( 496 j 
with gelatine or agar is fit for bacteriologie or enzyme experiments. 
The leaves of Phajus grandiflorus 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 experi- 
ment at low temperature without indican decomposition, the prepar- 
ation should be effected in presence of an enzyme poison which 
does not act on indican. To this effect the leaves are rubbed down 
in caustic lime or baryta, then filtered and carbonic acid passed 
through; after filtering again a very pure indican-solution is ob- 
tained !). The leaves can also be boiled in diluted ammoniac and the 
superfluous 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 the access of microbes, also remain 
unchanged 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, passing through carbonic acid or preci- 
pitating 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 witha 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 decoction or the crude indican 
prepared from it, either or not purified with lime, of Polygonum 
tinctorium and Indigofera leptostachya, cultivated partly in the 
garden of the Bacteriological Laboratory at Delft, partly at Wage- 
1) The extraction with caustic lime has also been applied by Mr, HazewiNkKeL for 
Indigofera. 
*) But after a very long time the amount of indican diminishes when air finds 
access, When air was excluded I could note no change in the solutions. 
