26 - Fixation (chap. 1) 



Calcium ions are insoluble at pH 4.5, so buffer solutions may be used. 

 Slower than other methods, but no perceptible tissue damage. 



e. Kristensen's (1948) fluid is highly recommended. 



8N formic acid (see page 408) 50.0 ml. 



IN sodium formate 50.0 ml. 



(pH 2.2) 24 hours. Wash in running water: 24 hours. 



Pros and Cons in Use of Acid Reagents 



Schajowicz and Cabrini (1953) observe that strong acids, such as nitric 

 acid, do alter histochemical behavior of bone and cartilage, and must 

 be used with care. Formic acid and citrate do not have this disadvantage 

 if used for only a few days. 



Case (1933) added 1% phloroglucinol to formic acid for improved 

 cell detail and preservation, and for staining qualities. Culling (1937) 

 disagrees and maintains that staining is poor after phloroglucinol. 



Morris and Benton (1936) found 1-2M hydrochloric acid the most 

 rapid decalcifier (approximately 3 hours). He foimd that this produced 

 adequate staining reactions if slides were mordanted in 5% aqueous 

 ammonium chloride for 30 minutes before staining. 



I on-exchange -re sin Method of Decalcification 



Dotti et al. (1931) theorized that replacing sodium citrate (formic acid- 

 citrate mixture) by a cation exchange resin might result in speedier de- 

 calcification. The liberated calcium coidd be removed more rapidly 

 from the solution — requiring about half the time necessary for formic 

 acid-citrate decalcification. They recommend 40% of resin in formic 

 acid. If speed is not essential, they recommend 10% of resin. The resin 

 is Win 3000, ammonium salt of sulfonated resin (Winthrop Stearns Inc., 

 N.Y.). 



Electrolysis Method of Decalcification 



The principle of electrolysis is based on the theory that there is an at- 

 traction of Ca ion to a negative electrode. The bone is suspended by a 

 platinum wire, becoming the anode, and a second platinum wire forms 

 the cathode. Then by electrolysis the calcium ions are freed from the 



