STAINS 199 



Material stained in DelafieJcPs ha^matoxylin may be counterstained with 

 alcoholic safranin, but very good results may be obtained with the tissues of 

 higher plants by staining first with safranin, as described in Sec. 185. 



184. Safranin. There are various kinds of safranin sold, some of which 

 dissolve more readily in water and some in alcohol. The stain should always 

 bfe placed in its appropriate solvent. A 1% solution in water is a good 

 strength, and a saturated solution in 95% alcohol mixed with an equal vol- 

 ume of water, making a 50% alcoholic solution, is also good. The alcohol 

 solutions are the most convenient. Anilin safranin is prepared from a satu- 

 rated solution in 95% alcohol mixed with an equal amount of anilin water 

 (made by shaking anilin oil in distilled water, when a small percentage of 

 the oil is taken up by the water). Anilin safranin is considered by some 

 to be the best of the safranin stains. 



Safranin colors cell walls as well as protoplasm. It is therefore a general 

 stain, but when properly extracted it may be made to differentiate certain 

 nuclear structures sharply (chromosomes and nucleolus), and is much used 

 in staining on the slide, especially in combination with gentian violet and 

 orange G (Sec. 199, D). Material may remain in safranin from one hour or 

 less to twelve hours or more. The stain is extracted in 50% alcohol until the 

 desired coloration is obtained, or, if very much overstained, the material may 

 be placed in acid alcohol (yV cc. hydrochloric acid in 100 cc. 70% alcohol). 

 The acid alcohol, if used, must be thoroughly washed out. 



185. Safranin and Delafield's haematoxylin. Safranin followed by Delafield's 

 hsematoxylin is an excellent stain for the tissues of higher plants, whether 

 in free-hand or microtome sections. Sections cut free-hand from fresh 

 material may be fixed for 10-15 minutes in absolute alcohol or medium 

 chrom-acetic acid (Sec. 194) ; those from preserved material may be stained 

 at once. They should remain in the safranin several hours (over night). 

 Wash in 50% alcohol (acid alcohol if desired) until the stain is extracted 

 from all parts except lignified cell walls, as in fibro-vascular bundles. 

 Remove the acid alcohol if used. Stain in Delafield's ha-matoxylin for 

 several minutes (1-30). AVash in tap w-ater, or extract the stain if necessary 

 in acid alcohol (as described in Sec. 183), which must be followed by tap 

 water until the stain is purple. Carry through 95% alcohol, then absolute 

 alcohol, clear in xylol, and mount in balsam. Sections cut on the microtome 

 are stained on the slide (Sec. 197) in the same manner as described above. 



186. Other anilin stains. There are numerous anilin dyes of great value 

 in special cases, but few of them have such general usefulness as eosin, 

 safranin, and gentian violet. The following, however, are important. 



A. Acidfuchsln. A 1% solution may be made in water or in 70'; alcohol. 

 The stain acts rapidly and is very brilliant. It may be extracted in 

 95% alcohol from overstained material or sections. 



