CASTLE: NORTH AMERICAN RHYNCHOBDELLID#. 19 
mended by Lee (94, p. 17) of stupefying with carbonated water.t The 
animals are placed in a Stender dish and covered with water from a 
“soda siphon.” As soon as they are thoroughly stupefied, they should 
be quickly transferred to the killing fluid, which is best used warm, not 
boiling hot, but heated to about 70°C. A stay of from two to five 
minutes in the carbonated water usually suffices to stupefy the smaller 
species enough for successful fixation, and indeed is better than more 
prolonged treatment. For if the animal still possesses a slight degree 
of irritability, it will usually straighten out in the warm killing fluid 
and die in a better state of extension than it was in before. The large 
species require a much longer treatment with the carbonated water. 
The best reagent to use in killing animals for whole preparations is, in 
my experience, Perenyi’s fluid, which leaves the animal well extended 
and renders it clear and transparent. It has the property of removing 
pigment from the body, particularly the darker sorts of pigment. For 
instance, I have noticed that in killing the beautifully variegated Glossi- 
phonia parasitica with this fluid, the green and brown spots often dis- 
appear entirely ; while the yellow and orange spots remain conspicuous. 
This quality is sometimes an advantage, sometimes a disadvantage. If 
one wishes to preserve the color-pattern unimpaired, he would do well 
to use a fluid containing picric acid, which seems to have the property 
of fixing the pigment ; or, better still, use formaldehyde both as the 
killing and as the preserving fluid. 
Flemming’s fluid is perhaps, on the whole, the best fixing fluid to use 
in preparing sections; corrosive sublimate is also good; Perenyi’s fluid 
is for this purpose not to be recommended, except for the study of the 
gross anatomy of the central nervous system, which it makes very clear 
by bringing out nerves and fibre tracts in strong contrast to their con- 
nective-tissue sheaths. 
Iron hematoxylin is the best stain which I have tried for sections. 
For whole preparations, animals should be heavily stained with carmine 
and then pretty thoroughly decolorized. I find Mayer’s hydrochloric 
acid carmine (70% alcoholic) very convenient and serviceable, as it stains 
powerfully and there is no danger of maceration of tissues, however long 
the stain is allowed to act. 
Decolorizing is best done with alcohol pretty strongly acidulated, as 
greater contrasts are thus obtained. I use 1% hydrochloric acid in 70% 
1 This method of stupefaction is also very useful in the study of the living 
animal, for the leech may be kept entirely motionless in the carbonated water 
within a live-box for hours, and then be revived by placing it again in fresh water. 
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