Appendix B 



PREPARATION OF MATERIALS 



1. Killing the specimens. Necturus is best killed by placing in hot water; turtles by 

 injection of ether or chloroform into the cloaca or better the trachea; birds and mammals by 

 inclosing them in a tightly closed vessel with a wad of cotton soaked in ether or chloroform. In 

 handling turtles pull the head forward by inserting a stout hook behind the jaw, and pry open 

 the mouth. 



2. Preparation of skeletons. Skeletons are best prepared from fresh materials or, in the 

 case of marine forms, those that have been preserved in brine. It is difficult to prepare skele- 

 tons from specimens that have been preserved in formalin. To prepare a skeleton remove the 

 skin, all of the viscera, and as much of the muscles as possible and soak the specimen in water. 

 The remaining flesh will decay and may be removed with a stiff brush or forceps. This process 

 of maceration in cold water takes some time. The process may be much shortened, and tough 

 specimens are more easily prepared by immersing the specimen for a few hours in hot or boiling 

 water to which gold dust or the following soap solution has been added. Kingsley gives the 

 following formula for the soap solution: 



75 gms. of hard soap 

 12 gms. of potassium nitrate (saltpeter) 

 150 c.c. of strong ammonia 

 2,000 c.c. of distilled or soft water. 



Mix thoroughly. In using, take one part of the soap solution to three or four parts of water. 

 The length of time required before the flesh will separate easily from the bones varies with 

 different animals and is shorter for small or young specimens than for large and old ones. Davi- 

 son gives the time for the cat as two to four hours in boiling water or better three to six hours in 

 water kept at 75 to 90 C. He also states that if the bones are heated for only one to two hours 

 at a temperature not above 85 C., the ligaments will be preserved, and when the skeleton is 

 dried the ligaments will harden and hold the bones together. Skeletons containing a consider- 

 able amount of cartilage should not, of course, be boiled but treated only with moderately hot 

 water. For fishes a few minutes' treatment with hot water is generally sufficient. Cartilagi- 

 nous skeletons should be preserved in weak formalin or in 70 per cent alcohol. 



3. Injection of the circulatory system. To render the blood vessels conspicuous and more 

 easily followed it is advisable and in fact practically necessary that the arteries at least be 

 injected with a colored solution. Injection syringes for this purpose may be obtained from 

 dealers in laboratory supplies, or an ordinary rubber atomizer bulb may be used. A glass 

 cannula is inserted in the vessel to be injected. A cannula is simply a piece of glass tubing 

 drawn out in the flame at one end to a size suitable for the blood vessel into which it is to be 

 inserted. It is also desirable that the end to be inserted in the vessel be slightly enlarged as it 

 will then hold more securely in the blood vessel. Loosen the vessel to be injected from the sur- 

 rounding tissues, pass a cord under it, and tie the cord in a loose single knot above the vessel. 

 With a fine scissors make a V-shaped cut into the blood vessel, having the cut extend not more 

 than halfway through the vessel, and immediately insert the cannula into the vessel. Tighten 

 the knot around the cannula and blood vessel by pulling on the two ends of the cord, but make 

 only a single knot. The cannula is connected with the injection syringe or rubber bulb by a 



