74 
METHODS IN PLANT HISTOLOGY 
are quite different and starch grains are well differentiated in the 
tube cells and the wall shows a violet intine contrasting sharply 
with a red exine, the mount is good. Anything intermediate is 
indifferent. If mitotic figures have been stained with cyanin and 
erythrosin, a first-class preparation should show blue chromosomes 
and red spindles; if stained with safranin and gentian-violet, the 
chromosomes should be red and the spindles violet. 
In staining growing points, apical cells, young embryos, anther- 
idia, archegonia, and many such things, the cell walls are the principal 
things to be differentiated, if the preparations are for morphological 
study. As a rule, it is better in such cases not to use double stain¬ 
ing, but to select a stain which stains the cell walls deeply without 
obscuring them by staining starch, chlorophyll, and other cell con¬ 
tents. For example, try the growing point of Equisetum. The 
protoplasm of such growing points is very dense. If Delafield’s 
haematoxylin and erythrosin be used, the haematoxylin will stain 
the walls and nuclei, and will slightly affect the other cell contents, 
but the erythrosin will give the cytoplasm such a dense stain that 
the cell walls will be seriously obscured. It would be better to use 
haematoxylin alone. For counting chromosomes, it is better to stain 
in iron-alum haematoxylin alone, or in safranin alone. The same 
suggestion may well be observed in tracing the development of 
antheridia, archegonia, embryos, and similar structures. 
In using combinations, it must be remembered that the second 
stain often affects the first, e.g., if safranin is to be followed by 
Delafield’s haematoxylin in staining a vascular bundle, it will not 
do to make the safranin just right and then apply the haematoxylin, 
for the acid which must be used to differentiate the haematoxylin 
and to avoid precipitates will also reduce the safranin, and the red 
will be too weak. You must overstain in safranin so that the reduc¬ 
tion will finally leave it just right. The same hint will apply if 
safranin is to be followed by anilin blue, since here, also, acid must be 
used; if light green is to follow the safranin, the stain itself is so 
acid that the safranin must be rather strong before the light green is 
applied. Orange, whether in water or in clove oil, reduces many 
stains and, consequently, such stains must be strong enough to allow 
the weakening. These hints are only samples: the student must 
observe the behavior of the various stains when used singly and when 
used in various combinations. 
