STAINING ON THE SLIDE 209 



Then after passing through tap water it may be placed in a solution 

 of safranin (Sec. 184) until the protoplasm and cell walls are slightly 

 stained, after which it is carried through absolute alcohol into xylol. 

 DelaHeld's hoematoxylin. This stain is excellent for tissues, since it 

 colors the cell walls sharply, as is not done by iron-alum haimatoxylin. 

 Follow the outline given in Sec. 183. 



Safranin, gentian violet, and orange G. It is not necessary to use 

 the orange G in this combination, known as Flemming's triple stain, but 

 the best results have been obtained with it. The technique of this 

 method of staining is difficult, but it gives perhaps the most effect- 

 ive staining for the study of protoplasmic structure, especially during 

 nuclear division. It is impossible to give more than a general programme 

 of the method, since the time limits necessary to obtain satisfactory 

 results vary with different material and must be tested experimentally. 



The slide is transferred from 95% alcohol to a well of safranin. 

 The alcoholic solution mixed with an equal part of water is good, as is 

 also anilin safranin (Sec. 184). After remaining in safranin from four 

 to twenty-four hours (over night is generally convenient), the slide is 

 placed in 50% alcohol and the stain extracted until it remains in the 

 nucleolus and chromatin alone. Acid alcohol (Sec. 184) may be used to 

 extract the stain more rapidly, but it is generally not necessary. 



The preparation is then placed in a well of gentian violet. A satu- 

 rated aqueous solution is good, or a 1% solution is generally strong 

 enough. The slide is left in gentian violet as short a time as possible to 

 obtain good results, and this can only be determined by trial. Some- 

 times merely dipping it in the stain is sufficient ; other material may 

 require a number of seconds, or even minutes. On removal from gen- 

 tian violet the slide is drained and rinsed in 50% alcohol, and then 

 absolute alcohol is poured over the sections, followed by a few drops 

 of oil of cloves placed in the center of the preparation. The oil of 

 cloves may be replaced with cedar oil or xylol to avoid possible fading 

 of the stain. 



The secret of success with gentian violet is not to stain the nucleolus 

 and chromatin so deeply that the stain will not wash out. They slunild 

 be left red and the other protoplasmic structures blue. If the nucleolus 

 and chromatin come out blue, the slide has been left too long in gentian 

 violet. In our practice the best results have come with very short treat- 

 ment in strong gentian violet (frequently only a dip, or a few seconds 

 timed by the watch), followed directly by absolute alcohol. The oil of 

 cloves may be depended upon to remove much of the gentian violet. 

 But, as previously stated, material dilTers very greatly in it« reaction to 

 gentian violet, and each subject recpires experimentation and a critical 



