If a more exact examination is desired, the tissues must be hardened and fixed. The material should be placed in the fluid used as soon as possible after it has been obtained. The point desired is that the conditions as they exist in the tissues during life shall be retained.

The different solutions vary greatly in their power of penetration and also in their effects upon different tissues. The action is facilitated by cutting the specimen in small pieces. After fixing and hardening it is generally necessary to thoroughly wash, so as to remove all traces of the agent employed.

The points to be observed are:

The specimens should not be more than 2 mm. in thickness.

The volume of reagent used should be from ten to fifteen times larger than the bulk of the specimen.

Place a layer of absorbent cotton or filter-paper in the bottom of the jar, so that the tissue may be acted upon by the fluid from all sides.

After sufficient hardening, remove the specimen and wash it in running water for twelve to twenty-four hours. It is then passed through alcohols of various strengths - 70, 80, and 90 per cent., about twenty-four hours in each.

1. Alcohol

It is used for rapid work and particularly if bacteria are suspected. It is not good for nervous tissue. Specimens should, as a rule, be put in weaker alcohol before being placed in absolute. This method is not used as much as formerly, on account of the shrinking and distortion of the tissues and the destruction of the red blood-corpuscles.

The so-called absolute alcohol is usually little more than 95 per cent. To extract the water, copper sulphate should be heated until the blue color disappears and then added to the alcohol. The alcohol should be filtered before using and the copper sulphate reheated when it begins to turn blue.

2. Formalin

This reagent is being used very greatly in place of alcohol. It has numerous advantages. The hardening takes place rapidly, the erythrocytes and other pigments retain their natural colors.

As formalin is bought it consists of a 40 per cent, solution of formaldehyd in water. The strength commonly used is a 1:10 or a 4 per cent, solution.

The tissues are left from four to six hours in the 4 per cent, solution, then thoroughly washed in water, and finally passed through alcoholic solutions of varying strengths.

Formalin is also used in combination with other mixtures, particularly as Orth's solution. This is made by adding 10 parts of formalin to 100 parts of Müller's fluid. This should be made fresh, as in the course of five or six days there is a crystalline precipitate formed. This fixes nuclear figures very well and hardens small pieces of tissue in from three to six hours. It is particularly important that they should be very carefully washed in running water. Is good for nervous tissues.

3. Miiller's Fluid

Miiller's Fluid is made up of:

Potassium bichromate....................... 2.5

Sodium sulphate............................ 1.0

Distilled water.............................. 100.0

This should be used in large quantities and should be changed every second day for about five times, and then be replaced whenever the solution becomes cloudy. To prevent the growth of mold 1 gm. of bichlorid of mercury should be added to 2 liters of the fluid.

For thorough hardening of small objects from ten to twelve weeks is required; for a large object, like the brain, a year. The process can be hastened by placing the preparation in an incubator and frequently changing the fluid.

After complete hardening the preparation is carefully washed in water, and then run through increasing strengths of alcohol. The sections stain well with hematoxylin and eosin. The red corpuscles are well preserved.

4. Erlicki's Fluid

Erlicki's Fluid consists of:

Potassium bichromate....................... 2.5

Sulphate of copper.......................... 0.5

Distilled water.............................. 100.0

This fluid has the advantage that preparations will harden in from eight to ten days; and if in the incubator, in from four to five days. Its disadvantages over Müller's fluid are that it does not prevent shrinking as well and that there is frequently a precipitate in the tissues.

5. Bichlorid Of Mercury

Bichlorid Of Mercury is of particular value in the fixation of cells and mitotic figures, but it has very little penetrating power. All the solutions that contain bichlorid have the drawback that there is a precipitation of mercury in the tissues that may be mistaken for pigment unless removed. These compounds may be dissolved by the addition of several drops of iodin to the 80 per cent, alcohol into which the specimens are put after having been washed. The iodin may be added to the alcohol in which the cut specimens are placed before being stained.

6. Zenker's Fluid

Bichlorid of mercury........................ 5.0

Potassium bichromate....................... 2.5

Sodium sulphate............................ 1.0

Distilled water.............................. 100.0

Glacial acetic acid........................... 5.0

The mercury and bichromate are dissolved in warm water and the sodium then added. It is best not to add the glacial acetic acid until the solution is ready to be used, as the acid rapidly evaporates.

After being in the fluid for twenty-four hours or less, according to the size of the specimen, it is thoroughly washed in running water twelve to twenty-four hours and then hardened in alcohol. The tissue should be passed through 80 per cent, alcohol containing iodin so as to remove the precipitate of mercury that forms.

Tissues prepared in this way stain according to all methods. The chromatin figures are well preserved as well as the erythrocytes.

7. Osmic Acid

Its penetrating power is very slight, so very thin pieces of tissue, not more than 5 mm. in thickness, can be used.

A 1 per cent, watery solution is usually employed. It should be kept in the dark, and when the specimen is fixed, well washed. The paraffin method of embedding should be employed, using chloroform or clove oil, as the celloidin will dissolve out the fat. In clearing, do not use xylol, as it also dissolves fat.

8. Flemming's Solution

Aqueous chromic acid solution (1 per cent.)....... 15

Aqueous osmic acid solution (2 per cent.)......... 4

Glacial acetic acid.............................. 1

The small bits of tissue are left in the fluid one to three days, well washed for several hours, then hardened in increasing strengths of alcohol. It is used for karyokinetic figures and for fat. Stains best with watery safranin.

9. Hermann's Fluid

Hermann's Fluid is a modification of the above. A 1 per cent, platinum chlorid solution is used instead of the chromic acid. The nuclear figures are especially well preserved. The method of employment is the same as with Flemming's.