538 POWDERED DRUGS 



The three-color method in which safranin, gentian violet, and orange G are suc- 

 cessively employed gives a most beautiful differentiation of the structures of the 

 dividing nucleus, and in tissues with resting nuclei there is a sharp differentiation 

 of the nucleus, nucleolus, and the cytoplasm, and of the cutinized, lignified, and 

 cellulose membranes. For embryonic tissues this' method of staining is unexcelled. 

 It is not, however, so simple as the methods given above, since the time relations 

 of the three stains employed must be accurately determined and adhered to for a 

 given material. This stain works best with material that has been fixed in Flem- 

 ming's fixative, q.v., or in a fixative containing chromic acid. Sections from material 

 which has been fixed in alcohol should be immersed for twenty-four hours in a I 

 per cent, solution of chromic acid, and then washed in water for a few hours, as 

 above suggested. The sections mounted on the slide are first immersed in a 

 safranin solution which has been prepared by adding to a concentrated alcoholic 

 solution of safranin an equal amount of water. Here the sections remain for 

 twelve hours, or over night. They are then rinsed in pure alcohol, and thereafter 

 immersed in alcohol to which is added about o.l per cent, of hydrochloric acid. 

 Just as the clouds of safranin cease to come from the sections, they are rinsed in 

 distilled water and placed in a saturated solution of gentian violet, remaining ten 

 minutes. They are quickly rinsed in water, and while the slide is held horizontally, 

 the sections are flooded with orange G, prepared by diluting a saturated solution 

 of the stain with about five times its bulk of water. After about four seconds the 

 orange G is drained off and the sections are quickly rinsed in water; then, while the 

 slide is held slanting downward, absolute alcohol from a drop-tube is flooded over 

 the slide, beginning at the upper edge of the rows of sections. This washes away 

 some of the surplus gentian violet and dehydrates the sections at the same time. 

 The slide is now again held horizontally and the sections are covered with clove 

 oil from a drop-tube. The process of decolorizing should be watched under the 

 microscope. Clouds of gentian violet come off in the clove oil, and when the 

 stain has passed from an opaque to a transparent color, the clove oil should be 

 drained off and the slide immersed in xylol, where the sections may be further 

 cleared without extracting more of the stain. When the sections appear clear and 

 without any milkiness in the xylol, they may be mounted in Canada balsam under 

 a cover-glass; or they may be first mounted in cedar oil under a cover-glass and 

 examined under high powers to see if the differentiation has been satisfactorily 

 attained in the finer structures. If the gentian violet should be found still too 

 dense, the oil may be washed off in xylol, the xylol rinsed off in alcohol, and the 

 sections again treated with the solution of orange G, alcohol, and clove oil, and there- 

 after brought into xylol and cedar oil for preliminary examination as before. If, on 

 the other hand, the gentian violet is found to be too faint, the sections should be 

 brought from the oil through xylol and alcohol to the gentian-violet bath for, say, 

 another ten minutes, and then the process onward to the examination in cedar oil 

 should be as at first described. The effect of the orange G is not only to impart its 

 own color to certain of the structures, but to cause the gentian violet, which stains 

 very intensely, to loosen its hold, to a certain extent, from the structures for which 

 it has a special affinity, so that the absolute alcohol and the clove oil may be able 

 to wash out the surplus stain. The critical part of the process is to allow the orange 

 to work long enough to cause the gentian violet to loosen its hold sufficiently, but 

 not too much. The time relations as above given produce the right result in 

 many instances, but it will be found that they must be altered for certain sub- 

 jects. In some instances the gentian- violet bath must be prolonged, and in others 

 the orange G solution must be stronger or must be allowed to act for a longer 

 time. 



It must be remembered that sections can not be transferred from water or an 



