66 Microscopic Histochemistry 



oxidative pretreatment (example: ceroid). Second, elastic 

 fibers in certain locations (mainly in the inner elastic coat of 

 small-caliber arteries) contain an unidentified nonlipid sub- 

 stance which behaves as an aldehyde whether or not an oxi- 

 dative pretreatment is employed. 



Differentiation between Schiff -positive substances —Al- 

 though normally the localization and morphology of glycogen 

 is quite different from that of mucin, the problem of distin- 

 guishing between the two substances may come up once in a 

 while. One of the oldest methods of differentiation is the 

 saliva test. It is based on the fact that saliva contains a dia- 

 static enzyme which will dissolve glycogen and starch but 

 not mucin, amyloid and other related substances which may 

 stain in a shade indistinguishable from that of glycogen. It 

 may be appied to all staining methods, whether histochem- 

 ical or not. Therefore, ff a substance stains without pretreat- 

 ment but fails to do so after an exposure to saliva of about 

 30-60 minutes, it must be glycogen or starch. On the other 

 hand, ff it persists in staining after the saliva test, it cannot 

 be glycogen. At present, more appetizing and sanitary tests 

 have replaced the time-honored saliva test. A number of 

 highly active diastase (amylase) preparations are available 

 which are specific and efficient. Being protein in nature, they 

 will not diffuse through collodion; therefore, if they are to be 

 used, sections should not be coated with collodion, and col- 

 lodion should be removed from celloidin-embedded material. 

 LiUie and Greco^^ recommend the use of a 1 per cent dilution 

 of extract of malt, U.S.P., in a phosphate buffer of pH 6.8-7.4. 

 A 1 per cent solution of malt or animal diastase (Nutritional 

 Biochemicals) is even more effective. These enzymes will re- 

 move glycogen and starch from sections not over 8 fjL thick 

 within 15 minutes at room temperature. Prolonged exposure 



(24-48 hours) may remove all carbohydrate material. 



A number of other specific enzymes for the hydrolysis of 

 various mucopolysaccharides have become available recently 



42. LilHe, R. D., and Greco, J.: Stain TechnoL, 22:67, 1947. 



