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 After the stain is sufficiently deep, it should be carefully withdrawn with blotting paper, and pure water run under the cover glass.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 removed from the slide and upper surface of the cover glass. It is generally best to gently wipe the edge of the cover glass with a small brush moistened with alcohol before applying the cement.

If the spore mother cells are still quite young, we shall find the nucleus (Fig.127, A, n) comparatively small, and presenting a granular appearance when strongly magnified. These granules, which appear isolated, are really parts of filaments or segments, which are closely twisted together, but scarcely visible in the resting nucleus. On one side of the nucleus may usually be seen a large nucleolus (called here, from its lateral position, paranucleus), and the whole nucleus is sharply separated from the surrounding protoplasm by a thin but evident membrane.

The first indication of the approaching division of the nucleus is an evident increase in size (B), and at the same time the colored granules become larger, and show more clearly that they are in lines indicating the form of the segments. These granules next become more or less confluent, and the segments become very evident, appearing as deeply stained, much-twisted threads filling the nuclear cavity (Fig.127, C), and about this time the nucleolus disappears.

The next step is the disappearance of the nuclear membrane so that the segments lie apparently free in the protoplasm of the cell. They arrange themselves in a flat plate in the middle of the cell, this plate appearing, when seen from the side, as a band running across the middle of the cell. (Fig.127, D, shows this plate as seen from the side, E seen from above.)

About the time the nuclear plate is complete, delicate lines may be detected in the protoplasm converging at two points on opposite sides of the cell, and forming a spindle-shaped figure with the nuclear plate occupying its equator. This stage (D), is known as the “nuclear spindle.” The segments of the nuclear plate next divide lengthwise into two similar daughter segments (F), and these then separate, one going to each of the new nuclei. This stage is not always to be met with, as it seems to be rapidly passed over, but patient search will generally reveal some nuclei in this condition.

Although this is almost impossible to demonstrate, there are probably as many filaments in the nuclear spindle as there are segments (in this case about sixteen), and along these the nuclear segments travel slowly toward the two poles of the spindle (Fig.128, A, B). As the two sets of segments separate, they are seen to be connected by very numerous, delicate threads, and about the time the young nuclei reach the poles of the nuclear spindle, the first trace of the division wall appears in the form of isolated particles (microsomes), which arise first as thickenings of these threads in the middle of the cell, and appear in profile as a line of small granules not at first extending across the cell, but later, reaching completely across it (Fig.128, C, E). These granules constitute the young cell wall or “cell plate,” and finally coalesce to form a continuous membrane (Fig.128, F).

The two daughter nuclei pass through the same changes, but in reverse order that we saw in the mother nucleus previous to the formation of the nuclear plate, and by the time the partition wall is complete the nuclei have practically the same structure as the first stages we examined (Fig.128, F).[15]

This complicated process of nuclear division is known technically as “karyokinesis,” and is found throughout the higher animals as well as plants.

The simple method of fixing and staining, just described, while giving excellent results in many cases, is not always applicable, nor as a rule are the permanent preparations so made satisfactory. For permanent preparations, strong alcohol (for very delicate tissues, absolute alcohol, when procurable, is best) is the most convenient fixing agent, and generally very satisfactory. Specimens may be put directly into the alcohol, and allowed to stay two or three days, or indefinitely if not wanted immediately. When alcohol does not give good results, specimens fixed with chromic or picric acid may generally be used, and there are other fixing agents which will not be described here, as they will hardly be used by any except the professional botanist. Chromic acid is best used in a watery solution (five percent chromic acid, ninety-five percent distilled water). For most purposes a one percent solution is best; in this the objects remain from three or four to twenty-four hours, depending on size, but are not injured by remaining longer. Picric acid is used as a saturated solution in distilled water, and the specimen may remain for about the same length of time as in the chromic acid. After the specimen is properly fixed it must be thoroughly washed in several waters, allowing it to remain in the last for twenty-four hours or more until all trace of the acid has been removed, otherwise there is usually difficulty in staining.

As staining agents many colors are used. The most useful are hÆmatoxylin, carmine, and various aniline colors, among which may be mentioned, besides gentian violet, safranine, Bismarck brown, methyl violet. HÆmatoxylin and carmine are prepared in various ways, but are best purchased ready for use, all dealers in microscopic supplies having them in stock. The aniline colors may be used either dissolved in alcohol or water, and with all, the best stain, especially of the nucleus, is obtained by using a very dilute, watery solution, and allowing the sections to remain for twenty-four hours or so in the staining mixture.

