DETECTION AND QUANTITATIVE DETERMINATION 31 



1 . Three per cent agar plates are prepared from agar which has been tested 

 previously and found to be free from growth substance. DuBuy (1931) 

 has shown that curvatures are reduced when higher concentrations of agar 

 are used. Two sizes of agar plates are in common use: 8 by 10.7 by 1.5 

 (Dolk, 1930) and 8 by 6 by 1.0 mm. (Went, 1935a). 



2. The coleoptile tips, portions of leaves, buds, or other plant parts to be 

 tested for growth substance are freshly severed from the plant and allowed 

 to stand proximal end downward on the rectangular agar plates for a period 

 of 2 hours (Fig. 8A). They should be covered with a bell jar lined with 

 moist paper throughout the period of diffusion. 



3. After diffusion the rectangular agar plates are cut up into 12 equal 

 blocks by means of a special cutting device: Dolk proposes that the plates 



Fig. 14. — Method for determining the degree of curvature in an Avena coleop- 

 tile. The transparent celluloid protractor is placed over a shadow picture 

 (Fig. 15) of the curved coleoptile and the angle of curvature is measured directly 

 by matching the thin line on the celluloid arm with the axis of the curved organ. 

 {Modified after Went, 1928a.) 



8 by 10.7 by 1.5 mm. be cut into 12 blocks, each 2.67 by 2.67 by 1.5 mm. 

 (10.7 mm.'); Went suggests that the plates 8 by 6.0 by 1.0 mm. be cut into 

 12 blocks, each 2 by 2 by 1 mm. (4.0 mm.'). Kogl and his associates cut 

 the agar into blocks 2 by 2 by 0.5 mm. or 2 mm.^ Since it has been shown 

 that size of block is not of great importance as long as the amount of contact 

 surface is the same, any of the foregoing sizes is satisfactory. The larger 

 blocks used by Dolk do not dry out so readily. 



4. These 12 blocks are applied unilaterally to 12 of the previously decapi- 

 tated coleoptiles (40 minute interval between decapitation and application 

 of blocks). The time allowed for curvature to take place is 110 (Dolk and 

 Thimann, 1932) to 120 minutes (Went, 1928a). The same procedure is 

 followed with agar blocks from Thimann's chloroform method (p. 26) or 

 with agar blocks made up with different dilutions of growth substance 

 (p. 24). In determining the growth-substance concentration of a solution 

 it is possible also to immerse the blocks in the unknown solution for 1 to 

 1 J^ hours and then proceed in the usual manner. 



