in a large volume of the fluid. If chromic acid be used, the bone must be first hardened in this fluid. Place it in .1 per cent. chromic acid for twenty-four hours, renew the fluid, but use .2 per cent., and after a week use .5 per cent.; shake the vessel from time to time, to bring new fluid into contact with the tissue.

If a more rapid process is desired, after the bone has been two or three days in dilute chromic acid (.2 per cent.), use the chromic and nitric acid fluid. Decalcification requires about fourteen days. To test for the removal of all the salts, push a needle into the bone, or make a section with a blunt razor. Obstruction in either case denotes that the bone has not been sufficiently decalcified. In most cases, the bony tissues should be hardened before they are decalcified. This is specially the case in connection with bone softened in chromic acid. It is better to harden them first in Müller's fluid, and then to decalcify them in chromic and nitric acid fluid (p. 36).

Bone decalcified in chromic acid must be thoroughly washed in running water for many hours to remove all the chromic salts, and is then transferred to 70 per cent. spirit and kept in the dark, otherwise there will be a copious deposit. Renew the spirit, and transfer the tissue to strong alcohol, still keeping it in the dark.

If a bone is to be softened in picric acid, it may be placed at once in this fluid, with the precaution indicated at p. 37. It need not be kept in the dark, but it is better to remove as much as possible of the yellow stain by means of alcohol. It decalcifies somewhat more slowly than chromic acid.

VIII. METHOD OF PREPARING TISSUES AND ORGANS FOR MICROSCOPICAL EXAMINA

TION.

As most of the tissues require to be hardened, and it is frequently impossible to obtain human tissues sufficiently fresh, recourse must be had to the fresh tissues of animals. As frequently as possible, however, human tissues should be secured. Most of the tissues may be obtained from a cat, rabbit, or guinea-pig, and for certain special purposes the dog, frog, newt, and salamander are used.

The cat, rabbit, or guinea-pig-or, better, all three-are killed. by chloroform. The animals are placed in an air-tight box-a large saucepan does very well-along with a sponge saturated with chloroform. Small animals may be chloroformed under a bell-jar. As soon as the animal is dead, open the thorax by a longitudinal incision through the costal cartilages-right and left--raise the

sternum, expose the pericardium, open it, and with a pair of scissors make a snip into the right auricle of the heart, and allow the animal to bleed freely.

It is best to begin by removing the brain and spinal cord. They are hardened in Müller's fluid or potassic bichromate (2 per cent.), and must be placed in a large volume of fluid. A few spinal ganglia should also be found and hardened in the same way.

Remove the trachea and lungs, and fill the lungs and trachea of the rabbit with a per cent. solution of chromic acid.

This is readily effected by tying a funnel into the trachea and pouring in the fluid. By squeezing the lungs gently much air is forced out, and the fluid gradually runs into and distends the lungs, which, when distended, are placed in a large volume of the same fluid. The chromic acid and spirit mixture may be used instead of pure chromic acid.

Fill the windpipe and lungs of the guinea-pig with per cent. silver nitrate. (See Lungs.)

Remove the heart, and harden it in alcohol, after washing away any blood with normal saline.

The central tendon of the diaphragm may be preserved for silvering. (Lesson IV.)

The omentum and mesentery, if desired, are silvered. IV.)

(Lesson

Open the abdomen, remove the liver, cut it into small pieces; harden some pieces in Müller's fluid, and others in spirit. Take out the tongue and oesophagus; harden them in Müller's fluid.

Open the stomach and intestine, wash away any food residues by means of normal saline. Harden part of the stomach-cardiac and pyloric-in absolute alcohol, other pieces in Müller's fluid, and others in corrosive sublimate, and small pieces in osmic acid. (See Lesson on Stomach and Intestine.)

The duodenum and small and large intestine are hardened in the same way, although the bichromate and chromic acid mixture (p. 29) is particularly good for the small intestine.

The salivary glands and pancreas are removed and hardened by the methods given under these headings, i.e., small pieces are placed in each of the following solutions :-Absolute alcohol, osmic acid, corrosive sublimate, &c.

Remove the lower jaw, cut it into short pieces, place it in .2 per cent. chromic acid for a few days, and then decalcify it in chromic and nitric fluid. This will yield sections of softened tooth.

Remove the kidneys, cut one longitudinally and the other transversely. Using the kidneys of different animals, harden pieces of each in the following fluids:--Müller's fluid, chromic acid and spirit,

ammonium chromate, and corrosive sublimate. Other methods of preparing the kidney are referred to. (Lesson on Kidney.)

The bladder is best hardened in chromic acid and spirit mixture, or in Müller's fluid.

Harden the spleen, without cutting into it, in Müller's fluid.

The suprarenals may be hardened in picro-sulphuric acid. (Lesson on Suprarenal Capsules.)

