FIXATION AND FIXATIVES 13 



This liberates almost all the bound picric acid and is much the best after- 

 treatment for picric-acid-stained materials which has appeared in the litera- 

 ture. It might be explained that the objection to the retention of bound picric 

 acid is that it interferes seriously with some forms of staining. 



Treating Hard Materials. It so happens that some materials, even after fix- 

 ation, are so hard that they cannot possibly be cut into sections. This hardening 

 is due either to the presence of calcium in the form of bone or calcareous 

 plates or to the presence of chitin. The removal of bony or calcareous material 

 is not as easy as it sounds, for if one were merely to hang the material in an 

 acid mixture there would be a great hydrolysis of the protein. Therefore, it is 

 necessary to have in the solution, besides acid, something which will prevent 

 hydrolysis and swelling. The most commonly used reagent for this purpose is 

 phloroglucinol. The following is a typical formula: 



Haug's Solution: 



96% Alcohol 70 ml. 



Water 30 ml. 



Phloroglucinol 1 Gm. 



Nitric acid 5 ml. 



Note: The phloroglucinol is dissolved in the warm acid, cooled, added to the 

 water, and the alcohol then mixed in. The acid should be heated very gently 

 under a stream of warm water: heating over a flame is likely to cause an 

 explosion. 



There is not much use in transferring objects to a vial containing this fluid, 

 for they will fall to the bottom and rapidly exhaust the acid around them. It 

 is much better to hang them with a fine thread of silk from the top of the 

 vial. The decalcification is complete if the object no longer feels hard when 

 pricked with a pin. If there is no part of the object where one may without 

 damage apply the pin, it is very easy to find out if decalcification is complete 

 by having a dentist or physician observe the specimen on a fluorescent screen 

 by x-ray. As soon as decalcification is complete the object should be washed 

 in large volumes of 70 per cent alcohol. Some people claim that phloroglu- 

 cinol interferes with afterstaining and prefer to restrain the swelling of the 

 tissues with mercuric chloride. The best mixture of this type is: 



McNamara, Murphy, and Gore's Solution: 



Water 80 ml. 



96% Alcohol 10 ml. 



40% Formaldehyde 8 ml. 



Mercuric chloride 2 Gm. 



Trichloroacetic acid 6 Gm. 



Nitric acid 1 ml. 



