Softening. All dry and hard substances should be softened before sections can be made properly. In the case of roots, rhizomes, tubers, corms, fruits, seeds, etc., they are soaked first in alcohol half an hour to expel air, then in water several hours or until saturated (hard tissues, shells, etc., may require several days); if now too soft for cutting, lay in alcohol 24 hours; if again too hard or brittle, place in a mixture of equal parts of alcohol and glycerin for 24 hours. In such roots as gentian, etc., that are much shrunken, we should use for water in second stage a 1 - 2 p. c. aqueous solution of potassium hydroxide or ammonia; this alkali, however, should always be washed out with water before hardening. Specimens thus prepared can be kept always in readiness by letting them remain immersed in a mixture of equal quantities of alcohol and glycerin.
Clearing. It is often necessary to clarify sections by having absorbed from them such substances as would prevent transparency - starch, resins, oils, etc. To accomplish this, sections should be boiled in water and put into diluted Labarraque's solution for 15 minutes, or placed for a short while into a mixture of 4 parts oil of turpentine + 1 of creosote, or into pure oil of cloves, then mounted in Canada balsam. When sections have been stained, should soak them first in alcohol for a few minutes and then in the clearing-mixture.
Staining Fluids. These make prominent and differentiate thin cell-walls, inconspicuous and uniform tissues, etc., thus making their differences in appearance very perceptible.
(a) Haematoxylin. Prepared by mixing 2 parts saturated alcoholic solution hematoxylin with 75 parts saturated aqueous solution ammonia alum; let stand a week in sunlight, filter, and to every 7 parts add 1 part each of glycerin and methyl alcohol, allow sediment to deposit by standing, filter. Used to stain lignified and cellulose walls - not cutinized ones; is also a good nuclear stain. Sections should soak several hours - those from alcohol should first be washed and all acids avoided.
(6) Fuchsin. A solution of fuchsin in water, used to stain lignified cell-walls, as these hold color better than non-lignified ones. When sections with fuchsin staining are washed with a mixture of saturated solution of picric acid 1 part + water 2 parts, the fuchsin is removed from unlignified cell-walls, while lignified ones remain beautifully stained. These may now be dehydrated and mounted, or double-stained with aniline blue, then dehydrated and mounted.
(c) Methyl-green. An aqueous solution of methyl-green sufficiently strong to give deep green color. It stains protoplasm, nucleus, also lignified and cutinized tissues better than it does cellulose. Tissues absorb color quicker if previously washed in weak acidified (HNO3) water.
(d) Iodine-green. Made by dissolving iodine-green in water until a deep green solution results. This stains lignified and cutinized tissues green, also proteids, amyloplasts attached to young starch-grains; acts on cellulose tissues slightly. Often used with carmine, eosin, or fuchsin for double staining.
Mounting. When for only temporary or immediate use, water or glycerin, or a mixture of the two, is employed. If it is to be permanent, then Canada balsam is the best medium. Mounting is accomplished thus: The sections, if stained in aqueous solution, should first be dehydrated by placing for a few minutes in 70 p. c. alcohol, then in 90 p. c, and finally in 98 p. c.; now put for a short while in clearing medium - oil of cloves or oil of turpentine - place a section on centre of slide, add to it a drop of balsam, apply cover-glass slantingly to avoid air-bubbles, slightly tapping same to a fixed position. If just sufficient balsam is used, we have simply to let it dry several days, then ring with a circle of colored cement around marginal contact of cover-glass with slide.