For the satisfactory examination of tissues it is necessary that they should be “hardened” in certain fluids. The object of this is to give the specimens greater consistence, so that thin sections may be more readily obtained and more safely manipulated, and also to “fix” the tissue element as far as possible in the same relative position as in the living body. The hardening process also acts on the protoplasm of the cells, and prevents their swelling up when placed in water, and in the various staining fluids.

The fluid used must be one which will not itself injure the specimen, and which can be thoroughly removed by washing, so that it may not interfere with staining operations. The specimens should be kept while hardening in wide mouthed bottles, on the bottom of which a little cotton wool or tow has been laid. This allows the hardening fluid to come freely in contact with the under surface of the pieces of tissue, and prevents their being flattened against the hard glass bottom.

The hardening fluid requires changing occasionally. This should always be done at the end of twenty-four hours, in order to get rid of any deposit of blood, &c., that may have accumulated. Besides this, the tissue when placed in the fluid contained a good deal of water which will have diluted it and consequently an early change is desirable. Afterwards the fluid requires to be changed only as often as it becomes turbid, or any deposit occurs, usually about once a week.

While hardening, specimens should be kept in a cool place, as warmth favours changes in the cells, &c.

In manipulating the portions of organs, forceps should always be used and these with great gentleness. The specimens should never be impaled with needles, or unsightly holes, which may even be mistaken for pathological appearances, will appear when a section is examined under the microscope.

It requires some practice to know when the tissue is sufficiently hardened. The object aimed at is to make them not really hard but tough. It is almost unnecessary to add that in testing this with the fingers the utmost gentleness must be observed, or serious damage may be done to the tissue.

When the tissue is sufficiently hardened the hardening fluid must be thoroughly dissolved out. This is most quickly effected by placing the specimen in a basin into which cold water from a tap is constantly running. The tissue may then be removed (forceps always being used and never the needle) and placed in an imbedding medium as subsequently directed; or, if it is not to be cut at once, into equal parts of methylated spirit and water, in which it may be kept indefinitely, the fluid being changed if it becomes at all cloudy.

It is unnecessary for ordinary work to have more than the following hardening fluids:—
MÜller’s fluid:—

Two drachms of carbolic acid are sometimes added to each pint of the fluid but as a rule it is not necessary.

MÜller’s fluid is the most generally useful of the various fluids employed, for the following reasons:—

1. It causes very little shrinking of the elements of the tissue, and hence may be employed for most delicate objects, e.g., the retina and embryos.

2. In consequence of its not making the tissues shrink, it does not squeeze the blood out of the vessels and where the organ has been congested before death, we may, by using MÜller’s fluid, preserve a natural injection of the capillaries.

3. There is comparatively little danger of over-hardening the tissue and rendering it brittle.

4. Sections of organs hardened in MÜller’s fluid are usually firm and easy to manipulate. They do not tend to curl up or adhere to one another as much as those hardened in spirit.

5. It readily permeates the tissues, and hence large portions of organs, or even the entire organ may be satisfactorily hardened in it.

6. It is very cheap. A gallon can be made up for about eightpence.

The fluid has however certain slight drawbacks:—

1. The hardening process is a slow one occupying four to eight weeks.

2. The fluid gives a permanent dingy colour to the tissue. This does not cause any inconvenience for microscopic purposes, but it is a disadvantage when it is intended to preserve the rest of the specimen, as a naked eye preparation. In such cases the organ should be hardened in spirit, carbolic acid, or formal.

MÜller’s fluid can be used for almost any tissue. It is especially useful for those which contain a large quantity of fluid, or of blood, and is essential for nerve tissues which it is intended to stain by Pal’s method (p.89).

To harden a specimen in it at least twenty times the bulk of fluid must be employed.

The fluid must be changed on the third day, and afterwards about every week as may be required.

Methylated spirit is a very useful hardening agent. It hardens in one to three weeks according to the size of the tissue and the quantity of spirit used. Its disadvantages are:—

1. It is more apt to overharden than MÜller’s fluid.

2. It causes a great deal of shrinking of the tissue and thus squeezes much of the blood out of the vessels.

It is most useful in hardening tissues containing much epithelium, e.g., kidney, epithelioma, &c.

Spirit is also frequently employed to complete the hardening by MÜller’s fluid and to preserve tissues after they have been hardened.

About ten or fifteen times the bulk of spirit should be used for one of the tissues. The fluid should be changed on the third day and afterwards as required.

MÜller’s fluid and spirit.—This is a useful combination for many purposes. It is made thus:—MÜller’s fluid, three parts; methylated spirit, one part.

The fluid must be allowed to cool after mixing before being used, and if necessary filtered. It will harden specimens satisfactorily in three weeks.

MÜller’s fluid and formal.—Is an extremely useful mixture made by adding one part of formal to nine of MÜller’s fluid. It hardens in a much shorter time than MÜller’s fluid and causes very little shrinkage.

Absolute alcohol.—Used as a hardening agent where the tissues are to be examined for micro-organisms, and for specimens to be stained by Nissl’s method (p.101). A cheaper and equally effective hardening medium is made by dehydrating methylated spirit by adding one ounce of fused carbonate of potassium to each pint of methylated spirit, and decanting.