Small lymphatic glands from the region of the neck or submaxillary region are hardened in alcohol, while others are injected with silver nitrate and osmic acid. (Lesson on Lymphatics.)

If desired, the large nerve-trunks may be removed and hardened as indicated in Lesson on Nerves, or the smaller branches of nerves may be used for showing the effects of the action of certain reagents on nerve-fibres.

Remove some of the long bones, leaving in each case the periosteum attached to the bone. Cut the bones into pieces about inch long, and place them for a week in per cent. chromic acid, and then decalcify them with picric acid, or chromic and nitric acid fluid, or Ebner's fluid. (Lesson XIII.)

In every case decalcify the ends of the bones, so as to have a section which will demonstrate the relation between the articular cartilage and the osseous tissue.

Place small pieces of striped muscle in per cent. chromic acid, and other pieces in alcohol.

Nerves, with the precautions given in Lesson on Nerves, are hardened in osmic acid, potassic bichromate (2 per cent.), alcohol, or picric acid.

For the methods of hardening the eye, ear, nose, see the Lessons on these subjects.

The testis-very small pieces-is best hardened in Flemming's mixture, and larger pieces in Müller's fluid.

For the methods of hardening the ovary, Fallopian tube, and uterus, see the Lessons on these subjects.

N.B.-Label every bottle, and write on the bottle the name of the hardening fluid used, and the dates on which it was changed.

I X.-EMBEDDING.

This is necessary for many tissues; the piece of tissue may be either too small to be conveniently held in the hand, or its parts may tend to fall asunder before or after they are cut.

There are two methods, one simple embedding, where the tissue is simply fixed or placed in another medium to hold it while it is

being cut, and the other interstitial embedding, where the substance used for the embedding process is made to penetrate into the interstices of the tissue.

A. Simple Embedding.-1. The tissue may be clamped between two pieces of carrot, scooped out to receive it, or in elder pith, or (what is very convenient) between two pieces of amyloid or waxy liver hardened in alcohol.

2. Paraffin. - It is sometimes desirable to surround the tissue with paraffin or some such medium. The embedding medium should be about the same degree of hardness as the tissue.

Two paraffins are required, a hard paraffin melting at 60° and a soft one at 45° C. For use they may be mixed as follows :

:

Two parts of the hard paraffin and one of the soft yield a mixture which cuts well when the temperature of the room is 21° C. (70° F.), but a softer paraffin is easily made by mixing two parts of the hard paraffin with one part of chrisma or vaseline. The mixture can be made softer by the addition of a little more vaseline, and harder by adding more paraffin. The paraffin mixture is heated on a water-bath or sand-bath until it melts, but its temperature is raised as little as possible above its melting-point. It is convenient to melt it in a porcelain dish with a wooden handle. The tissue is removed from alcohol, the surplus alcohol removed by wiping it with blottingpaper, until the surface is dry. It is then placed in melted paraffin, and retained in it until the paraffin solidifies. The melted paraffin can be run into embedding boxes of paper (fig. 30), or the embedding L's may be used (p. 44), but this simple method is now but rarely used. It has been almost entirely displaced by the following method.

B. Infiltration Method or Interstitial Embedding.

1. Embedding in Gum.-The tissues after being hardened must have all their alcohol removed by prolonged soaking in water. They are then transferred to gum mucilage, or a mixture of gum and syrup, in which they can be preserved until they are required for freezing, if freezing is to be the process used for cutting the sections.

Tissues saturated with and embedded in gum mucilage may be hardened in alcohol and then cut. The sections are placed in water, which dissolves out the gum.

2. Saturation with, or Infiltration with, and Embedding in Paraffin Interstitial Embedding. In this case the embedding

medium is made to penetrate into the tissue, and when it sets, it thus supports all its component parts. This method is extremely valuable, especially for brittle and friable tissues, and is largely used. Moreover, the tissues once embedded can be kept in a box, each duly labelled, for any length of time.

Make a mixture of two parts of hard paraffin and one part of soft; place the mixture in a small copper pan or capsule in a hotair oven, kept at a constant temperature by means of a gas regulator. The gas supply must be so arranged that the thermometer

FIG. 29.-Mayer's Paraffin Embedding Bath, as made by Jung of Heidelberg.

steadily registers at most 1° C. above the melting-point of the paraffin. Or the paraffin may be melted and kept melted in a little copper vessel, placed in a hot-water bath, kept at a constant temperature, as shown in fig. 29. The temperature is kept constant by means of a gas regulator, R; Z is for filling the instrument with water; a, b, c, are embedding vessels and pots.

The tissues to be saturated and embedded should not be large, and they must be thoroughly dehydrated; keep them, therefore, several hours in absolute alcohol. Place them direct into turpentine, creosote, benzol, toluol, or xylol-some use chloroform, bu