48 
METHODS IN PLANT HISTOLOGY 
passing the slides through the alcohols, staining in a solution of saf- 
ranin in 50 per cent alcohol from the alcohol of a concentration 
nearest that of the stain; and 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 or 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¬ 
toxylin 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. 
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 
