42 GROWTH HORMONES IN PLANTS 



maximum concentration generally has been reached, i.e., as 

 much as 400 WAE per Uter. 



The method of concentration was as follows : 



(1) The culture fluid was filtered into portions of 10 liters, 

 acidified with citric acid and concentrated under reduced pressure 

 to 200 cc. Concentration also may be carried out in large 

 enameled dishes at 50 to 60°C. Growth substance is fairly 

 stable in the impure condition. (2) The concentrated filtrate 

 was again filtered and extracted with an equal amount of 

 peroxide-free ether for 24 hours, being rotated slowly; this 

 process usually was repeated four times. (3) The purified ether 

 extracts were evaporated over 100 cc. distilled water. (4) The 

 aqueous solution was neutralized by the addition of 2 g. sodium 

 bicarbonate and shaken out three times with 100 cc. peroxide- 

 free ether, to remove various ether-soluble compounds. The 

 growth substance, which is an acid, remained in the aqueous 

 solution. (5) The aqueous solution was acidified with citric 

 acid and shaken out three times with 100 cc. peroxide-free ether. 

 (6) The ether extract was evaporated, and the residue dried over 

 calcium chloride in a vacuum desiccator. The residue was 

 extracted by boiling petroleum ether for 20 minutes. (7) The 

 growth substance did not pass over into the petroleum ether, and 

 after the latter was evaporated off it was extracted from the 

 residue by treatment with 100 cc. cold water for one hour. (8) 

 The growth-substance solution thus obtained contained some 

 citric acid, which was removed by neutralization with sodium 

 bicarbonate, weak acidification with acetic acid, and extraction 

 three times with 100 cc. peroxide-free ether. (9) The ether was 

 distilled over 100 cc. distilled water, and the growth substance 

 thus was obtained in aqueous extract. This was kept at low 

 temperatures. 



Purification entailed appreciable losses of growth substances ; if 

 there were 2,000 WAE present in the culture fluid after the original 

 concentration (step 1), only about 800 to 1,000 WAE remained in 

 the purified solution. One milligram of the dry substance con- 

 tained 4 to 10 WAE, and the product was sufficiently pure for 

 physiological investigations. 



Substratum Content and Growth-substance Production. — Boysen 

 Jensen (1932) subsequently found that Aspergillus could convert 

 tryptophane and other amino acids (lysine, leucine, tyrosine, 



