Stains and Staining 59 



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, 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. 



B. The following formulae and method seem to be better for 

 mitotic phenomena in plants : Make a 1 per cent solution of alcoholic 

 safranin in absolute or 95 per cent alcohol, and after the safranin is 

 completely dissolved, add an equal volume of a 1 per cent solution of 



