R XXI.

HISTOLOGICAL METHODS.

In the more exact investigations of the tissues, it is often necessary
to have recourse to other reagents than those we have used hitherto,
in order to bring out plainly the more obscure points of structure.
This is especially the case in studies in cell division in the higher
plants, where the changes in the dividing nucleus are very
complicated.

For studying these the most favorable examples for ready
demonstration are found in the final division of the pollen spores,
especially of some monocotyledons. An extremely good subject is
offered by the common wild onion (_Allium Canadense_), which flowers
about the last of May. The buds, which are generally partially
replaced by small bulbs, are enclosed in a spathe or sheath which
entirely conceals them. Buds two to three millimetres in length
should be selected, and these opened so as to expose the anthers.
The latter should now be removed to a slide, and carefully crushed
in a drop of dilute acetic acid (one-half acid to one-half
distilled water). This at once fixes the nuclei, and by examining
with a low power, we can determine at once whether or not we have
the right stages. The spore mother cells are recognizable by their
thick transparent walls, and if the desired dividing stages are
present, a drop of staining fluid should be added and allowed to act
for about a minute, the preparation being covered with a cover
glass. After the stain is sufficiently deep, it should be carefully
withdrawn with blotting paper, and pure water run under the cover
glass.

The best stain for acetic acid preparations is, perhaps, gentian
violet. This is an aniline dye readily soluble in water. For our
purpose, however, it is best to make a concentrated, alcoholic
solution from the dry powder, and dilute this as it is wanted. A
drop of the alcoholic solution is diluted with several times its
volume of weak acetic acid (about two parts of distilled water to
one of the acid), and a drop of this mixture added to the
preparation. In this way the nucleus alone is stained and is
rendered very distinct, appearing of a beautiful violet-blue color.

If the preparation is to be kept permanently, the acid must all be
washed out, and dilute glycerine run under the cover glass. The
preparation should then be sealed with Canada balsam or some other
cement, but previously all trace of glycerine must be