Fig. 127.—A, pollen mother cell of the wild onion. n, nucleus. B–F, early stages in the division of the nucleus. par. nucleolus; acetic acid, gentian violet, × 350.
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.
Fig. 128.—Later stages of nuclear divisions in the pollen mother cell of wild onion, × 350. All the figures are seen from the side, except B ii, which is viewed from the pole.
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 per cent chromic acid, ninety-five per cent distilled water). For most purposes a one per cent 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.