84 MICROSCOPIC TECHNIQUES 



60° oven during the course of 1 hr. If air bubbles are present in the 

 leaves, apply suction to remove them. 



4. Embed in paraffin colored red by stirring a few grains of Sudan 

 IV in the molten material. In uncolored paraffin it is difficult to see 

 the tissue in the sections. 



5. Cut sections, mount on slides, remove paraffin, and protect 

 with collodion film just as in steps 9, 10, and 11 under A. 



IT, Preparation of frozen sections. A, Kernel sections (rootlet and 

 leaf sections of the sprout are too fragile to permit satisfactory 

 frozen-section technique) : 



1. Soak kernels 4-6 hr. in water. 



2. Mount in a drop of water on freezing head of microtone. 



3. Cut sections 15 fi thick, keeping knife cold with Dry Ice, and 

 transfer, with a needle cooled by Dry Ice, into 80% alcohol. (The 

 80% alcohol is used rather than water since, in the latter medium, 

 the starch endosperm disintegrates, and separates from the rest of 

 the section.) 



4. Float the section onto a glass slide immediately. If wrinkled, 

 straighten section in a drop of 70% alcohol. 



5. Dehydrate by covering section with five successive drops of 

 absolute alcohol, draining off after each drop is added. 



6. Cover section with a small drop of 0.5-1.0% collodion soln. 

 and harden film by dipping slide in 80% alcohol. Wash in distilled 

 water. 



Ill, Demonstration of enzyme activity: 



1. Remove preformed mineral deposits by placing sections in dis- 

 tilled water for 24 hr. at room temperature. Citrate buffer could 

 probably be used too (page 000) . 



2. Place sections in substrate medium at 37°, for the following 

 digestion periods: 



Kernel, paraffin sections — Embryo, 1 hr.; non-embryonic part 30 

 min. 



Kernel, frozen sections — Embryo, 15-30 min.; non-embryonic part, 

 5-10 min. 



Rootlets, paraffin sections — 3 hr. 



Epicotyl, paraffin sections — 24 hr. 



3. Wash sections with three changes of distilled water, dip into 

 2% acetic acid, and wash well with distilled water. 



