46 CULTURE METHODS AND TECHNIQUE 



hours, and then with absolute alcohol. It is desirable to change the 

 absolute alcohol once, permitting the material to remain each time 

 from four to six hours. 



The infiltration methods, that is, methods by which the penetra- 

 tion of paraffin into the tissues and cells is effected, are various, and 

 biologists do not agree as to which is most practicable. The chief 

 difference lies in the nature of the solvent employed to precede the 

 paraffin. After having tried for years chloroform, xylol, and cedar 

 oil in turn, it is preferred with the majority of tissues to employ 

 the chloroform method, a method at once simple and sure. It is 

 as follows : 



The chloroform infiltration method. The material from absolute 

 alcohol is put into a mixture of equal volumes of absolute alcohol 

 and chloroform. It is permitted to remain in this mixture for from 

 twelve to twenty-four hours, and then pure chloroform is sub- 

 stituted. In the pure chloroform readily penetrable tissues will soon 

 sink and will be thoroughly penetrated within twenty-four hours. 

 Many tissues will require two days, and two days may be most 

 desirable. At the end of this period, whether the tissue is sunken 

 or not, it is poured out into an open dish (small porcelain vessels 

 2 or 3 centimeters broad and deep being very desirable), and into 

 this dish is cut more than enough hard paraffin (53 to 54 C.) 

 finally to cover the material with paraffin alone, or, better, suffi- 

 cient in which finally to imbed the material. These dishes are then 

 put into the oven at 55 to 56 C. and the chloroform evaporates 

 within a day or two. If stirred once or twice, it will evaporate more 

 promptly, and the material is then in excellent condition to be 

 imbedded in the papers or special imbedding trays commonly used. 

 Paraffin used in this process, if the chloroform has all evaporated, 

 is excellent from the standpoint of viscosity, and consequently it 

 will cut more evenly than fresh paraffin. 



Cedar oil and xylol infiltration. Some prefer to use cedar oil as 

 a solvent with the paraffin. That method, however, is somewhat 

 more taxing and seldom to be recommended. The cedar oil is 

 more difficult to remove from the tissues, and I have found it 

 desirable only in cases where the material is exceptionally brittle. 

 When cedar oil is employed, it seems desirable to pass from 

 absolute alcohol to a mixture of cedar oil and alcohol, the tissues 



