Part 21 (1/2)

HISTOLOGICAL METHODS

In the ations 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 co these the most favorable examples for ready demonstration are found in the final division of the pollen spores, especially of soood 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 theth 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 exa with a loe can deteres The spore nizable 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 lass After the stain is sufficiently deep, it should be carefully withdraith 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 perlycerine run under the cover glass The preparation should then be sealed with Canada balsalycerine must be relass It is generally best to gently wipe the edge of the cover glass with a s the cement

[Illustration: 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 , we shall find the nucleus (Fig 127, _A_, _n_) coranular appearance when strongly ranules, which appear isolated, are really parts of filaether, but scarcely visible in the resting nucleus On one side of the nucleus e nucleolus (called here, from its lateral position, paranucleus), and the whole nucleus is sharply separated fro protoplasm by a thin but evidentdivision of the nucleus is an evident increase in size (_B_), and at the saer, and showthe forranules next beco as deeply stained,127, _C_), and about this time the nucleolus disappears

The next step is the disappearance of the nuclear ments lie apparently free in the protoplase themselves in a flat plate in the , when seen fro across the127, _D_, shows this plate as seen from the side, _E_ seen from above)

About the time the nuclear plate is complete, delicate linesat two points on opposite sides of the cell, and for its equator This stage (_D_), is known as the ”nuclear spindle” The segthwise into two sioing to each of the new nuclei This stage is not always to be met with, as it seeenerally reveal some nuclei in this condition

[Illustration: FIG 128--Later stages of nuclear divisions in the pollen ures are seen from the side, except _B_ ii, which is viewed froh this is almost impossible to demonstrate, there are probably asthese the nuclear seg 128, _A_, _B_) As the two sets of segments separate, they are seen to be connected by very nu nuclei reach the poles of the nuclear spindle, the first trace of the division wall appears in the form of isolated particles (s of these threads in the ranules not at first extending across the cell, but later, reaching coranules constitute the young cell wall or ”cell plate,” and finally coalesce to forhter nuclei pass through the saes, but in reverse order thatin 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 sa 128, _F_)[15]

[15] The division is repeated in the same way in each cell so that ultiinal mother cells

This complicated process of nuclear division is known technically as ”karyokinesis,” and is found throughout the higher ani and staining, just described, while giving excellent results in many cases, is not always applicable, nor as a rule are the permanent preparations soalcohol (for very delicate tissues, absolute alcohol, when procurable, is best) is the enerally very satisfactory Specimens may be put directly into the alcohol, and allowed to stay two or three days, or indefinitely if not wanted iood results, specienerally 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 re on size, but are not injured by reer Picric acid is used as a saturated solution in distilled water, and the specith of time as in the chromic acid After the specihly 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 re

As staining agents many colors are used The most useful are hae which entian violet, safranine, Bismarck brown, methyl violet Haematoxylin and carmine are prepared in various ways, but are best purchased ready for use, all dealers inthem 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 re mixture

Haelycerine or balsam (Canada balsa lycerine it is so the water to slowly evaporate, as otherwise the speci to the too rapid extraction of the water frolycerine, 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-lass cap covering the neck, and contains a glass rod for applying the balsa in balsam, the specimen must be completely freed from water by means of absolute alcohol (Soradually 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 (bergaood), which renders it perfectly transparent, when a drop of balsalass placed over the preparation The chloroform in which the balsa the object embedded in a transparent fillass No further treatment is necessary