298 Leake . — The Localization of the Indigo-producing 
The first experiments to be undertaken with the definite object of 
localizing the indigo-forming substance within the tissues of the plant 
are those of Molisch, published in 1893 (5 and 6). In these experiments 
he uses two methods for ascertaining whether a particular tissue contains 
this substance. The first, or macro-chemical, method consists in boiling 
the tissue with dilute ammonia (2 c.c. commercial in 98 c.c. water) ; indigo 
is thus formed and will become evident on filtering, when the masking 
colour of the filtrate is removed. Chloroform may be added to the 
unfiltered ammoniacal extract, when the chloroform will separate out as 
a blue layer, for indigo is slightly soluble in chloroform. In the second, 
or micro-chemical, method the tissue is exposed in a confined atmosphere 
to the action of alcohol vapour for a period of twenty-four hours, after which 
the chlorophyll may be removed by immersion in absolute alcohol. The 
presence of indigo, and hence of the indigo-forming substance, will be in- 
dicated by the blue coloration of the tissue. In a later paper in 1899 ( 7 ) 
he suggests chloroform as an alternative for alcohol, and later again, in the 
same year (8), strong ammonia. Direct immersion in 40°/ o alcohol also 
results in a formation of blue within the tissues. Sections can now be cut, 
or the material teased and examined under the microscope. 
Beijerinck, in 1899 ( 9 ), in applying the 4 alcohol test’ of Molisch to 
hidigofera arrecta, Hochst. (termed by him /. lep tost achy a), found that 
indigo-formation occurred in only the youngest leaves. In the ‘woad,’ 
again, only a slight blue deposition occurs. As an alternative and more 
satisfactory method, he immersed the tissue used for experiment in 
mercury, or exposed it in a vacuum, for a few hours, after which he 
exposed to ammonia vapour as before, finally extracting the chlorophyll 
with alcohol. 
In 1900 ( 10 ) he uses as a macro-chemical method the addition of 
hydrochloric acid and ferric chloride to a decoction of the tissue to be 
examined, and adapts this reaction for micro-chemical examination by 
boiling a section of the living leaf or tissue in a mixture of strong hydro- 
chloric acid and ferric chloride. As an alternative he uses in the same 
manner a solution of isatin in hydrochloric acid, by which reagent the indigo- 
forming substance is supposed to yield indigo-red, which is deposited 
in situ within the tissue. The former method of indicating the presence 
of an indigo-forming substance by the formation of indigo as the result of 
the action of an acid and ferric chloride is also used by Hazewinkel ( 11 ). 
Methods. 
The above methods are in no case of such a character as to give to 
the tissue a condition at all comparable to that of material treated by the 
ordinary fixing and hardening reagents, and the microscopic examination 
