Examination Of Fresh Material

The examination of fresh material may be made by teasing the tissue in water or, preferably, 0.6 per cent, saline solution. This, however, may not be satisfactory unless the tissue has been allowed to remain in some fluid long enough for the cells to become separated from the basement membrane. This is known as maceration; the following fluids are used for this purpose:

1. Alcohol, 33 per cent. (Ranvier), in which soak the specimen twenty-four hours.

2. Very weak chromic acid solutions, 1:10,000, or its salts. Müller's fluid is especially useful for nervous tissue. Leave in the acid twenty-four hours; in the latter, three to five days.

3. Osmic acid, 1 per cent., for twelve to twenty-four hours. Is useful if there is any fat present.

4. Potassium hydrate, 33 per cent., for from fifteen to twenty minutes. The specimen should be examined in the same fluid, as water distorts the cells. To preserve the tissue, wash in 50 per cent, acetic acid, then in water, and after staining in alum carmin can be mounted in glycerin. Is good for the examination of tissues or tumors that contain smooth, involuntary muscle-fibers.

5. Arnold's method: The small pieces of tissues are placed for five to ten minutes in 1 per cent, acetic acid, then for twenty-four to forty-eight hours in the weak chromic acid solution. They may finally be stained with picrocarmin.

Various reagents may be used in the examination of fresh specimens to render them transparent, to bring out certain details, or to cause various substances to disappear:

1. Glycerin clears the tissues and has the advantage of not changing chemically nor getting thin. Permanent mounts may be made by sealing the edges of the cover-glass with paraffin.

2. Potassium acetate in a saturated watery (50 per cent.) solution has a clearing action similar to, but less marked than, glycerin.

3. Acetic acid: Has the advantage that it causes the nucleus to shrink and the connective tissue to swell and become transparent. It does not affect fat, but dissolves the protein granules, so differentiates the two processes. Elastic fibers and micro-organisms are unaffected, so stand out prominently against the changed connective tissue. The acid may also be used to dissolve calcium salts. Solutions of 1 to 2 per cent, are generally employed, but the pure glacial acetic acid may be used.

A solution of acetic acid with fuchsin may be employed and in that way stain the nuclei.

4. Weak watery solutions of iodin. The following solution (Lugol's) is mixed with 3 to 5 parts of water:

Iodin........................................ 1

Potassium iodid............................... 1

Distilled water................................ 100

This brings the nucleus and the cell contour more plainly to view and also stains glycogen and amyloid particles brown.

5. Potassium and sodium hydrate solutions of from 1 to 3 per cent, have the power to dissolve most tissues, but do not affect elastic tissue, fat, bone, pigment, bacteria, or amyloid. Thirty-three per cent, solutions dissolve the cement substance and isolate the cells. This reaction takes place in a few minutes.

6. Osmic acid in 1 per cent, watery solution will stain fat black or brown.

7. Hydrochloric acid in from 3 to 5 per cent, is used for the recognition of lime salts, either in bone or in the tissues which it dissolves, with the production of bubbles of C02.

8. Fresh preparations may be stained by allowing a few drops of watery stains to pass under the cover-glass and then washing out the excess. Methyl-green, Loffler's methyleneblue, or acetic acid fuchsin may be used. Hematoxylin is unsuitable.