tion, 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 twelve 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 4 to 6 mm. 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 can be removed by means of iodin, which forms a colorless, soluble compound. For this purpose it has usually been advised to add iodin to the alcohol in which the tissues are preserved. Prolonged treatment, however, is required, and iodin exerts an injurious effect on the staining properties of the cells, so that in general it will be found best to embed the tissues and cut sections without removing the precipitate, and to treat the sections, just before staining, with Lugol's solution or a 1 per cent. alcoholic solution of iodin for ten to twenty minutes, followed by alcohol to remove the iodin. In old Zenker preparations the areas in the tissues occupied by the mercuric precipitate often stain with alum-hematoxylin, although the precipitate itself has been removed.

Zenker preparations stain slowly but beautifully in alumhematoxylin. Excellent results can also be obtained with eosin, followed by Unna's alkaline methylene-blue solution. Excellent results are also obtained after staining in phosphotungstic-acid hematoxylin, and by the anilin-blue method. They bring out fibrin and various kinds of fibrils in addition to nuclear details. Fuchsin and safranin stains are sometimes useful.

Corrosive Sublimate.-Use a saturated solution (made

by heat) in normal salt solution. The addition of 5 per cent. of glacial acetic acid is usually advisable. I. Harden thin pieces of tissue (2 to 5 mm.) for six to twenty-four hours; 2. Wash in running water twenty-four hours; 3. Preserve in 80 per cent. alcohol.

Tissues hardened in corrosive stain quickly and brilliantly in nearly all staining solutions. It is the only fixative after which the Heidenhain-Biondi triple stain gives good results.

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, but the histological detail is not so good as after Zenker's fluid. The addition of 5 per cent. of acetic acid would unquestionably improve it.

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.,

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. 286).

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

Formaldehyde.-The gas formaldehyde (HCOH) is soluble in water to the extent of 40 per cent. Solutions of this strength are manufactured by different commercial houses under the names of formaline, formol, and formalose. The best strength of formaldehyde to use for fixing tissues is a 4 per cent. solution; that is, 10 parts of the aqueous 40 per

cent. solution, no matter what name is given to it, to 90 parts of water. The addition of 5 per cent. by volume of glacial acetic acid to this solution is said to improve its fixing properties.

This fixing reagent penetrates very quickly. Its hardening action is not understood. It does not precipitate albuminous bodies, but makes them quite firm. It also hardens nerve-sheaths, acting toward them and red globules like the chrome salts. Formaldehyde is very useful for preserving gross specimens, because it gives them a rather tough elastic consistence and preserves the normal color better than other hardening fluids and also the transparency of many parts, such as the cornea. Tissues, such as muscles or the contents of a multilocular cystoma, which have been made very hard by the fixing reagent used, can be softened even when mounted in celloidin, so that they can be cut perfectly by placing in a 4 per cent. solution of formaldehyde.

In histological work it is chiefly useful in the preservation of nervous tissue and as a fixing reagent for tissue from which frozen sections are to be cut, because it improves the cutting qualities of the tissue. As a fixative for specimens that are to be imbedded in paraffin or celloidin it is not recommended unless combined with other reagents, such as bichromate of potassium in Orth's fluid, because it does not appear to harden the tissue elements sufficiently to enable them to resist the shrinking effects of prolonged exposure to alcohol and other fluids in the process of imbedding. In frozen sections, however, prepared by the method described below, this shrinkage of the tissue elements is not apparent, probably because prolonged exposure to dehydrating, clearing, and imbedding agents is avoided.

Wright's Method for Frozen Sections.-This method has given such satisfactory results as a histological procedure, and has proved, after an exhaustive testing in practical work, to be such a saver of time, labor, skill, and expense in obtaining satisfactory sections adequate for most routine purposes, that it seems destined to replace extensively the celloidin and paraffin methods. The essential feature of

the method is the fastening of the section to the slide immediately after cutting, thus keeping even the most fragile or irregularly shaped sections intact and smooth during the processes of staining, dehydrating, and clearing which are carried out on the slide. The fixation of nuclei and cells is superior to that effected by alcohol, and there is much less shrinkage than in sections prepared by the usual methods. Inasmuch as the success of the method depends to a considerable extent upon the frozen sections being as thin as good celloidin sections, special attention is called to the points given on p. 236 in regard to the technique of frozensection cutting. The method is as follows:

1. Fix pieces of the tissue not more than 1 cm. thick in 4 per cent. aqueous solution of formaldehyde for fifteen to twenty-four hours. Thicker masses of tissue should be soaked longer. The tissue may remain indefinitely in the formaldehyde solution. If the tissue is in small fragments or is too soft and friable to yield coherent sections, it may be imbedded in gelatin, as described on p. 272.

2. Trim the piece of tissue with a knife in such a manner that it will present a thickness to be frozen of not more than 5 mm. The other dimensions of the piece of tissue may be as large as the freezing box of the microtome will accommodate.

3. Rinse the piece of tissue for a few seconds in water, and place it on the freezing box of the microtome with a few drops of water beneath it; then freeze and cut frozen sections, giving careful attention to the directions for cutting such sections set down on p. 236. The sections should not be over 10 or 15 microns in thickness.

4. Float the sections off the knife into water, and select a good one. Spread this section smoothly on a slide. This is easily done by passing the slide into the water and under the section, while at the same time the section is manipulated by means of a small glass rod drawn out to a small probepointed extremity. The slide should be free from grease. 5. Drain off superfluous water.

6. Press the section upon the slide with a piece of smooth