Stains and Staining 45 



staining after the final absolute alcohol, if the stain is dissolved in 

 clove oil. 



A stain of 3 or 4 minutes in safranin adds an excellent differentia- 

 tion in- case of many algae and does not obscure nuclear details. 

 The exine of pollen grains may take a brilliant red with safranin 

 in 5 to 10 minutes, contrasting sharply with the mouse gray of the 

 intine. Orange G, in clove oil, often gives a pleasing contrast. 



Delafield's Haematoxylin. "To 100 c.c. of a saturated solution 

 of ammonia alum add, drop by drop, a solution of 1 g. of haema- 

 t'oxylin dissolved in 6 c.c. of absolute alcohol. Expose to air and 

 light for one week. Filter. Add 25 c.c. of glycerin and 25 c.c. of 

 methyl alcohol. Allow to stand until the color is sufficiently dark. 



v 



Filter, and keep in a tightly stoppered bottle" (Stirling and Lee). 

 The addition of the glycerin and methyl alcohol will precipitate 

 some of the ammonia alum in the form of small crystals. The last 

 filtering should take place 4 or 5 hours after the addition of the 

 glycerin and methyl alcohol. 



The solution should stand for at least two months before it is 

 ready for using. This " ripening" is brought about by the oxida- 

 tion of haematoxylin into haematin, a reaction which may be secured 

 in a few minutes by a judicious application of peroxide of hydrogen. 

 However, we prefer to let the haematoxylin ripen naturally. There 

 is no objection to making this stain in considerable quantity, since 

 it does not deteriorate. We have used Delafield's haematoxylin 

 which had been in a cork-stoppered bottle for twenty years, and 

 it still gave the rich characteristic stain. 



Transfer to the stain from 50 or 35 per cent alcohol or from water. 

 The length of time required is exceedingly variable. Sometimes 

 sections will stain deeply in 3 minutes, but it is often necessary to 

 stain for 30 minutes or even longer. This stain may be diluted 

 with several times its own volume of water; when this is done, the 

 time required is correspondingly long, but the staining is frequently 

 more precise. The length of time required will be fairly uniform for 

 all material taken from the same bottle. This fact indicates that the 

 washing process, which follows killing and fixing, is an important 

 factor; if the washing has been thorough, the material will stain 

 readily; but if the washing has been insufficient, the material may 



