CHAPTER II.

ON PREPARING TISSUES FOR EXAMINATION.

THE most essential point in pathological investigation is the proper hardening of the material to be examined, and this must be done gradually, as, if any tissue is placed at once in a strong solution, such as 1 per cent. chromic acid or absolute alcohol, the elements of which it is composed shrink on the outside and undergo such alterations as to make it impossible to form a correct idea of the pathological change that has taken place. Bad hardening, combined with imperfect histological knowledge, will account for most of the extraordinary fallacies that have been made public of late years.

In making these hardening solutions French weights and measures will be found the most simple to use; but, as many are not accustomed to them, the proportions will also be given in English weights when practicable.

One gramme is equivalent in weight to one cubic centimetre of water; it will therefore be seen at once how easy it is to make percentage solutions when French weights and measures are used.

For example, to make a 1 per cent. solution, 1 gramme is weighed out and dissolved in 100 c.c. of water. The only apparatus required is a set of gramme weights and two measured glasses, one of 100 c.c., the other 500 c.c.

Hardening solutions, as a rule, do not require filtering.

The best plan is to make a large quantity at a time. Take a bottle holding about 2000 c.c. of water-this amount should be measured into the bottle, and the height of the fluid marked on it with a diamond; the amount of the chemical used should be written on the label; so that, when a new supply is wanted, the bottle can be held under the tap until the water reaches within a few inches of the mark; the amount of the hardening agent is then weighed out and put into the funnel, and the bottle filled up to the mark.

This method can be used with substances like chromic acid, which dissolve readily; others, such as bichromate of potash, require rubbing up with hot water in a mortar.

1. CHROMIC ACID MIXTURE.-The most useful hardening agent is a mixture of chromic acid and spirit.

Make a solution of chromic acid in water, 15 grains to the pint, or 1 gramme to 600 c.c.; ordinary water may be used.

Take of this 2 parts, and ordinary alcohol 1 part-stir.

The material must be cut into small pieces-about half-inch cubes, and a large quantity of fluid used; a wide-mouthed stoppered bottle holding from six to ten ounces, according to the quantity of material, is best; change the fluid at the end of twenty-four hours, and again every third day, and the material will be hardened in from eight to twelve days; this can be easily proved by taking out a piece and feeling it. If allowed to remain too long it gets brittle. When it is found to be moderately hard, usually after about eight to ten days, pour off the chromic-acid mixture, and wash well in water for some hours; the water should be changed several times, then place it in dilute spirit made thus: Take alcohol 2 parts, and water 1 part.

About half the quantity of this spirit mixture may be used, but the material must be well covered.

Let the material remain in this for from twenty-four to thirty-six hours, never longer than three days, and then replace it by pure alcohol, enough to cover the material; it may remain in this for an indefinite time, but it will often be found that the spirit becomes cloudy and full of deposits in a few days; in this case it is only necessary to change the spirit until it remains clear.

A large quantity of one-sixth per cent. solution of chromic acid should be kept on hand, and it should be mixed with the spirit as required; it will be found the most useful of all the hardening agents, if it is changed at the proper time.

In some cases a one-sixth per cent. solution of chromic acid may be used without the spirit with advantage. In other cases it may be necessary to use a solution much weaker, as a one-tenth per cent. These are, however, not required for the ordinary work of the pathologist, but when special investigation is being made of some particular organ, it is better to try the effect of different strengths of the hardening agent to see which gives the best result in that particular case.

2. MULLER'S FLUID is a good hardening mixture, but requires a much longer time, taking weeks to do what the chromic-acid mixture will do in days. It is made thus: Take potass. bichrom. 2 parts, soda sulphat. 1 part, water 100 parts.

In making this solution, the ingredients should be pounded up in a mortar, and then warm water added until they are dissolved.

A large quantity of the fluid should be used, about ten times the bulk of the tissue.

The advantage of this mixture is that larger pieces can be hardened in it, and it does not require changing after the first week or two; but it will take from five to seven weeks to harden anything, according to its size. When it becomes cloudy it requires changing. The material, when sufficiently hardened, should be well washed and then placed in dilute spirit in the same manner as recommended after hardening in the chromic acid mixture.

