66 METHODS IN PLANT HISTOLOGY 



staining mitotic figures, one stain may stain the chromosomes, while 

 another of a different color may be used to stain the spindle. 



One stain may affect another, the second stain often weakening the 

 first. There is not likely to be much success without patience and con- 

 stant observation. 



Flemming's safranin, gentian violet, orange. — Safranin has long 

 been a famous stain for mitosis. This triple combination was pubhshed 

 in 1891, but its value in plant cytology was not thoroughly appreciated 

 until five or six years later, when its application was developed to a 

 high degree of perfection by various investigators of the Bonn (Ger- 

 many) school. Three methods, which may be designated as A, B, and 

 C, will be described. 



A. According to Flemming, stain 2 or 3 days in safranin (dissolve 

 0.5 g. safranin in 50 c.c. absolute alcohol, and after 4 days add 10 c.c. 

 distilled water) ; rinse quickly in water; stain 1-3 hours in a 2 per cent 

 aqueous solution of gentian violet; wash quickly in water, and then 

 stain 1-3 minutes in a 1 per cent aqueous solution of orange G. Trans- 

 fer from the stain to absolute alcohol, clear in clove oil, and mount in 

 balsam. We are indebted to Flemming for the chromo-acetic-osmic 

 fixing agent. No cytologist has made a greater or more permanent con- 

 tribution to cytological technique, but his method of using the triple 

 stain is mentioned only as a matter of history : no one uses it today. 



B. The following formulas and method seem to be better for mitotic 

 phenomena in plants : Make a 1 per cent solution of safranin in 50 per 

 cent alcohol, a 1 per cent aqueous solution of gentian violet, and a 

 1 per cent aqueous solution of orange G. 



Transfer paraffin sections to the stain from 50 per cent alcohol after 

 the xylol or turpentine used in dissolving away the paraffin has been 

 rinsed off. Stain from 3 to 24 hours. If the period be too short, the 

 washing out is so rapid that it is difficult to stop the differentiation 

 at the proper point, and, besides, the red is likely to be less brilliant. 

 Rinse in 50 per cent alcohol until the stain is washed out from the 

 spindle and cytoplasm, but stop the washing before the chromosomes 

 begin to lose their bright red color. If the washing out takes place 

 too slowly, treat with shghtly acidulated alcohol (one drop of HCl 

 to 50 c.c. of 50 per cent alcohol) for a few seconds. The acid must be 

 removed by washing for 15-30 seconds in alcohol which has not been 

 acidulated. 



