300 Leake —The Localization of the Indigo-producing 
effected which gives rise to a loss of indigo by oxidation. This loss is 
avoided by the use of sulphuric acid. Even now the proportion of 
persulphate to sulphuric acid is of considerable importance, since an excess 
of the former produces a further oxidation of indigo to isatin. Experiment 
has shown that in the above proportions a loss does not occur 1 . 
The hardened material is now embedded in paraffin wax and ribbon 
sections cut in the normal manner. The thickness of the section depends, 
to a certain extent, upon the nature of the material. Thus, for Indigofer a sp. 
a thickness of 4-5 /x was found to be most suitable on account of the small 
size and dense arrangement of the cells. For Polygonum tinctorium the 
most suitable thickness is 8 /x ; while for Isatis , Strobilanthes , Phajus , and 
Calanthe a thickness of 10-12 /x is advisable. 
Staining. For this purpose a combination of Haematoxylin and 
Eosin was found most suitable. The sections, after successive immersion 
in xylol and absolute alcohol, are placed in a solution of Haematoxylin 
consisting of: — 
Delafield’s Haematoxylin . . . 50C.C. 
Water 300 c.c. 
in which they are left for at least twelve hours. They are then transferred 
to acid alcohol (1 °/ o HC 1 in 50 °/ o alcohol) until sections are almost colourless 
to the naked eye. After the removal of all traces of the acid and alcohol 
by washing in water, the sections are transferred to eosin — 1 °/ o solution of 
Griibler’s water-soluble eosin — for at least an hour, after which they are 
rapidly dehydrated by absolute alcohol, passed into xylol and mounted 
in balsam. By this method a delicate differential coloration of cell-walls 
and cell contents is obtained in which the grains of indigo-blue stand out 
prominently and are hence readily localized. 
Examination of Material. 
The material employed in the preliminary experiments was obtained 
from two species of Indigofera , /. sumatrana , Gaert., and /. ai'recta , 
Hochst. These two species were made the subject of the most minute 
investigations, for the two reasons that they are the two indigo-yielding 
plants cultivated in Behar, where the work was commenced 2 , and that 
the indigo-yielding species of the genus Indigofera , being inhabitants of 
1 In the ‘Report to the Government of Bengal on the Research Work carried out at the 
Dalsing Sarai Research Station,’ the above-described reagent is dealt with more fully from a macro- 
chemical standpoint by W. P. Bloxam. This Report is, at the time of writing (Sept. 1904), still 
in the press. 
2 At the Dalsing Sarai Research Station. Here the plant was grown and the material pre- 
pared. Examination was deferred and finally carried out at the Botanical Laboratory, Cambridge. 
