1 86 BOTANICAL MICROTECHNIQUE. 



e. Safranin and Gentian Violet. 



322. According to the methods recommended by Her- 

 mann (I, 60), the sections are first placed for 24-48 hours in 

 a solution of safranin which contains I gram of the dye to- 

 10 ccm. of alcohol and 90 ccm. of aniline-water. They are 

 then treated successively with water, acid alcohol, and alco- 

 hol, but so that they remain still too deeply stained for 

 direct observation. From the alcohol, the sections are then 

 transferred for 3 to 5 minutes to a solution of gentian violet 

 containing I gram of that dye to 10 ccm. of alcohol and go 

 ccm. of aniline-water. The sections are then quickly rinsed 

 off in alcohol and placed in a solution of iodine and potas- 

 sium iodide prepared as for Gram's method ( 321), in which 

 they remain for one to three hours until they are quite 

 black. Then they are differentiated in alcohol, cleared in 

 xylol, and mounted in Canada balsam. 



In successful preparations the nucleoli of resting nuclei 

 are vivid red, the nuclear framework blue-violet. Of the 

 karyokinetic figures the spirem and dispirem are blue, while 

 the intermediate stages are red. The achromatic figure is- 

 lightly stained yellow-brown by the iodine. 



C. Safranin-Gentian-violet-Orange. 



323. According to Flemming (III, 685, note), objects 

 fixed with chrom-osmic-acetic acid (cf. 309) or with Her- 

 mann's platinum-chloride-osmic-acetic acid (cf. 313) may 

 best be placed for 2 or 3 days in a concentrated alcoholic so- 

 lution of safranin which is diluted with about an equal volume 

 of water and a little aniline-water. They are then washed in 

 water and then extracted with alcohol containing at most ,\% 

 of hydrochloric acid, or with pure alcohol. After a brief 

 washing in water, the objects are placed for I to 3 hours in a 

 concentrated aqueous solution of gentian violet, and then, 

 after another short washing in water, in a concentrated aque- 

 ous solution of orange,* in which its dark color is gradually 

 dissolved out. After a few minutes, while blue clouds of 



* This may be obtained from Dr. G. Griibler under the name " Orange G.'* 