HÆmatoxylin and carmine preparations may be mounted either in glycerine or balsam. (Canada balsam dissolved in chloroform is the ordinary mounting medium.) In using glycerine it is sometimes necessary to add the glycerine gradually, allowing the water to slowly evaporate, as otherwise the specimens will sometimes collapse owing to the too rapid extraction of the water from the cells. Aniline colors, as a rule, will not keep in glycerine, the color spreading and finally fading entirely, so that with most of them the specimens must be mounted in balsam.

Glycerine mounts must be closed, which may be done with Canada balsam as already described. The balsam is best kept in a wide-mouthed bottle, specially made for the purpose, which has a glass cap covering the neck, and contains a glass rod for applying the balsam.

Before mounting in balsam, the specimen must be completely freed from water by means of absolute alcohol. (Sometimes care must be taken to bring it gradually into the alcohol to avoid collapsing.[16]) If an aniline stain has been used, it will not do to let it stay more than a minute or so in the alcohol, as the latter quickly extracts the stain. After dehydrating, the specimen should be placed on a clean slide in a drop of clove oil (bergamot or origanum oil is equally good), which renders it perfectly transparent, when a drop of balsam should be dropped upon it, and a perfectly clean cover glass placed over the preparation. The chloroform in which the balsam is dissolved will soon evaporate, leaving the object embedded in a transparent film of balsam between the slide and cover glass. No further treatment is necessary. For the finer details of nuclear division or similar studies, balsam mounts are usually preferable.

It is sometimes found necessary in sectioning very small and delicate organs to embed them in some firm substance which will permit sectioning, but these processes are too difficult and complicated to be described here.

The following books of reference may be recommended. This list is, of course, not exhaustive, but includes those works which will probably be of most value to the general student.

• 1. Goebel. Outlines of Morphology and Classification.
• 2. Sachs. Physiology of Plants.
• 3. De Bary. Comparative Anatomy of Ferns and Phanerogams.
• 4. De Bary. Morphology and Biology of Fungi, Mycetozoa, and Bacteria.

These four works are translations from the German, and take the place of Sachs’s Text-book of Botany, a very admirable work published first about twenty years ago, and now somewhat antiquated. Together they constitute a fairly exhaustive treatise on general botany.—New York, McMillan &Co.

• 5. Gray. Structural Botany.—New York, Ivison &Co.
• 6. Goodale. Physiological Botany.—New York, Ivison &Co.

These two books cover somewhat the same ground as 1 and 2, but are much less exhaustive.

• 5. Strasburger. Das Botanische Practicum.—Jena.

Where the student reads German, the original is to be preferred, as it is much more complete than the translations, which are made from an abridgment of the original work. This book and the next (7 and 8) are laboratory manuals, and are largely devoted to methods of work.

• 7. Arthur, Barnes, and Coulter. Plant Dissection.—Holt &Co., New York.
• 8. Whitman. Methods in Microscopic Anatomy and Embryology.—Casino &Co., Boston.

For identifying plants the following books may be mentioned:—

• Green algÆ (exclusive of desmids, but including CyanophyceÆ and VolvocineÆ).
• Wolle. Fresh-water AlgÆ of the United States.—Bethlehem, Penn.
• Desmids. Wolle. Desmids of the United States.—Bethlehem, Penn.
• The red and brown algÆ are partially described in Farlow’s New England AlgÆ. Report of United States Fish Commission, 1879.—Washington.
• The CharaceÆ are being described by Dr.F.F. Allen of New York. The first part has appeared.
• The literature of the fungi is much scattered. Farlow and Trelease have prepared a careful index of the American literature on the subject.
• Mosses. Lesquereux and James. Mosses of North America.—Boston, Casino &Co.
• Barnes. Key to the Genera of Mosses.—Bull. Purdue School of Science, 1886.
• Pteridophytes. Underwood. Our Native Ferns and their Allies.—Holt &Co., New York.
• Spermaphytes. Gray. Manual of the Botany of the Northern United States. 6th edition, 1890. This also includes the ferns, and the liverworts.—New York, Ivison &Co.
• Coulter. Botany of the Rocky Mountains.—New York, Ivison &Co.
• Chapman. Flora of the Southern United States.—New York, 1883.
• Watson. Botany of California.