SPECIAL METHODS: 439 



10. Methods of Decalcification. Tissues which contain calcified 

 nodules or bone must be freed from lime-salts before they can be 

 cut. It is difficult to do this rapidly without injury to the softer 

 tissue-elements. When good results are desired, and the necessary 

 time can be afforded, the tissues should first be fixed and hardened, 

 small pieces being selected. Zenker's fluid fixes w r ell for this pur- 

 pose, but Orth's fluid or alcohol may be used. If Zenker's or Orth's 

 fluid is used, the tissues must be washed in water and hardened in 

 alcohol for at least a day before they are decalcified (see Methods of 

 Fixing and Hardening, pp. 403, 408). 



Decalcification is accomplished by treatment with acids. Five 

 per cent, nitric acid will decalcify small pieces of bone in from one 

 to five days. The progress of the decalcification may be deter- 

 mined by pricking the tissue with a needle, but after it appears 

 to be soft it is well to continue the action of the acid for a day or 

 two, lest some undissolved particles should remain and injure the 

 edge of the microtome-knife. A saturated aqueous solution of 

 picric acid is sometimes used for decalcifying. Its action is very 

 slow, though not injurious to the tissues, which require no prelimi- 

 nary treatment, the picric acid acting as a fixing and decalcifying 

 agent. 



After decalcifying in nitric acid the tissues should be thoroughly 

 washed in running water for twenty-four hours and then rehardened 

 in alcohol, after which they may be embedded. After decalcifying 

 in picric acid the tissues are placed in 70 per cent, alcohol and hard- 

 ened without previous washing in water. 



When rapid decalcification is necessary nitric acid and phloro- 

 glucin, which restrains the destructive action of the acid, may be 

 used. The solution is prepared by dissolving 1 gram of phloro- 

 glucin in 10 cc. of pure nitric acid. To this 100 cc. of 10 per cent, 

 nitric acid are added. Decalcification takes place within a few hours 

 in this solution, which contains about 20 per cent, of nitric acid. 

 The tissues should then be washed and hardened. 



Another rapid method which combines decalcification with hard- 

 ening is to place the fresh tissues in a large bulk of 5 per cent, 

 nitric acid in 80 per cent, alcohol. After decalcification has taken 

 place the tissues are hardened in alcohols of increasing strength, 

 large quantities being used in order to remove the acid. Before 

 staining, the sections should be washed thoroughly in water to get 

 rid of any residual traces of acid. 



