CASTLE : NORTH AMERICAN RHYNCHOBDELLID/E- 19 



mended by Lee ('94, p. 17) of stupefying with carbonated water,* 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 hsematoxylin 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 tlie living 

 animal, for the leech may be kept entirely motionless in the carbonated water 

 within a live-box for hours, and tlien be revived by placing it again in tVcsh water. 



