STAIN TECHNIQUE 21 



weaker counterstain should be tried or the first stain should be 

 changed to a more intense dye of the same color. Blue dyes that 

 do not act so rapidly as Aniline Blue are Niagara Sky Blue and 

 alum Haematoxylin. If Rhodamine 6G does not give enough 

 color, the more intense Saf ranine or Acridine Red should be tried. 

 Combinations of three stains. The technique of applying 

 three of four dyes is nearly the same as that employed with com- 

 binations of two stains. Only two solutions are needed when two 

 or more dyes from the same group, either acid or basic, are mixed 

 and used as one stain. The proportionate amount of each dye in 

 a mixed stain is calculated either by the spot test or the time test. 



1. Spot test using basic dyes. On filter paper note the contrast 

 between the red from a drop of Safranine and the violet from a 

 drop of Methyl Violet. For the first test try equal quantities of 

 Safranine and Methyl Violet ; compare a drop of this mixed stain 

 with the spots of the separate dyes on the filter paper. If the spot 

 given by a drop of mixed dyes shows a color intermediate between 

 the colors of the two original stains, the mixture contains the 

 proper amount of each. Should one color predominate in the test 

 on paper, the proportions are changed until the result is a blend of 

 the two original colors. As a rule, three parts of Safranine will 

 neutralize one part of Methyl Violet. 



Staining procedure for mature plant tissues. Stain the mate- 

 rial for 24 hours in Orange II, rinse in distilled water, and follow 

 with the mixture of three parts Safranine and one part Methyl 

 Violet. Again rinse the section in distilled water, transfer it to 

 95 per cent alcohol, complete the dehydration in absolute alcohol, 

 and clear in xylene. During the counterstaining, a longer interval 

 in the mixed stain yields more violet while less time increases the 

 red. 



2. Time test using acid dyes. Note the different time intervals 

 needed for staining a section of an earthworm in Sun Yellow, 

 Orange II, and other acid dyes. 



Sun Yellow, C. I. No. 620, 10 minutes. 

 Orange II, C. I. No. 151, 5 minutes. 

 Crocein Scarlet MOO, C. I. No. 252, 5 minutes. 

 Biebrich Scarlet, C. I. No. 280, 2 minutes. 



