DECALCIFICATION 731 



ground. The end of the slide with the drop of blood is then touched to 

 the middle of a second slide, the drop spreads out between the two7 and 

 the first slide is rapidly drawn over the surface of the second, while 

 being held at an angle of about 45 degrees. A broad smear is thus 

 left upon the surface of the second slide,, some portions of which are 

 sufficiently thin, other portions too thick for use. Like the former 

 maneuver the success of this depends upon rapidity, cleanliness, and the 

 use of a sufficiently small drop of fluid. 



Fixation by Vapors. Smears of fluids or very thin pieces of tissue 

 may be fixed by a very brief exposure to the vapor of osmium tetroxid, 

 formalin, etc. This method is only useful in occasional instances. Os- 

 mium tetroxid vapor has been shown to preserve faithfully the mito- 

 chondria in young cells from tissue cultures (M. R. and W. H. Lewis, 

 Amer. Jour. Anat., 17, 3, 1915). 



In all methods of fixation where pieces of tissue are immersed 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 decalci- 

 fication before they can be sectioned for examination. If the calcareous 

 deposit is limited in amount, as in early fetal tissues, this can be ac- 

 complished and the tissue fixed at the same time by the use of Kleinen- 

 berg's fluid, a saturated aqueous solution of picric acid, or a 5 to 10 

 per cent, aqueous solution of sulphurous acid, the tissues being per- 

 mitted, to remain in the decalcifying fluid until a needle or slender scal- 

 pel can be readily pushed to the most central portions without producing 

 great resistance or any grating sensation. 



For well developed and mature bones the above methods are in- 

 sufficient, 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, Miiller-formol, Zenker's fluid, 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 decalcifi- 

 cation is complete. They are then thoroughly washed in running water 

 for twelve to twenty-four hours and hardened in graded alcohol. 



