and Laboratory Methods. 1737 



insertion than an- ordinary cannula attached to a syringe. The injection which 

 was found to give the best results was the cold gelatine mass recently devised by 

 Tandler^ for microscopic work. The mass was colored with a small amount of 

 a saturated aqueous solution of anilin blue-black. 



In detail the procedure was as follows : Large specimens of the common 

 earthworm {L^imbricus agricola) were stupified in the ordinary way by the use of 

 alcohol. They were not allowed to become entirely stupified, but just as soon 

 as they had become sufficiently quiet they were opened with fine scissors along 

 the dorsal side just to one side of the mid-dorsal line. The cut began just be- 

 hind the clitellum and was carried forward in some cases clear to the anterior 

 end, in other cases only to a point about two centimeters in front of the clitel- 

 lum. After making this first cut the septa were cut down close to the body wall 

 for a short distance only and the specimen stretched lengthwise and pinned out 

 in a wax-bottomed dissecting pan in normal salt solution (.75 per cent. NaCl). 

 No attempt should be made to dissect the septa down far enough to admit 

 of pinning the specimen out flat, as it is almost impossible to avoid cutting 

 some of the larger vessels. It was only found necessary to spread the worm 

 enough so that the vessels on the intestine could be seen. 



The glass tube was partially filled by suction with the injection mass, and its 

 pointed end was inserted into the dorsal vessel, the point being directed for- 

 wards. It was found best to introduce the tube into the vessel at a point just 

 under the clitellum, as here the dorsal vessel is considerably larger than at any 

 other point in its course. It is necessary to get the tube into the vessel very 

 soon after the worm is opened or it will not be possible to insert it at all, as the 

 vessels contract strongly soon after they are exposed. It is not necessary to 

 make any cut in the vessel for the insertion of the tube as the wall is so thin as 

 to admit of its being pushed in directly. No ligature was used. After the tube 

 is inserted, a steady, even pressure may be applied to the upper end of the tube 

 by blowing, and in this way the mass may be started into the vessels. It was 

 necessary to use a very gentle pressure at first, or otherwise the vessel would 

 break immediately in front of the point of the tube. When once started the 

 mass will usually run very freely and the pressure may be increased without 

 rupturing the vessels. The injection should be continued till all visible vessels 

 are distended. The tube may then be removed and the specimen put in 4 per 

 cent, formalin for preservation and to harden the injection mass. 



Specimens injected in this way have the dorsal vessel and all the vessels which 

 come off from it in the region in front of the clitellum well filled. The parietal, 

 intestinal and lateral oesophageal vessels fill completely and the excretory plexus 

 shows very well. The " hearts " present a very striking appearance. For some 

 reason which was not apparent the injection mass could not be made to 

 pass from the " hearts " into the subintestinal vessel. Instead this vessel be- 

 came distended with blood. No practical difficulty is caused by this lack of 

 injection of the subintestinal vessel, as it is sufficiently large to be traced with 

 ease in any ordinary specimen. By dissecting and pinning the specimens out in 

 glass dishes containing a layer of black wax on the bottom, permanent prepara- 

 tions may be made. Raymond Pearl. 



Zoological Laboratory, University of Michigan. 



