Double Staining.—When a section is passed through methyl green solution and afterwards one of carmine, the lignified portion is coloured green and the unlignified red. Acid green may be used in the place of methyl green, with a similar result. When picric acid is used with carmine, ingrosine, or Hoffmann’s blue, the picric acid dyes the ligneous portion and the others colour the unlignified structure, red, black, and blue respectively.
Eosin stain is the most useful for sieve-tubes and plates. Make a strong solution of eosin in equal parts of water and alcohol, and stain the section for five or ten minutes. Wash well in methylated spirit, dehydrate, clean in oil of cloves, and mount in Canada balsam.
Bleaching Process.—The bleaching and clearing of vegetable structures before staining is a very necessary process, especially so if starch be present in any quantity. Clearing agents are of two kinds—those which act by virtue of their property of strongly refracting light, and those which disintegrate and dissolve the amyloid cell contents. To the first class belong the essential oils, as oil of cloves, Canada balsam, glycerine, and other similar bodies; to the second class, solutions of potash, phenol, and chloral hydrate. The actual value of some of these agents is questionable. The process usually preferred is as follows: Place the sections in a fresh clear solution of chlorinated lime, allowing them to remain until quite bleached, say from two to four or five minutes; then gently warm in a test-tube for a few seconds, and quickly replace the solution with distilled water and boil for two or three minutes; repeat the treatment with boiling water three times; wash with a one per cent. solution of acetic acid, and finally with distilled water. The sections are then quite ready for staining operations.
When the stem is hard and brown, a solution of chloride of lime should be used—a quarter of an ounce of chloride dissolved in a pint of water, well shaken and stood by to settle down, then pour off the clear fluid for use. For hard tissues this solution answers well, but it is not suitable for leaves, as they require not only bleaching, but the cell contents should be dissolved out to render them transparent. A solution of chlorinated soda answers well for both stems and leaves. It is prepared as follows:—
To one pint of water add two ounces of fresh chloride of lime, shake or stir it well two or three times, then allow it to stand till the lime has settled. Prepare meanwhile a saturated solution of carbonate of soda—common washing soda. Now pour off the clear supernatant fluid from the chloride of lime, and add to it, by degrees, the soda solution, when a precipitate of carbonate of lime will be thrown down; continue to add the soda solution till no further precipitate is formed. Filter the solution, and keep it in a well-stoppered bottle in the dark, otherwise it speedily spoils.
Sections bleached in chlorinated soda must, when white enough, be washed in distilled water, and allowed to remain in it for twenty-four hours, changing the water four or five times, and adding a few drops of nitric acid, or at the rate of eight or ten drops to the half-pint, to the water employed before the final washing takes place. From water transfer them to alcohol, in which they must remain for an hour or more.
Although alkaline glycerine has been recommended for several purposes in micro-technique, it is not so well known as it should be how serviceable it is as an extempore mounting solution in vegetable histology. The best mixture for general use is composed of glycerine 2 ozs., distilled water 1½ oz., solution of potash, B.P., ½ oz. This combines the refringent property of the glycerine with the clearing action of the caustic potash, while the swelling action of the potash is considerably diminished.
Cutting Sections of Hard Woods.—The lathe and circular saw will be found as useful for cutting sections of the harder kinds of woods, as for bone structure. It may be necessary to subject the older and consequently harder pieces of wood to the action of steam for a few hours to soften them, and afterwards transfer them to methylated spirit, before making an attempt to cut sections. But the more open woods, of one, two, or three years’ growth, will show all that may be required, and these can be cut by hand, or with the microtome, as already described.
With a little practice the finest and thinnest possible slices may be cut by hand. It is usual to take off the first slice to give a smooth and even surface to the specimen. Then turn the screw to raise it a little, sprinkle the surface with spirit and water, and cut with a light hand. Remove the cut sections with a fine camel’s-hair brush or a section lifter ([Fig. 250]) to a small vessel containing water, when the thinnest will float on the surface, and remove to methylated spirit and water, where they should remain until they can be mounted. Sections of hard woods, and those containing gum-resins, or other insoluble material, must first be kept in methylated spirit or alcohol, and finally transferred to oil of cloves, to render them sufficiently transparent for mounting in Canada balsam.
If the structure of an exogenous wood is required to be examined, the sections should be made in at least three different ways: the transverse, the longitudinal, and the oblique, or, as they are sometimes called, the horizontal, vertical, and tangential, each of which will exhibit different appearances, as seen in [Fig. 245]: b is a vertical section through the pith of a coniferous plant, and exhibits the medullary rays known to the cabinet-maker as the silver grain; e is a magnified view of a part of the same; the woody fibres are seen with their dots l, and the horizontal lines k indicating the medullary rays cut lengthwise; c is a tangential section, and f a portion of the same; the medullary rays m m, and the woody fibres with vertical slices of the dots, are shown. Instructive preparations will be secured by cutting oblique sections of the stem. The sections seen are made from the pine. All exogenous stems, however, exhibit three different appearances, according to the direction in which the section is made.