3. DILUTE SPIRIT.-Many tissues can be hardened in spirit alone if they are placed in dilute spirit at first, so that the elements of which they are composed are not shrunk. This process is also used after hardening by any of the others.

Dilute spirit is made by adding one part of water to two parts of alcohol.

The material to be hardened should not be left in this mixture more than from twenty-four to forty-eight hours.

It is then transferred to pure spirit, and after remaining in it for from three to five days, it is ready for cutting.

4. BICHROMATE OF POTASH.-Make a 2 per cent. solution and keep it on hand, as it is very useful for many tissues that require slow hardening. A solution can be made much more quickly with warm water than cold. This fluid is also very useful to place portions of morbid material in, on their removal from the body in the post-mortem room; they can afterward be transferred to the chromic acid mixture for more rapid hardening. This solution takes from three to seven weeks to harden, according to the size of the specimen, and the frequency with which the fluid is changed.

5. BICHROMATE OF AMMONIA.-A 2 per cent. solution is used in precisely the same manner as the former, and is applicable to the same tissues.

6. When any tissue is obtained which it is thought desirable to examine for microorganisms, a small portion should be removed and placed at once in absolute alcohol; the remainder can then be hardened more slowly, so that the natural condition of the elements is not altered. Material hardened in chromic acid is not good for bacteriological research, as the acid interferes with the staining of the organism.

7. SILVER NITRATE.-Nitrate of silver possesses the property of forming a compound with the intercellular cement between the cells of serous membranes and other parts. For this purpose it is very

useful for demonstrating the existence of squamous epithelium in such situations as the surface of the mesentery, or the lining of blood vessels. A per cent. solution is used, made by dissolving 1 gramme of the salt in 200 c.c. of distilled water.

In the case of the mesentery, it is merely necessary to immerse it for two or three minutes in the solution, and then expose it to the light in distilled water for two or three days. It is necessary to change the water several times.

When the lining membrane of a blood vessel is to be demonstrated, or in any similar case where it would damage the part to remove it at once, the silver solution may be poured on it in situ, and after being allowed to remain for two or three minutes, washed off with distilled water, the tissue being afterward dissected out.

In making specimens of mesentery it is necessary to be careful that they should be spread out without being stretched, which will destroy the silver lines.

The whole mesentery may be removed with the intestine and stained in silver, and then hardened in spirit. Some portions of mesentery will then be found stretched out in a natural manner, and these can be cut out and mounted in Canada balsam solution.

They may with advantage be stained in a 5 per cent. watery solution of eosin or a strong solution of soluble aniline blue; in this way the structure will be brought out.

8. PICRIC ACID.-A saturated solution of picric acid will decalcify small bones. It is also used in some cases as a hardening agent by adding 1 part of water to 2 parts of a saturated solution; but it does not give such good results as the chromic acid mixture.

9. OSMIC ACID.-This can be procured as a 1 per cent. solution in water, and it is then diluted to various strengths as required. It blackens fat and the medullary sheath of nerves.

A piece of mesentery placed in a weak solution for half an hour will show the fat cells lying along the course of the blood vessels, as round black bodies.

It is also used for hardening the internal ear.

It is quite as important that pathological specimens should be properly hardened as normal tissues, but how seldom is this done. In the first place it is difficut to get the morbid tissues fresh enough, and yet they are often put on one side, or at most placed in the lump in a small quantity of spirit and water, and it is expected that good sections can then be prepared from them.

Nothing is more erroneous than this idea. The subject has been

dead probably twenty-four hours at the least when the post-mortem is made, often longer, and in summer especially this means utter ruin to many organs. How important is it, therefore, that these organs should be put in the hardening medium at once, when as fresh as possible. For this purpose a wide-mouthed bottle of Muller's fluid should be taken to every post-mortem examination, and small bits of any organ that may seem interesting on any account may be put in. A small paper label may be tied on, and they can be separated afterward.

They may with advantage be allowed to remain in the Muller's fluid for a week; they are then cut into small pieces and placed in the chromic acid mixture in separate bottles duly labelled. The same remarks apply to tissue removed by operation; this should be placed in the hardening fluid at once, without more handling than necessary.