76 MICROSCOPIC TECHNIQUES 



Another method was published by Hackmann (1942), who em- 

 ployed the freezing-drying technique for the fixation of the tissue, 

 prior to the preparation of paraffin sections. Colored sulfonamides 

 were observed directly in the sections, and colorless ones were 

 visualized by forming a red azo dye in the following manner: The 

 sections were exposed to nitrous acid vapor for 30 sec. by placing 

 the slide over a measuring cylinder 20 cm. high containing several 

 milliliters of 0.1 A^" hydrochloric acid and a few milligrams of sodium 

 nitrite. The diazotized sulfonamide was coupled with a-naphthyl- 

 amine by immersing the slide in a 5% solution of the amine in xylol. 



The detection of sulfonamides by fluorescence microscopy is 

 discussed on page 108. 



Method of MacKee et ah for Sulfonamides 



SPECIAL REAGENTS 



Sulfa Reagent. Dissolve 1 g. pure p-dimethylaminobenzaldehyde 

 in a soln. of 95 ml. absolute alcohol and 5 ml. cone, hydrochloric 

 acid. Store in a glass-stoppered amber bottle, and do not use after 

 2-3 weeks or when the soln. becomes yellow. 



5% Concentrated Hydrochloric Acid in Absolute Alcohol. 



PROCEDURE 



1. Fix the tissue for 2-24 hr. with formaldehyde gas by covering 

 the bottom of a beaker with paraformaldehyde and the top with a 

 piece of gauze, placing the tissue on the gauze, setting the whole in 

 a glass jar whose floor has also been covered with paraformaldehyde, 

 and closing the jar tightly with a glass lid. 



2. Cut frozen sections of the fixed tissue 10-20 /^ thick and place 

 directly on glass slides. 



3. Cover each section with a drop or two of the sulfa reagent, 

 and after 3-5 min. add a drop or two of the alcoholic hydrochloric 

 acid soln. 



4. Dry quickly without heat by absorbing excess fluid on filter 

 paper and holding in a current of air. 



5. Cover at once with a drop of damar resin in xylol (10 g. 

 resin dissolved in 10 g. xylol) and fit cover slip, taking care to re- 

 move air bubbles. Seal edges with melted paraffin. 



