DECALCIFICATION AND INJECTION 647 



in the fixing and hardening fluids, it is desirable to prevent the 

 distortion of the object from the pressure of contact with the glass 

 container. This is accomplished by suspending the object by means 

 of a thread, or by resting the tissue upon a thin layer of cotton 

 placed in the bottom of the jar. 



DECALCIFICATION 



Tissues containing bone or other calcareous material require 

 decalcification before they can be sectioned for examination. If 

 the calcareous deposit is limited in amount, as in early fetal tissues, 

 this can be accomplished and the tissue fixed at the same time by 

 the use of Kleinenberg's fluid, a saturated aqueous solution of picric 

 acid, or a 5 to 10 per cent, aqueous solution of sulfurous acid, the 

 tissues being permitted to remain in the decalcifying fluid until a 

 needle or slender scalpel can be readily pushed to the most central 

 portions without producing great resistance or any grating sen- 

 sation. 



For well developed and mature bones the above methods are 

 insufficient, and stronger acids must be relied upon. Nitric acid is 

 the one most generally used for this purpose. The tissue should 

 have been previously fixed, Muller-formol or mercuric chlorid being 

 the preferable fixatives. The fixed and washed tissues are placed 

 in 2 to 5 per cent, nitric acid, and the fluid changed daily until 

 decalcification is complete. They are then thoroughly washed in 

 running water for twelve to twenty-four hours and hardened in 

 graded alcohol. 



INJECTION 



Injection is used either for the rapid dissemination of fixing 

 fluids through whole organs, embryos, etc., or for the demonstra- 

 tion of blood or lymphatic vessels. For the former purpose mer- 

 curic chlorid is the most useful fixative, since it may be immediately 

 followed by the injection of a hardening fluid, alcohol, by which 

 the remaining mercury is dissolved out of the tissue before over- 

 fixation occurs. For the latter purpose a colored fluid, either 

 aqueous or gelatinous, is forced into the blood or lymphatic 

 vessels. Berlin blue, carmin, vermilion, and lampblack are the 

 coloring matters most frequently used. The last two merely 

 require suspension in a gelatinous or an aqueous menstruum ; the 

 preparation of Berlin blue and carmin is somewhat more compli- 

 cated. 



