46 MICROSCOPIC TECHNIQUES 



wash with water and counterstain if desired (it is well to use a red 

 eounterstain such as f uchsin) . 



6. Rapidly dehydrate, clear in xylol, and mount in Canada 

 balsam. 



Result. Acid polysaccharide is indicated by the blue color. 



MUCOPROTEINS* 



Toluidene blue will stain quite a variety of acid substances, but 

 the metachromatic staining by this dye of mucoid compounds 

 containing polysaccharide esters of sulfuric acid is specific for 

 these compovmds, provided that the method of Lison (1935) is 

 strictly adhered to (Sylven, 1941, 1945). Sylven (1941) has made a 

 thorough study of the staining and he emphasized that "false" 

 metachromatic staining can be obviated by the prompt removal, of 

 water by alcohol after the staining, the alcohol assuring a ''true" 

 reaction which is the red stain characteristic of, and specific for, 

 the polysaccharide sulfates in tissue. Holmgren and Wilander ( 1937) 

 found that basic lead acetate solution was a superior fixative for 

 tissues to be subjected to the metachromatic toluidene blue stain, 

 but Sylven now employs a mixture of this fixative with formalin to 

 reduce the time required for the fixation. The staining time can be 

 reduced by aging the dye solution, and the greater the alcohol 

 concentration in the dye solution the paler the resulting stain will 

 be. In order to bring out mast cell granules properly, the dye is 

 made up in alcohol of a concentration of 30% or higher (Sylven). 



According to the claim of Hempelmann ( 1940) , chondroitin and 

 mucoitin sulfuric acid proteins can be differentiated from one 

 another in histological preparations by means of the toluidene blue 

 stain. In a dilution of 1 : 1,280,000 an aqueous solution of toluidene 

 blue is supposed to stain the chondroitin material in paraffin sections 

 a violet-red color, while the mucoitin protein complex remains un- 

 stained. Differentiation is also claimed when the dye is used in a 

 1 : 410,000 dilution in a solution of 10 vol. alcohol and 45 vol. water. 

 The alcohol concentration is stated to be critical, presumably both 

 mucoproteins will be stained if the proportion of alcohol is less, 

 and neither if it is greater. No confirmation of these claims has been 

 made; in fact, to the writer's knowledge several attempts to do so 

 have failed. 



* See Bibliography Appendix, Refs. 8 and 10. 



