32 Microscopic Histochemistry 



The methods used for the demonstration of insoluble Ca 

 fall into two classes: ( 1 ) those which are specific for Ca itself 

 (the gypsum, oxalate, and lake-dye reactions) and (2) those 

 which demonstrate the anions of calcareous deposits (heavy- 

 metal methods ) . 



The only absolutely specific reaction is Schujeninoff's.^ This 

 is based on the formation of insoluble crystals of gypsum 

 (CaS04) by the action of H2SO4 in a dilute alcoholic me- 

 dium. 



Method 



Cover the section with 50 per cent alcohol; place a drop of 

 5-10 per cent H2SO4 on the under surface of a cover slip and 

 put it on the section. Watch slide under the microscope; 

 almost immediately typical rhombic crystals, very prone 

 to aggregate in swallowtail or rosette-like formations, will 

 appear. 



The sections cannot be counterstained, and the crystals 

 are far too coarse to permit exact localization. 



Several hydroxyanthraquinone dyes ( alizarinsulf onic acid, 

 purpurin,^ anthrapurpurin ) ^^ give intensely colored ( reddish 

 or purple ) insoluble lakes with calcium and can be used for 

 the demonstration of deposits of a medium particle size. 

 Relatively large, dense structures such as bone spicules are 

 poorly penetrated; in the case of fine, dustlike deposits the 

 shades are not intense enough to be seen distinctly. This 

 applies also to gallamin blue,^^ a lake dye of another chemi- 

 cal group. Anthraquinone lake dyes can be used in the form 

 of a 0.1-0.5 per cent solution in 50 per cent alcohol; gallamin 

 blue in 0.1-0.2 per cent aqueous solution in an M/5 borate 

 buffer of pH 7.6. Staining time is several hours. For counter- 

 staining, one of the plasma stains ( anilin blue or light green 



8. SchujeninofF, S.: Ztschr. f. Heilk. 18:79, 1897. 



9. Grandis, V., and Mainini, C: Arch. ital. de biol., 34:73, 1900. 



10. Salomon, H.: Jahrb. f. wissensch. Bot., 54:308, 1914. 



11. Stock, A.: J. Roy. Micr. Soc, 69:20, 1949. 



