Extraction and Measurement Techniques 47 



An assay for the capacity of substances to produce epinastic re- 

 sponses has been described by Hitchcock (1935). This test is carried 

 out using plants such as the tomato high in epinastic sensitivity. Lano- 

 lin pastes containing the substances to be tested are applied to one 

 side of the petiole or sometimes to the whole plant, and arbitrary 

 ratings of the responses are used to estimate the quantitative differ- 

 ences. 



Auxins are involved in the movements of leaves, and this property 

 has been used as the basis for an assay technique by Ferri and Camargo 

 (1950). 



The capacity of auxins to stimulate root formation has been 

 utilized as an auxin assay (Raalte, 1950). Placing petioles of Ageratum 

 in auxin solutions for 24 hours causes the production of roots, the 

 logarithm of the number of which is proportional to the logarithm of 

 the concentration of indoleacetic acid between 10 ♦^ and 10-^ M or 

 about 0.001 to 0.1 mg./liter. 



CHEMICAL AND PHYSICAL ASSAYS 



Two color tests for indoleacetic acid were proposed by Mitchell 

 and Brunstetter in 1939, one utilizing a reaction with nitrous acid and 

 the other a reaction with ferric chloride. The nitrite reaction has not 

 found widespread use as a quantitative test, but is a good indicator for 

 use in paper chromatographic separations. The ferric chloride re- 

 action, however, has been developed into a good quantitative assay by 

 Tang and Bonner (1947) and by Gordon and Weber (1951). Each of 

 these workers has described an acid ferric chloride reagent which forms 

 a color complex with indoleacetic acid (lAA). The color is measured 

 quantitatively with a colorimeter. The components of each of the two 

 described reagents are as follows: 



Tang and Bonner Gordon and Weber 



15 ml. 0.5 M FeCla 1 ml. 0.5 M FeCU 



300 ml. H^SO, (Sp. gr. 1.84) 50 ml. 35^0 HC10« 

 500 ml. H^O 



These reagents differ only in the selection of mineral acids, and in 

 each case apparently the same color complex with lAA is formed. 

 The procedures used in each of these techniques are as follows: 



Tang and Bonner Gordon and Weber 



Take 1 ml. of aqueous lAA sample. Take 1 ml. of aqueous lAA sample. 



Add 4 ml. acid reagent. Add 2 ml. acid reagent. 



Read with colorimeter after 30 min. Read with colorimeter after 25 min. 



(Maximum absorbancy at 525 m,j.) (Maximum absorbancy at 530 m^) 



