Decalcification 25 



edge. If deposits are sparse, overnight soaking in water will soften them 

 sufficiently for sectioning. This is undertaken when the tissue is blocked 

 and ready for sectioning (page 55). Heavy deposits may be removed by 

 any of several methods. Opinions are varied as to preferred method, 

 ijut do not leave tissues in any of the fluids longer than necessary. If any 

 doubt arises concerning completion of decalcification, check for calcium 

 by the follo^ving method. 



To 5 ml. of the solution containing" the tissue, add I ml. of 5% so- 

 diimi or ammonium oxalate. AIIoav to stand 5 minutes. If a precipitate 

 forms, decalcification is not complete. A clear solution indicates it is 

 complete. Sticking needles in the tissue to check hardness is sloppy 

 technic which can damage the cells. 



Acid Reagents 



After using an acid for decalcification, transfer directly to 70% alcohol 

 to prevent swelling in the tissue and impaired staining reactions: 3-4 

 hours or overnight. 



a. formic acid 5.0-25.0 ml. 



formalin 5.0 ml. 



distilled water to make 100.0 ml. 



With 5 ml. formic acid content, 2-5 days. If increased to 25 ml. less 

 time is required, but with some loss of cellular detail. 



b. formic acid, 50% aqueous (50 ml./50 ml. water) 50.0 ml. 

 sodium citrate, 15% aqueous (15 gm./lOO ml. 



water) 50.0 ml. 



c. formalin 10.0 ml. 



nitric acid, concentrated 5.0 ml. 



distilled water 85.0 ml. 



If acidity is a problem for staining, treat with 2% aqtieotis lithitnn 

 carbonate solution, or 5% sodium sulfate: 6-12 hours, then into 70% 

 alcohol. 



d. Citrate-citric acid buffer, pH 4.5 (controlled hy- 

 drogen ion concentration) (Culling, 1957) 

 citric acid monohydrate, 7% aqueous (7 gm./ 



100 ml. water) 5.0 ml. 



ammonium citrate, anhydrous, 1 Ab% aqueous 



(7.45 gm./lOO ml. water) 95.0 ml. 



zinc sulfate, 1% aqueous (1 gm./lOO ml. water) 0.2 ml. 



chloroform few drops 



