Substance in Indigo-yielding Plants. 
299 
must consequently be somewhat imperfect ; moreover, with the exception 
of one plate (Ber. der D. B. Gesell., Bd. xviii, 1899, Taf. 18), in which 
the distribution in the cells of Isatis and Calanthe , as indicated by the 
deposition of blue induced by exposure to alcohol vapour, is figured, the 
results are not illustrated. It seemed, therefore, that if a suitable method 
could be obtained for precipitating the indigo within the cells of the tissue 
and at the same time producing all the results of a fixing reagent, thus 
leaving the material in a suitable condition for hardening and staining 
in the ordinary manner, there would be room for further examination. 
This especially applies to examples of the genus Indigofera. By far the 
greater portion of the previous work has been carried out in Europe, and 
hence this genus, which is chiefly tropical in its habit, has received com- 
paratively little attention. Such a method was eventually found and will 
be briefly described. The tissue to be examined is cut into small pieces 
and rapidly immersed in a solution of the following constitution : — 
Acetic Acid, Glacial 
Sulphuric Acid, Concentrated 
Ammonium Persulphate 
Water to . 
2 C.C. 
I C.C. 
o-5 g. 
100 C.C. 
In compact tissues, such as those of Indigofera , where the intercellular 
spaces are small, penetration is slow and the pieces must be relatively 
small — J in. square at most ; in those tissues having relatively large inter- 
cellular spaces considerably larger pieces may be employed. 
As penetration takes place a deposition of indigo within the cell is 
effected, and at the same time the protoplasmic contents are fixed in the 
normal manner. After penetration is completely effected — the tissue 
should be cut in pieces of such a size that penetration is complete in 4 -6 
hours, or after twelve hours’ immersion at most — the solution is poured off, 
and the material, after 3-4 washings of twenty-four hours each in 5o°/ o 
alcohol, is in a condition to be taken up through the alcohols to absolute 
alcohol in the usual manner. 
As far as I am aware the only reference for the use of a persulphate 
in the formation of indigo in plant extracts or tissues occurs in a paper 
on the Fermentation of the Indigo Plant by Bergtheil (12). Here the 
author says : ‘ The best method for precipitating indigotin from an extract 
of the plant is that devised by Rawson in 1901 for the analysis of indigo- 
yielding plants. The extract is made strongly acid with hydrochloric acid, 
and a solution of ammonium persulphate is gradually added, the indigotin 
being precipitated in a finely crystalline form.’ The method here stated to 
be devised by Rawson has not, as far as I can ascertain, been published by 
him. Its employment as here indicated, without modification, is not to be 
recommended, since with this combination a liberation of chlorine is 
