STAINS AND STAINING 
63 
Bismarck Brown.—Use a 2 per cent solution in 70 per cent al¬ 
cohol. 
This is a good stain for cellulose walls, although it is not so precise 
as haematoxylin. Embryo sacs stained in one of the carmines are 
improved by 1 or 2 minutes’ staining in Bismarck brown. Material 
fixed in alcohol stains better than that which has been fixed in reagents 
containing chromic acid. A faint background of Bismarck brown is 
quite effective in staining sections containing bacteria. 
Nigrosin.—Make a 1 or 2 per cent solution in water. A few 
drops of this solution to a watch glass full of water stains filamentous 
algae or fungi in 1 to 3 hours. It keeps well in glycerin or Venetian 
turpentine. It also keeps well in balsam, but it is of little value in 
staining microtome sections. 
COMBINATION STAINS 
Sometimes preparations are stained with a single stain, selected 
to emphasize some particular feature, but in the great majority of 
cases two or more stains are used. In staining a vascular bundle, 
one stain may be selected which stains the xylem, but not the phloem, 
while another of a different color stains the phloem, but not the 
xylem, thus affording a sharp contrast. In staining mitotic figures, 
one stain may stain the chromosomes, while another of a different 
color may be used to stain the spindle. 
Success in double staining can be obtained only by noting the 
effect of each stain upon the various plant structures. 
Flemming’s Safranin, Gentian-Violet, Orange.—Safranin has 
long been a famous stain for mitosis. 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. Three methods, which may be desig¬ 
nated as A, B, and C, will be described. 
A. According to Flemming, 1 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 to 3 hours in a 
2 per cent aqueous solution of gentian-violet; wash quickly in water, 
and then stain 1 to 3 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. 
1 Flemming’s original method is included only as interesting history. 
