifty to seventy hours and no further evidences of them 

 were seen. In the preparations made by shaking up the finely 

 cut embryo with Ringer's solution, a greater or less number of 

 single cells were found. In the course of a few days these were 

 greatly increased in number with a distinct massing together of 

 the cells, usually along the outer border of the drop of albumen. 

 Owing to accidents of various kinds these were not carried along 

 far enough to show the tissue formation noted by Carrel. 



The most marked instance of tissue formation was that appar- 

 ent in a culture made from the heart of a fourteen day chick, 

 which, at the end of twenty days was encircled by a new forma- 

 tion five times the diameter of the original piece of tissue. This 

 new formation was several cells in thickness and composed of 

 fusiform and polygonal cells, sometimes massed together, forming 

 a network, or in other places showing distinct cell boundaries. 

 Among these cells many showed division figures at various stages. 

 Around the outer margin of the mass of cells and extending nearly 

 three-fourths of the entire distance around it, the cells had taken 

 on a different character. Here they had become flat, thin and 

 elongated in a direction parallel with the margin of the circle. 

 This formation was several cells in thickness with the cells closely 

 matted together and forming a distinct boundary that was 

 conspicuous without the aid of a lens. The remaining one-fourth 

 of the margin was occupied by cells actively pushing outward. 



To test the effects of cold on the growth of the tissues, the 

 embryo was sealed up in a stender dish containing Ringer's 

 solution and placed in the ice box of the refrigerator with the 

 temperature but a few degrees above zero, Centigrade. The 

 first of these was used the second day and behaved like normal 

 tissue. Most of those kept in the refrigerator for a number of 

 days became infected with bacteria. The longest period of cold 

 storage which gave successful cultures was four days, from Jan- 

 uary 31 to February 4. One half hour after making the cultures 

 from this embryo the cells were moving out in an active condition 

 in four out of the sixteen cultures made. The subsequent history 

 of these cultures was the same as that of unrefrigerated tissue. 



The longest period during which tissues have been kept alive 



