SULFONAMIDES AND UREASE 77 



6. Run controls by repeating the above steps but omitting the 

 treatment with the sulfa reagent, or repeat the complete procedure 

 on a portion of the same kind of tissue known to be free of sulfona- 

 mides. 



Result. Sulfonamides are indicated by the presence of a pre- 

 cipitate that ranges in color from lemon-yellow to orange. However, 

 the color fades rapidly, particularly in the presence of air, making 

 it necessary to examine the sections as early as possible. Colored 

 photomicrographs should be taken not later than 3-4 hr. after the 

 reaction has occurred. 



D. ENZYMES 



UREASE 



Sen ( 1930) elaborated a method for the localization of urease in 

 tissue sections which he employed for a study on the jack bean. The 

 carbonic acid formed on decomposition of urea is precipitated as 

 calcium carbonate, which may be visualized by conversion to silver 

 carbonate and reduction of the latter to a black deposit of metallic 

 silver; or the carbonic acid may be converted to cobalt carbonate and 

 the latter changed to a brown or black precipitate of cobalt sulfide. 

 The latter method is to be preferred. This principle was later employed 

 by Gomori for the localization of the phosphoric acid hberated by 

 phosphatases, pages 78 and 80. However, Sen digested the tissue 

 in the substrate medium before paraffin infiltration and sectioning, 

 and only treated the deparaffinized sections with sulfide to convert 

 the colorless cobalt salt to the black sulfide. This procedure has many 

 disadvantages; the schedule of Gomori, in which the sections are 

 prepared prior to digestion, should be used instead, if the enzyme 

 can stand the dehydration, paraffin embedding, and deparaffinization. 



For jack bean tissue. Sen employed a preliminary treatment for 

 1 hr. with 1% cobalt nitrate in 80% alcohol followed by a 48-60 

 hr. digestion with a substrate medium consisting of 0.5% urea and 

 0.5% cobalt nitrate in 80% alcohol. The cobalt carbonate was con- 

 verted to sulfide by the action of either dilute sodium sulfide or a 

 saturated solution of hydrogen sulfide. For animal tissues, Sen used 

 cobalt-urea solns. in graded alcohols from 60 to 80%. 



