342 



The indican caii be precipitated as indigo-blue or indigo-red, and both ways point 

 out that it is present in the protoplasm and wanting in the cell-walls, cell-nuclei, and 

 cell-sap. To demonstrate this a not too thin microscopic transverse section of 

 a leaf is put in living condition in a boiling mixture of streng hydrochloric acid and 

 ferrichloride. The indican is suddenly decomposed and the freed indoxyl as quickly 

 oxidized into indigo-blue, which is easily detected under the microscope as a precipi- 

 tate in the shape of small blue granules in the colourless protoplasm of the green 

 parenchyma and the epidermis. I could not tracé it with certainty in the chloro- 

 phyll-granules. 



If the sections, in living condition, are put in a boiling mixture of hydrochloric 

 acid and isatine, the indican passes into indigo-red, which sets off in the protoplasm 

 as very characteristic red crystal needies '). 



The enzyme, on the contrary, is exclusively accumulated in the chlorophyll-granu- 

 les as is proved by the following. 



If living microscopic sections of leaves of Pliajiis are put in an indican-solution 

 (e. g. in a decoctof Indigofera or Polygonum) they become blackish blue in a short 

 time, which colour is exclusively caused by indigo-blue precipitated in the chlorophyll- 

 granules. In the epidermis much indigo is precipitated only in the cells of the stomata, 

 elsewhere none at all. If the microscopic sections are beforehand killed and extracted 

 with alcohol, the enzyme spreads in the cell but remains confined within the cell- 

 walls, so that, by putting them into an indican solution they become of a uniform 

 intense blue, in which only the bast bundies remain colourless. 



The accumulation of enzyme in the chlorophyll-granules is perhaps connected 

 with the formation of starch from the glucose of the indican. 



As to the localisation of indoxyl in the leaves of woad I have acquired no cer- 

 tainty, but I suppose that, like indican, it occurs only in the protoplasm. 



The hypothesis of Mr. Molisch^) according to which indoxyl and indican should 

 be in close relation to the decomposition of carbonic acid in the chlorophyll, appears 

 contrary to the great accumulation of indoxyl in the root-peridermis, which is com- 

 pletely free from chlorophyll, and in the colourless root-buds of the woad, which seems 

 unnoticed by Mr. Molisch. Nor do I think his arguments and figures convincing for 

 the occurrence of indoxyl and indican in the chlorophyll-granules; moreover was Mr. 

 Molisch unacquainted with the existence of indigo-enzymes and their localisation. 



Elsewhere than in the indigo-plants indigo-enzymes seem but seldom to occur. 

 Like Dr. van Romburgh') I observed that emulsine of almonds decomposes indican, 

 and in § 6 the intensity of this action is graphically represented in connection with 

 temperature. 



The said fact may serve to demonstrate in a simple way the localisation of emuls- 

 ine in almonds. If thin sections of the seedlobes are put in an indican-solution at 



') The presensc of indoxyl in urine may bc shown with much more certainty and 

 exactness in the form of indigo-red than of indigo-blue. To this end the urine is boiled 

 with hydrochloric acid and isatine by which the colour grows red. At cooling the 

 indigo-red crystallises in characteristic microscopic ncedks. These are easily filtcrcd 

 and dissolve beautifully red in alcohol (best is to boil out the whole filter with alcohol). 



-) Berichte der deutschcn Bot. Gescllschaft, Bd. 17, p. 230, 180Q. 



') Communicated by Mr. Hazcwinkel, Maandelijksch Buiktin N°. i, pag. 8. 



