DETECTION AND QUANTITATIVE DETERMINATION 27 



poured from the residue into, a small separatory funnel where the 

 aqueous layer is drawn off and placed again with the ground tissue. 

 A small amount of chloroform is added, and the mixture is 

 ground again; the extract is placed in the separatory funnel as 

 before, and the aqueous layer is again returned to the ground 

 tissue. The same procedure is repeated a third time. The 

 total chloroform-water mixture then is shaken thoroughly, and 

 the chloroform layer separated off. The latter contains the 

 growth hormone and is transferred to a small test tube and 

 evaporated off. The minute amount of hpoidal material which 

 remains is taken up in a very small volume of water, to which an 

 equal volume of 3 per cent agar is added. If 0.15 cc. each of 

 water and agar are used, the resulting 0.3 cc. may be cast into a 

 small block 8 by 10.7 by 1.5 mm. (there is always some loss in 

 volume, and the amount left will approximately fill a mold of this 

 size), which in turn may be cut into 12 smaller blocks of equal 

 size if quantitative determinations are desired (see Went's 

 quantitative method) . 



8. EXTRACTION OF GROWTH SUBSTANCE WITH WATER. Thi- 



mann also tried water extractions with fair success but found that 

 the growth substance was rapidly inactivated by oxidizing 

 enzymes. 



QUANTITATIVE DETERMINATION OF GROWTH SUBSTANCES 



Determination of Growth Curvature Produced by the Uni- 

 lateral Action of a Growth Substance. — After demonstrating that 

 growth substance would "diffuse out" of hving parts of plants 

 into agar, Went (1928a) showed that if agar blocks were placed 

 unilaterally on decapitated coleoptiles (after the method of Stark, 

 19216), there woidd result a curvature proportional within certain 

 limits to the amount (later shown to be concentration) of growth 

 substance in the block (Fig. 2) (Went). The investigations of 

 Nielsen (1930a, h) and Dolk and Thimann (1932) have shown a 

 similar direct relationship between concentration of growth 

 substance and coleoptile curvature (Fig. 11), and it is upon this 

 simple fact that quantitative determinations depend. 



The tests should be carried out in a darkroom under photo- 

 tropically inactive light. 



Boysen Jensen's Quantitative Method. — This involves a deter- 

 mination of the difference in length of the convex and concave 



