( 49s) 
From the acid alcohol the object is placed for a day at least, rather 
for a week, into an alcoholic solution of methylene-blue, to which 
1°/, hydro-chloric acid has been added. It is sufficient when 1/, gram 
of methylene-blue is dissolved in 100 ce. alcohol of about 70°/,. If 
more coloring-matter is taken, a sediment remains on the bottom of 
the bottle. After the addition of the hydro-chlorie acid, blue erystal- 
line needles separate themselves from the liquid. For this reason it is 
desirable that this addition should be made not at the moment of 
using, but some time before. 
The object when taken from the pigment, should not show 
any sediment. If it does, it has not been extracted long enough 
with acid aleohol. Although it is not lost yet, it may cost months 
hefore the sediment is removed. The intensely blue-colored object 
is treated in the usual manner in the above mentioned acid alcohol, 
which is renewed several times on the first day and once daily 
afterwards. The renewal is continued until the aleohol shows no 
blue tinge the next day. The time required for this is, of course, 
dependent on the size of the embryo. This time can be shortened 
by taking alternately alcohol of about 70°/, and a stronger one and 
hanging the object one day in the stronger alcohol, whereas the 
next day it is allowed to settle on the bottom of the bottle; this 
is not necessary however. In about a week the stain has been removed 
from all the tissues, except from the fundamental substance of 
the cartilage. It is not necessary anxiously to observe the day 
when the alcohol shows no more coloring; objects kept for a year 
and longer in the colorless acid alcohol, showed the cartilage still 
distinctly blue. { 
The object is now dehydrated in absolute alcohol, in the usual 
way, and rendered transparent in xylol. To avoid wrinkles, it 
is not put immediately from the alcohol into xylol, but first in 
a mixture of two parts of absolute alcohol with one part of xylol, 
then in a mixture of one part of absolute alcohol with two parts of 
xylol and only after that in xylol only. Larger-sized embryos are 
cut in halves or in different pieces with the razor. After that the 
objects are put first in a thin, afterwards in a thick solution of 
canada-balsam in xylol and finally in a solution, which in ordinary 
temperature is solid, but liquid at 60°. In this solution they remain 
in the thermostat at 60° during a couple of hours and are then 
enclosed in glass-cells under a covering-glass. 
The glass-cells in trade are usually too low, higher ones can easily 
be obtained by fixing stripes of window-pane with canada-balsam 
on an object-glass. 
+ 
Proceedings Royal Acad. Amsterdam. Vol. V, 
