Staining Paraffin Sections 63 



suggested above. Use a smear microscope, preferably one that has 

 only lOx (16 mm.) and 20x (8mm.) objectives and no condenser. 

 The magnification is adequate and the objectives have such long 

 working distance that they are not likely to be dipped into reagents 

 on the slide. The tissues must not become dry during this quick 

 examination. Nuclei should be blue-black. Cellulose cell walls should 

 be black, whereas lignified cell walls should be nearly colorless. 

 Plastids may be pale blue to blue-black, and cytoplasm blue-gray. 

 If the foregoing structures do not have a deep enough color, transfer 

 the slide from the water to hemalum and give it another interval 

 in the stain, ustially as long as the first immersion. Rinse and wash 

 in tap water, examine again and if satisfactory, proceed with de- 

 hydration as in Chart I. 



If a slide is left in hemalum longer than the optimiun period, 

 the contents of the cell may become black, and the details of the 

 wall and protoplast may be obscured. The slide can be destained 

 by brief immersion in dilute acid. The preferred destaining agents 

 are 1 to 5% acetic acid, 0.5% hydrochloric acid, or a saturated 

 aqueous solution of picric acid. Try one minute in acid, wash, alka- 

 lize and re-examine with a microscope. When the stain is satisfactory, 

 proceed with dehydration according to Chart I. 



Staining Chart I now calls for progressive dehydration of the 

 tissues and the surface of the slide, followed by "clearing." Consult 

 the reference manuals for the various clearing agents in common 

 use. An inexpensive agent is carbol-xylene, the formula of which is 

 given on page 61. Both ingredients must be of high purity. Phenol 

 has a great affinity for water and removes the last traces of water 

 from the preparation. Xylene has nearly the same index of refraction 

 as glass, thus rendering the tissues transparent. High-grade phenol 

 and xylene should not affect the stain even after several days of im- 

 mersion. Equal volumes of xylene and cedar oil may replace the 

 carbol-xylene. 



The final operation consists of cementing a cover glass on the 

 preparation. Have ready a supply of newly cleaned and dried cover 

 glasses. Use a cover glass of generous, but not wasteful, size, with 

 shape and dimensions in keeping with the material to be covered 

 (Fig. 6.6) . Discoloration of resin and fading of stain with age proceed 

 from the edges of the cover inward. Have a margin of at least 5 mm. 

 between the sections and the edge of the cover glass. For mounting 

 one section on a slide, or a few sections in a single row, use a 1/2-. %-, 



