DECALCIFYING FLUIDS 493 



Phloroglucin i 



Nitric acid 5 



Alcohol, 95% 70 



Water 30 



The addition of i or 2 per cent, of nitric acid to the 80 per cent, alcohol 

 will decalcify small embryos. The specimen should then be thoroughly 

 washed in fresh 80 per cent., in order to remove the acid. 



2. Imbedding. 



Most of the fixatives employed are in aqueous solution. After fixa- 

 tion and the removal of the fixative by washing in water or alcohol, as 

 directed, the specimen must not be left in water, but must be dehydrated. 

 Dehydration has a double purpose: (i) to remove the water, which espe- 

 cially favors post-mortem decomposition, and (2) to prepare the tissue 

 for infiltration with the imbedding substance or, in the case of objects 

 to be mounted whole, for infiltration with the mounting medium. 



All the fixation methods given above end with placing the block of 

 tissue in 80 per cent, alcohol. Here they may be left until wanted, 

 although immersion for a considerable time causes a gradual loss in stain- 

 ing qualities. Stronger alcohol causes an overhardening, while macera- 

 tion may occur in weaker alcohols. 



Dehydration is accomplished by immersing the specimen in gradually 

 increasing strengths of alcohol. Those commonly employed are 50 per 

 cent., 70 per cent., 80 per cent., 95 per cent, and absolute. The lower 

 grades may be prepared from the ordinary barrel alcohol, of about 95 per 

 cent, strength, as follows: 



80 per cent. 425 c.c. 95 per cent, alcohol mixed with 75 c.c. distilled water 

 70 per cent. 370 c.c. " 130 c.c. 



50 per cent. 265 c.c. " 235 cc. 



The specimen is left in each grade long enough to be saturated. The 

 time required varies from 3 to 24 hours. Objects of average size require 

 about 6 to 12 hours. Prolonged immersion in 95 per cent, or absolute is 

 very injurious to the tissues. 



In imbedding, the tissue is surrounded and infiltrated with a firm sub- 

 stance which can be cut into thin sections, supporting and holding firm the 

 fragile tissue. Celloidin, which is solid upon the evaporation of its solvent, 

 and paraffin, which is solid at ordinary temperatures, are the substances 

 used, each having its particular advantages. 



Paraffin Imbedding. Specimens cannot be passed directly from al- 

 cohol to paraffin, since alcohol dissolves only a very little paraffin and the 

 specimen would not be thoroughly infiltrated. So the specimen must first 

 be passed through some fluid which mixes with absolute alcohol and will 

 dissolve paraffin. Of a host of reagents possessing this property, chloro- 

 form is recommended for general use. 



After thorough dehydration (12-24 hours in absolute alcohol), the 



