BISMUTH 31 



ization of the bismuth as a mure reddish, rather than a yellowish- 

 orange deposit. More recently Wachstein and Zak ( 1946) employed 

 a modified Castel method in which the black sulfide, the form in 

 which bismuth appears in tissues, is converted to the white sulfate 

 by treatment with hydrogen peroxide and the sulfate is then trans- 

 formed to the brucine iodide salt. 



In the procedure of Wachstein and Zak (1946), iron, which may 

 be present as the black sulfide, is oxidized to golden brown hemosid- 

 erin, which does not react wath Castel reagent, but which does give 

 the typical iron reactions. Wachstein and Zak pointed out that lead 

 sulfide would deposit in tissues in the same fashion as bismuth but 

 differentiation may be made by the fact that, after the lead sulfide 

 is converted to the sulfate by the peroxide, it will yield the slightly 

 yellowish lead iodide on treatment with Castel reagent in contrast 

 to the brilliant orange-red bismuth product. Similarly silver and 

 mercury will give yellow, and copper brown, iodides that can be 

 differentiated from the color of the bismuth precipitate. Melanin in 

 tissue is not bleached by the short treatment with peroxide and does 

 not react with Castel reagent. Wachstein and Zak emphasized that 

 melanin never impregnates capillary walls while bismuth does. 



Wachstein and Zak Method for Bismuth 



SPECIAL REAGENTS 



Modified Castel Reagent. Dissolve 0.25 g. brucine sulfate in 100 

 ml. distilled water containing 2-3 drops cone, sulfuric acid. After 

 the brucine salt has dissolved add 2 g. potassium iodide. Store in 

 a brown bottle and filter before use. 



Diluted Castel' s Reagent. Add 3 vol. distilled water to 1 vol. 

 reagent. 



30% Hydrogen Peroxide. (Superoxol, Merck). Store in a refrig- 

 erator. 



Levulose Solution. Dissolve 30 g. levulose in 20 ml. water by warm- 

 ing at 37'^ for 24 hr. and add a drop of the diluted Castel reagent. 



Counterstain Solution. Add 1 ml. 1% aqueous light green SF 

 {Hartman-Leddon) to 100 ml. undiluted Castel reagent. Filter 

 before use. 



PROCEDURE 



1. Prepare either frozen or paraffin sections of formalin-fixed 

 tissue. 



