190 GLUCOSIDES 



ammonia. The addition of chloroform to the filtered extract 

 may be made to separate the indigo ; the chloroform will sink 

 to the bottom of the solution, carrying with it the indigo. 



2. Tissues containing indican on exposure to the vapour 

 of alcohol for twenty-four hours will turn blue ; the reaction 

 will be better marked if the chlorophyll be subsequently dis- 

 solved out with absolute alcohol. 



3. The tissue, in bulk or in section, may be boiled in 

 strong hydrochloric acid and ferric chloride added. The in- 

 digo will separate out. 



4. The tissue is cut up into pieces and quickly immersed 

 in the following mixture : 



Glacial acetic acid 2 c.c. 



Strong sulphuric acid I c.c. 



Ammonium persulphate *5 gram. 



Water to 100 c.c. 



As this fluid penetrates the cells, the indigo is precipitated in 

 blue granules. When penetration is effected fully, the material 

 is washed for twenty-four hours in water, after which sections 

 may be cut and stained in the usual way. 



Indican is hydrolysed by indimulsin, with which it is as- 

 sociated in the plant, into glucose and indoxyl according to 

 the equation : 



COH 



C 7 H 6 NC.O.C 6 H n O g + H 2 = C 6 H 12 O 6 + C 6 H 4 <^)cH 



NH 



Indican Indoxyl 



The same reaction can also be effected, though more slowly, 

 by emulsin. 



The resulting indoxyl, by exposure to air, is oxidized to 

 the deep blue colouring matter indigotin. 



COH CO CO 



2C 6 H 4 / VH + O = C 6 H 4 C : C>C 6 H 4 + H 2 O 



NH NH NH 



Indoxyl Indigotin 



The production of indigotin from the indigo plant is based 

 on these two reactions and consists in fermenting the plant 

 material by steeping it in slightly acidified water for a few 

 hours, and then exposing to the air the fermented extract to 

 which a little ammonia has been added to facilitate oxidation. 



