STAINS AND STAINING 59 



deeply stained, and sufficient differentiation is not secured within 5 or 

 10 minutes, a drop of hydrochloric acid added to 50 c.c. of the alcohol 

 will hasten the extraction of the stain. If staining vascular tissue, 

 draw the stain from the cellulose walls, but stop before the lignified 

 walls begin to fade. If a contrast stain is to be added, like light green, 

 which weakens the safranin; or anihn blue or Delafield's haematoxylin, 

 which need to be followed by an acid; the safranin should be strong 

 enough to allow the necessary reduction. If staining mitotic figures, 

 draw the stain from the spindle, but stop before the chromosomes be- 

 gin to weaken. When the desired differentiation has been reached, 

 wash out the acid in 50 per cent alcohol, if acid has been used. About 

 5 minutes should be sufficient for the washing. 



If safranin is to be used alone, pass through 50, 70, 85, 95, and 100 

 per cent alcohol, through the xylol-alcohol, then through xylol to bal- 

 sam. If clove oil is used, omit the xylol-alcohol, but follow the clove 

 oil with xylol to hasten the hardening of the preparation. 



If a second stain is to be added, transfer from the 50 per cent alcohol 

 to any alcoholic stain. If the second stain is an aqueous stain, rinse 

 the slide or sections for a minute in water before applying the stain. 



Safranin is the most generally useful of all the red stains, and, for- 

 tunately, it is quite permanent. Lignified, suberized, cutinized, and 

 chitinized structures stain red, as do also the chromosomes, nucleoli, 

 and centrosomes. 



Directions for using safranin in combination with other stains will 

 be given in connection with various objects. 



Acid fuchsin. — Use a 1 per cent solution in water or in 70 per cent 

 alcohol. The solution in alcohol is preferable if sections are to be 

 mounted in balsam. This stain often acts with great rapidity, 2 or 3 

 minutes being sufficient. The method for using acid fuchsin with 

 woody tissues is given in the chapter on "Freehand Sections" (chap, 

 vi). In staining embryo sacs, pollen grains, and such structures, longer 

 periods are better. Stain 1 or 2 hours, and then differentiate in a sat- 

 urated solution of picric acid in 70 per cent alcohol. This may require 

 30 seconds, or even several minutes. Rinse in 70 per cent alcohol until 

 a bright red replaces the yellowish color due to the acid, and then pro- 

 ceed as usual. 



Basic fuchsin. — This stain has become valuable to botanists 

 through the researches of Gourley, who used it to stain the vascular 



