mounting. Most fixatives are mixtures of different reagents so combined that all the desirable properties may be present in as large a degree as possible.

The choice of the proper fixing reagent for a given tissue is often difficult, and must depend largely on the nature of the pathological lesions present or suspected, and on the purposes for which the tissue is preserved. For diagnosis, for general bacteriological study of tissues, and for many valuable and important chemical reactions alcohol is to be preferred to any other reagent. Although by no means an ideal fixative, it will be found exceedingly useful.

For finer histological study it is important to preserve valuable tissue in some more perfect fixative than alcohol. Zenker's and to some extent Orth's fluid will be found the most generally useful. Flemming's solution is especially to be recommended for the study of renal lesions with fatty degeneration. For general pathological work, aside from the nervous system, these four fixatives will be found the most valuable.

It is strongly advised that pieces of tissue in all important cases be hardened both in alcohol and in Zenker's fluid— in alcohol for bacteria and for chemical reactions; in the other for bacteria, nuclear figures, blood, and general histological study. For special investigations other fixatives are sometimes desirable.

Tissues fixed in alcohol or in a solution of formaldehyde may remain as long as desirable in those fluids. Tissues hardened in most of the other fixatives must be transferred, after thorough washing in water, to alcohol for preservation. It is usually recommended to pass the specimens through graded alcohols, either through 30, 60, 90, and 96 per cent., or through 50, 70, and 96 per cent., allowing them to remain from a few hours to a day in each strength. For most purposes it will be found sufficient to transfer the specimens directly from water to alcohol of 70 to 80 per cent., in which they may remain until it is desired to imbed them.

Alcohol extracts chrome salts from tissues hardened in solutions of them. As these salts are precipitated in the alcohol under the action of light, it is desirable, although by no means necessary, to keep all such specimens in the dark.

Alcohol. The strength of alcohol ordinarily used in laboratories is 95-96 per cent. Absolute alcohol is much more expensive. Tissues hardened in either of these strengths shrink a great deal. The exposed surface becomes extremely hard, and the outer layers of the cells of tissues like a rabbit's kidney, for example, are as shrunken and flattened as though dried in the air. It is only inside of this hard casing, where the alcohol has penetrated more slowly and has been somewhat diluted by the fluid of the tissue, that the cells are better preserved. Moreover, this extreme hardening of the surface hinders the penetration of the alcohol into the deeper parts.

Tissue which is to be hardened in absolute or 95 per cent. alcohol should be cut into thin pieces, preferably not over 1⁄2 cm. thick. The volume of alcohol used for hardening should be fifteen to twenty times as great as the specimen, and should be changed after three or four hours. The tissue should be kept in the upper part of the alcohol by means of absorbent cotton, or the jar may be frequently inverted and the alcohol thus kept of even strength.

The advantages of strong alcohol, 95 per cent. and absolute, are that the tissue is more quickly fixed than with weaker strength, and that at the same time it is made quite hard a quality more necessary formerly than now when tissues are so generally imbedded. Tissues hardened in strong alcohol should later be transferred to 80 per cent. alcohol for preservation or the staining properties will gradually become impaired.

For general purposes it will be found better to place tissues. at first into 80 per cent. alcohol, which should be replaced in two to four hours by 95 per cent. alcohol. In this way less shrinkage is caused and the surface of the tissues is not made so hard.

The solution is practically Müller's fluid saturated with corrosive sublimate, plus 5 per cent. of glacial acetic acid. It is advisable not to add the acetic acid to the stock solution, but only in the proper proportion to the part taken for hardening pieces of tissue, because the acid evaporates so readily.

Directions for Use.-1. Fix tissues in the solution one to twenty-four hours, rarely forty-eight hours, according to thickness.

