338 



of 0.5 to I pCt. indicaii, which as such, or after mixiiif; with gelatine or agar is fit 

 (or bacteriologie or enzyme experiments. 



The leaves of Phajus grandiüorus decompose the indican at high temperatures 

 with so much energy, that the extraction by boiling does not produce indican but 

 indoxyl, so that I first took Phajus for an indoxyl-plant. In this case, in order to 

 perform the experiment at low teniperature without indican decomposition, the pre- 

 paration should be eiïected in presence of an enzyme poison which does not act on 

 indican. To this effect the leaves are rubbed down in caustic linie or baryta, then 

 filtered and carbonic acid passed through; after filtering again a very pure indican- 

 solution is obtained^). The leaves can also be boiled in diluted ammoniac and the 

 superflupus ammoniac be removed by evaporation. Another method is to crush the 

 leaves under alcohol by which the enzyme, though not destroyed, precipitates in the 

 cells, while the indican dissolves in the alcohol and after evaporation of the latter 

 can be taken up in water. 



By evaporating the solutions to dryness, the impure indican results as a brown 

 mass, resembling sealing-wax, which can be powdered and, in dry condition, be kept 

 /Unchanged an unlimited length of time. The crude, neutralized or feebly alkaline- 

 solutions, when sterilized and preserved from access of microbes, also remain un- 

 changed for many months"). 



A purified indican-preparation is obtained from the decoctions by evaporating 

 them to dryness with caustic lime or baryta, dissolving in little water, filtering, pas- 

 sing through carbonic acid or precipitating the baryta with aluminium sulphate, then 

 again filtering and evaporating to dryness. The thus formed preparation contains 

 fewer pigments and fewer proteids than the crude solutions. 



The impure or thus purified indican is fit for mixing with a solid medium destined 

 for microbe-cultures. On such «indican agar« or «indican gelatine « poured out 

 to plates, colonies or streaks of microbes produce or do not produce indigo, according 

 to the species. Of this later more. 



For our experiments we used the decoctiun or the crude indican prepared from 

 it, either or not purified with lime, of Polygoiiuin tinctorium and Indigofera lepto- 

 stachya, cultivated partly in the garden of the Bacteriological Laboratory at Delft, 

 partly at Wageningen, and kindly procured by Mr. van Lookeren Campagne. I also 

 received from Mr. Hazewinkel of Klaten, Java, perfectly well preserved extracts of 

 Indigofera in tins, together with crude enzyme prepared from this plant. 



2. Preparation of the Ensymes. 



For this preparation I foliowed the method pointed out before (1. c. pag. 124). 

 The plants are rubbed fine in a mortar under alcohol and during the rubbing the al- 

 cohol is a few times renewed. In the beginning alcohol of 96 pCt. is taken, which is 

 sufificiently diluted by the juice of the plant, but afterwards some water is added 



') The extraction with caustic linie has also been applied by Mr. H a z .■ « i n k .■ I 



for Indigofera. 1 



') But after a very long time the ainount of indican diminishes whon air liiuls 

 access. When air was excludcd I could note no change in the solutions. 



