394 Journal of Applied Microscopy. 



This is a good combination for general work, and if properly used is excellent 

 for mitotic phenomena and the most delicate cytological work. Delafield's 

 haematoxylin may be used wdth er}-throsin. Stain first with the haematoxylin, 

 and after the purple color has replaced the red due to the acid, stain lightly with 

 erythrosin. Eosin may be used instead of erj'throsin, but is less transparent. 

 Eosin will be mentioned later in connection with special methods for algae and 

 fungi. 



Flenwiing's Safranin-Gentiaii Violet- Orange. — Safranin has long been a 

 famous stain for karj'okinesis. This triple combination was published 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 (Germany) school. 



According to Flemming, stain two to three days in safranin (dissolve 0.5 gr. 

 safranin in 50 cc. absolute alcohol, and after four days add 100 cc. distilled 

 water) ; rinse quickly in water ; stain one to three hours in a 2 per cent, aqueous 

 solution of gentian violet ; wash quickly in water, and then stain one to three 

 minutes in a 1 per cent aqueous solution of orange G. Transfer from the stain 

 to absolute alcohol, clear in clove oil, and mount in balsam. 



The following method seems to be better for mitotic phenomena in plants : 

 Transfer to safranin from 35 per cent, alcohol and stain sixteen to twenty-four 

 hours. If the stain acts for only a few hours, it washes out too rapidly to be 

 controlled with any precision. (The safranin may be made up according to the 

 formula given in the preceding paragraph or according to the general formula.) 

 Rinse in water for a minute and then in 50 per cent, alcohol until only nucleoli 

 and the chromosomes of dividing nuclei retain the red color. If the alcohol 

 does not wash out the stain sufficiently, add a few drops of hydrochloric acid 

 (not more than 0:1 cc. HCl. to 100 cc. alcohol.) If acid has been used, wash for a 

 moment in pure 50 per cent, alcohol and then stain in gentian violet (aqueous 

 solution, or made up according to the general formula). Rinse for a few seconds 

 in water and then stain about thirty seconds in a 1 per cent, aqueous solution of 

 orange G. Transfer from the stain directly to absolute alcohol and hasten the 

 dehydrating by gently rinsing the slide in the fluid. As a rule, not more than 

 ten seconds can be allowed for dehydrating because the gentian violet washes 

 out so rapidly. Treat with clove oil for five to ten seconds. The clove oil not 

 only clears, but it rapidly extracts the gentian violet, producing an elegant dif- 

 ferentiation. Replace the clove oil by cedar oil. Cedar oil does not extract the 

 stain. The preparation should now be examined under a microscope, and if the 

 stain is still too deep the clove oil may be applied a second time. Mount in 

 balsam. If mounted directly from clove oil, the gentian violet is almost sure to 

 fade. Chromosomes should take a clear red and the spindle fibers a bright 

 violet. This combination works best after Flemming's solution, but does fairly 

 well after other members of the chromic acid series. Achromatic structures do 

 not seem to stain well after corrosive sublimate or picric acid. 



Fuchshi. — Use a 1 or 2 per cent, solution in water or in TO per cent, alcohol. 

 Transfer to the alcoholic solution from 70 per cent, alcohol ; stain one to two 

 hours ; differentiate the stain in 1 per cent, solution of picric acid in 70 per 



