198 J. B. Neilands 



medium. The flasks were sterilized by autoclaving and aliquots of sterile 

 solutions of ferric chloride plus itoic acid, itoic acid alone and ferric chloride 

 alone were added to duplicate flasks. The remaining two flasks served as 

 controls. After adjusting the volume to 10 ml with sterile distilled water the 

 flasks were inoculated and incubated at 25° under conditions of vigorous 

 aeration. The turbidity was determined from time to time with a Klett 

 colorimeter equipped with a green filter. 



Utilization of Itoic Acid by Bacillus Subtilis in the Presence of Iron. Cultures 

 of B. subtilis were grown in duplicate flasks with the usual medium. After 

 36 hr, when the itoic acid production had reached a maximum value, 1-0 ml 

 of a sterile solution of ferric chloride was added to one flask. To the control 

 flask was added 1-0 ml of sterile distilled water. At suitable time intervals, 

 aliquots of each culture were aseptically withdrawn, the cells removed by 

 centrifugation and the itoic acid determined by the ferric chloride reaction. 



Release of Hydroxylamine from the Ferrichrome Compounds. Recrystal- 

 lized samples of ferrichrome and ferrichrome A were heated at 100° in 

 3 N H2SO4 and the hberated "hydroxylamine" determined by the method of 

 Csaky (1948). 



RESULTS 



Properties of the Ferric Complex of Itoic Acid 



The colour reaction with ferric chloride exhibited by itoic acid as a function 

 of pH is illustrated in Table 1. It is apparent from these data that the reaction 



Table 1. The pH dependence of the colour reaction of 

 ITOIC acid with ferric chloride 



is strongly pH-dependent and that the structure of the complex undergoes 

 several transformations in the region pH 2-10. At neutral pH the visible 

 spectrum of the ferric complex in excess itoic acid showed general absorption 

 in the region 500-700 m/t with a mM extinction coefficient of 3-7 per g atom 

 of iron at 560 m/t (Table 2). 



