[Vor. 1, 1914] 
242 ANNALS OF THE MISSOURI BOTANICAL GARDEN 
the leaf to dry in the air, remove the area desired from the 
balance of the leaf, and place in a killing fluid. Тһе best com- 
bined killing and tissue-clearing mixture for this purpose is 
one recommended by Dr. Duggar, composed of glacial acetic 
acid and 95 per cent aleohol. I have used equal parts of these 
agents most advantageously. This dissolves the chlorophyll, 
renders the leaf transparent or nearly so, and at the same time 
fixes the fungus with little plasmolysis. Allow the killing mix- 
ture to act for 24-36 hours; wash in 50 or 70 per cent alco- 
hol, to remove the acid; and pass successively through the stain 
(15-30 minutes), water (2 minutes), acid alcohol (as short a 
time as possible), carbol-turpentine (until clear), xylol (until 
clearing agent is removed), and then mount in balsam. This 
process of differential staining has been successfully used 
with Ascochyta Pisi on pea, Helminthosporium sativum on 
barley, and Phoma Brassice on cabbage. 
Pianeze’s stain has not given as good results with the rusts 
as Durand’s combination of Delafield’s haematoxylin and eosin. 
Durand's stain! was not uniformly successful, however, and it 
was found that one of the chief diffieulties often experienced 
finds its explanation in the killing solution which the stain 
follows. Flemming's solution, which was first used, gave 
very poor results. А modification of Gilson's mercuric chloride 
solution was found most satisfactory. This solution, as recom- 
mended by Dr. Durand, is made up as follows: 
bic AD 007 КАССИНИ ee 60 ce. 
Alcohol, 95 рег сепі........................... 42 се. 
Acetic acid, шізсізі............................ 18 ce. 
Nitric acid, concentrated...................... 2 ce. 
Mercurie chloride, sat. ад. өо!................... 11 ce. 
Diseased tissue may be fixed from 6 to 24 hours, then washed 
in 65 per cent alcohol, run through the alcohols, infiltrated 
with cedar oil, and imbedded in paraffin. This method is 
undesirable for nuclear structures, but gives excellent prepara- 
tions for gross histological work. 
Graduate Laboratory, Missouri Botanical Garden. 
! Durand, E. J. The differential staining of intercellular mycelium. Phyto- 
pathology 1: 129-30. 1911. 
