DAVID L. CLAYBROOK 235 



stirred gently to bring them into contact with the Hydras tentacles. 

 The effect of the period of heating on the subsequent growth 

 rate is depicted in Table 1. The reduction in the growth rate is 

 seen to be progressive with time of heating. This indicated to us 

 that some substance was being inactivated by the heat treatment 

 so that its availability to the Hydra became limiting to the growth 

 process. 



TABLE 1 



Relation of growth rate of Hydra littoralis. Ham strain, to period of 



heating of Artemia (70°) 



Growth Rate 



Heating time Doul:)hng time Growth Rate Constant 



(min.) (days) (/c)** 



.36 

 .21 

 .20 

 .15 

 .13 



On the assumption that replacement of the growth-limiting fac- 

 tors to a nutritionally deficient diet would increase the rate of 

 ]:)udding, we assayed numerous biochemical and biological sub- 

 stances for their capacities to stimulate budding when added as 

 supplements to a diet of heated shrimp. Artemia heated for 7 

 minutes at 70 were fed to Hydra cultures for at least a week 

 before the Hydra were used for bioassays. This period served to 

 deplete the animals of any reserve of the growth factor. 



The heated Artemia diet was first supplemented with defined 

 and complex substances dissolved in the salt solution, bathing the 

 Hydra. Natural extracts, vitamins, amino acids, and other possible 

 growth factors, alone and in various combinations, were tested in 

 this system. No stimulatory effect on the growth rate was observed 

 in any expriment. 



Since the lack of growth response to external supplements 

 could have been due to relative impermeability of the ectodermal 

 cells to dissolved materials, it was necessary to devise a technique 

 for introducing the test materials directly into the coelenteron 