Small pieces must be used. The depths of the block should not exceed 3/8 inch. The fluid should be changed on the third day. Hardening will be completed in about ten days or even earlier.

Osmic acid.—For rapidity of action, and for rapid fixing of all the tissue elements in their natural position osmic acid is one of the best hardening reagents we possess.

Its disadvantages as a hardening agent are:—

1. Its expense.

2. Its irritating and corrosive vapour.

3. The fact that only small pieces of tissue can be hardened in it, since the external surface is very rapidly hardened and thus the fluid is prevented from penetrating into the centre of the lump.

It is most frequently used as a hardening agent for very delicate structures, such as the retina, or embryos, or for fresh sections of brain (p.95).

The acid itself may be procured in sealed tubes, each containing one gramme. These should be broken in a bottle under sufficient distilled water to make a one per cent. solution. The bottle containing it should be covered with brown paper to exclude the light. For hardening purposes small pieces of the tissue, not much larger than a pea, should be placed in the acid, the one per cent. solution being diluted with five to ten volumes of distilled water. The tissues may be left in this for from three to five days. They must then be thoroughly washed in distilled water and may afterwards be preserved in methylated spirit.

Both the hardening and the subsequent washing must be carried on in the dark.

Osmic acid is also a most valuable staining reagent (see p.81).

Carbolic acid (5 per cent.).—May be used to harden almost any tissue, but is particularly useful for hardening nervous tissues such as brain or spinal cord which are afterwards to be preserved as museum specimens. It does not discharge the colour of a specimen so rapidly as spirit.

Three or four times the bulk of fluid should be used. It requires changing at the end of twenty-four hours, and again at the end of the first week.

Saturated aqueous solution of corrosive sublimate is one of the most convenient hardening reagents for small pieces of delicate tissue, e.g., embryos. It hardens them in a few days. When they are sufficiently hardened the mercurial salt should be removed by washing first in methylated spirit for a few hours and then in running water.

Formal.—An aqueous solution containing about 35 per cent. of formaldehyde. It is a rapid hardening agent, causes very little shrinkage of the tissues, and does not discharge the colour of the specimens as much as alcohol. For hardening formal should be used as a two to five per cent. solution in distilled water. It may also be used as a ten per cent. solution for mounting museum preparations, but there is some tendency for a cloudy deposit to form on the glass after a time. It is the most suitable hardening agent at our disposal for eyes. It rapidly fixes the tissue elements, but does not cause much contraction. It may also be used for hardening the brain and spinal cord. Large quantities of fluid must be used for the latter purpose and it must be frequently changed. As soon as they are sufficiently hardened they should be transferred to methylated spirit.

Marchi’s fluid.—This consists of:—

It is used for hardening specimens as a preliminary to Golgi’s method for staining nerve cells (p.97), and also to complete the hardening of sections of spinal cord, &c., before employing SchÄfer’s modification of the Weigert Pal hÆmatoxyline method (p.91).

It is also used as a stain for recently degenerated nerve tracts and fibres, especially after experimental lesions.

The fluid has little penetrating power, and therefore tissues must be cut into small pieces, about 3/8inch cube. It is not necessary to place them in this fluid at once on removal from the body, but the preliminary hardening must be in MÜller’s fluid and not in alcohol, &c.

Special Hardening Reagents for Rapid Fixation in Order to Study Cell Structure.

1. Alcohol.

2. Flemming’s solution (modified by Friedmann):—

Small pieces should be hardened in this fluid for twelve to twenty-four hours, and then washed and transferred to alcohol for some days before staining.

3. Nitric acid.—A ten per cent. solution in distilled water. It hardens the tissue in three to four hours, and should be followed by 70 per cent. alcohol, the hardening being completed in absolute alcohol.

In using any of these methods it is necessary that the tissue be removed from the body during life or immediately after death. They are employed for revealing the changes in the cells and their nuclei in rapidly growing or inflamed tissue, for studying karyokinesis in cancer cells, and investigating the appearance of nerve cells and gland cells at rest, when actively employed and when fatigued; and they are also most useful in preparing specimens of the “parasitic bodies” which have been described in many cancer cells.

Decalcifying Fluids.

Used in the preparation of bone, tooth, osseous tumours, &c. The two best fluids for general use are:—

Chromic and nitric fluid.—This is made as follows:—

If the bone is not very compact the fluid may be used diluted with an equal quantity of water. A large quantity of fluid should be used, and like all decalcifying fluids, it should be frequently changed.

As soon as the specimen is sufficiently flexible, it should be thoroughly washed in running water for some hours, and then transferred to spirit until it is convenient to cut sections.

Von Ebner’s solution:—

It is a very useful decalcifying agent, but causes the fibrous elements to swell up rather more than chromic and nitric fluid. A large quantity of the fluid must be used, and it should be changed daily. It must be very thoroughly washed out in running water when the decalcification is completed.

Bleaching solution (eau de Javelle).

Shake up well.

Mix the two solutions. Allow them to stand for an hour and filter.

It is used chiefly for clearing vegetable sections but may also be used for sections containing a large quantity of pigment. It is particularly useful in decolourizing sections of “madura foot” due to the presence of a black fungus.