Decalcificntion 27 



bone.^ Case {1953) considered the electrolysis method to be inferior to 

 the use of either nitric or formic acid. (Unyden [19^2) and Lillie et al. 

 [1951) demonstrated that an increase in temperature accomplished the 

 same thing. Probably the rise in temperature was the principal reason 

 for speed of decalcification. Culling (1957) agreed that heat speeds up 

 decalcification but causes severe swelling and is not to be recommended. 

 Lucas (1952) found no evidence that the passage of a current by itself 

 accelerated decalcification. Experiments by Lillie et al. (1951) indicate 

 that in respect to temperature there is another story. Decalcification at 

 room temperature or even cooler produces the best results for staining. 

 The present author finds nothing to be recommended about the elec- 

 trolysis method. 



Chelating Agents for Decalcification 



These agents during decalcification offer the advantage of maintaining 

 good fixation and sharp staining. They are organic compounds that have 

 the power of binding certain metals, such as calcium and iron. Versene 

 (Dow Chemical Company) or Sequestrene (Geigey Chemical Company), 

 the disodium salt of ethylene diamine tetracetic acid (EDTA), is the 

 most commonly used agent. The method does have two disadvantages; 

 the tissue tends to harden and decalcification is slow. 



Hilleman and Lee (1953) 



200 ml. of a 5.5% solution of either Versene or Sequestrene in 10% 

 formalin, for pieces 40 X 10 X 10 mm. It may require up to 3 weeks. 

 Renew the soliuion at the end of each week. Transfer directly to 70% 

 alcohol. 



Vacek and Plackova (1959) 



0.5M solution of EDTA at pW 8.2-8.5 yields better results in silver 

 methods than does decalcification -^vith acids. 



Schajowicz and Cabrini (1955) 



Versene is here considered the better of the two solutions. The so- 

 lution should be adjusted to a pH 7.0 with NaOH and HCl. Hema- 

 toxylin and eosin stains as usual, but glycogen is lost, and alkaline 

 phosphatase has to be reactivated after chelating agents. 



^ Bone Decalcifier, Portable, Chicago Apparatus Co. Catalog #28-712. 



