Of the workers applying the von Kóssa test to calcium oxalate crystals in animal tissue, about half reported blackening in silver nitrate on exposure to light. We found that "pure" calcium oxalate crystals, produced in rats by the administration of ammonium oxalate or ethylene glycol, gave a negative von Kóssa reaction. These crystals, however, were blackened when treated with a solution of silver nitrate in concentrated hydrogen peroxide. Other von Kóssa negative salts, such as CaF2 or CaSO4·2H2O, do not respond to the H2O2 and AgNO3 test.

Tannic acid in aqueous solution is bound to mucins in formalin-fixed and formalin-free fixed tissues and its presence can be detected with ferric chloride as a dark gray, blue-black to black complex. This colored compound is readily extracted by acids and some chelating and bleaching agents and is changed to a reddish brown by alkalis. Hydrolysis in 1.2 N hydrochloric acid at 60#{176}Cfor 4 hr or hot trichloroacetic acid prevents the tannin-iron reaction. Acetyl chloride or bromide is able to inhibit the binding of tannic acid to the mucosubstances and saponification restores the characteristic reaction. Several mechanisms for the attachment of tannic acid to the mucins appear possible. Mayer (15) in his classical study of 1896 on mucus observed that mucus could be stained by a tannic acid-fernic chloride sequence. No furthen studies were made on this reaction until a series by Salazaar (21, 22), who reported similar findings. Gray (4) has alluded to numerous references to tannic acid as a mordant for tissue Alcian Blue at various pH or azure A-eosin B stains were used to check the efficiency of the blockages. Unless otherwise stated, all liquid solutions are made of v/v.

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