2. Wash in running water twelve to twenty-four hours. 3. Preserve in 80 per cent. alcohol until used.

Tissues float at first in this solution, which will be found a most admirable general fixative. It penetrates quickly, so that pieces of tissue do not need to be so thin as with most other fixatives, but it is advisable not to let them exceed cm. in thickness. Nuclear figures, red blood-globules, and protoplasm are all perfectly preserved. The greatest drawback to the fluid is the precipitation of mercury which takes place to a varying degree in the tissues. This precipitation may be removed by adding a little tincture of iodin (up to per cent.) to the alcohol in which the specimens are preserved. As soon as the color of the iodin disappears, on account of its forming a colorless, soluble compound with mercury, more iodin must be added until the alcohol remains stained faintly yellow. It will sometimes be found difficult to get rid of all the deposit even after fairly prolonged treatment (two weeks) with iodin in this manner, so that it is sometimes necessary to treat sections, after cutting, with Lugol's solution for a short time. Sometimes, indeed, it is preferable to reserve the treatment with iodin for the sections only.

Zenker preparations stain slowly but beautifully in alumhematoxylin. Excellent results can also be obtained with eosin, followed by Unna's alkaline methylene-blue solution. Fuchsin and safranin stains are sometimes useful.

Orth's Fluid. Recently Orth has highly recommended as a general fixative a solution consisting of the well-known Müller's fluid plus 4 per cent. of formaldehyde:

The formaldehyde should be added only at the time of using, for in two days the solution becomes darker, and by the fourth day a crystalline deposit begins to take place. As fixation is ordinarily complete in three to four days, this deposit does not matter. The tissue should not be over I cm. in thickness. Small pieces to cm. in thickness can be readily hardened in the incubator in three hours. The specimens should be washed thoroughly in running water six to twenty-four hours before placing in 80 per cent. alcohol.

The method is particularly recommended for mitosis, red blood-globules, bone, and colloid material (in cystomata, etc.), as it gives a very good consistency to the tissues. Flemming's Solution.

Osmic acid, 2 per cent. aqueous solution,
Chromic acid, I per cent. aqueous solution,
Glacial acetic acid,

4;

15;

I.

I. Fix in the solution one to three days. 2. Wash in running water six to twenty-four hours. 3. Alcohol, 80 per cent.

It is best to keep the osmic acid in a 2 per cent. solution, and the chromic acid in a 1 per cent. solution. The mixture can then be quickly made up fresh at the time it is needed. The best stains after hardening in Flemming are Babes' safranin, aniline-gentian-violet, and carbol-fuchsin.

Pieces of tissue for hardening in Flemming's solution should not be over 2 mm. in thickness, because it has very slight penetrating properties.

Hermann's Solution.

Osmic acid, 2 per cent. aqueous solution,

4;

Platinic chlorid, I per cent. aqueous solution, 15;
Glacial acetic acid,

I.

This modification of Flemming's solution is, perhaps an even better fixative than the model on which it is based, but is more expensive. It should be employed in the same manner. Pianese's Solution.—

Chlorid of platinum and sodium, I per cent.

aqueous solution (platinic),

Chromic acid, per cent. aqueous solution,
Osmic acid, 2 per cent. aqueous solution,
Formic acid, C. P.

15 C.C.

5

5

I drop.

Fix small pieces of tissue, not over 2 mm. thick, in the solution for thirty-six hours. Wash in running water for twelve hours, then 80 per cent. alcohol. Stain paraffin sections by Pianese's special methods (see p. 250).

This fixative and the special staining methods are particularly recommended for the study of karyomitosis and of the so-called cancer bodies.

Rabl's Chromo-formic Acid Solution.

Chromic acid, 0.33 per cent. aqueous solution, 200;
Formic acid,

4 to 5 drops,

to be added just before the solution is used.

Directions for Use.-1. Harden in the fixing solution twelve to twenty-four hours; 2. Wash in running water twelve to twenty-four hours; 3. Dehydrate in 80 per cent. alcohol.

Rabl used after this fixative a very faint stain with hematoxylin, followed by safranin.

Corrosive Sublimate.-Use a saturated solution (made by heat) in normal salt solution. The addition of 5 per cent. of glacial acetic acid is sometimes advisable. 1. Harden thin pieces of tissue (2 to 5 mm.) for one to six hours; 2. Wash in running water twenty-four hours; 3. Preserve in 80 per cent. alcohol.