The principle of staining depends upon the different affinity of certain portions of the tissue for special dyes, so that they become more evident for purposes of study. There are certain stains which show a distinct affinity for the nuclei, while others select the cell protoplasm and the intercellular substance. By employing two stains a double coloring is obtained. In some conditions a single color may affect different portions of the tissue differently.

According to their reaction, stains are divided into the basic, which are commonly nuclear or chromatin stains, and the acid, those that affect the cell protoplasm or the intercellular tissue. Neutral stains are generally artificial combinations of some of the above two.

After being stained it is generally well to differentiate. Although a stain may be a nuclear one, yet there is usually some effect upon the other substances, the same holding true in regard to the acid stains. To remove this color, certain fluids are used, as water, weak solutions of acid in water or alcohol, alcohol, anilin oil, and tannic acid.

It is also necessary that the sections shall be rendered transparent, and this is brought about by placing them in xylol, carbol-xylol, oil of cloves, creosote, or bergamot.

Certain general rules should be observed:

1. All staining fluids should be filtered before use to avoid precipitates in the tissue. Good stains should be used; the best being those of Dr. Grübler, of Leipzig.

2. The sections should be spread out in the stain and should not lie upon each other, as the fluid is then likely to stain unevenly. Large amounts of stain in large dishes should be employed. It is also an advantage to carefully move the sections to and fro.

3. The time required for staining varies, as a rule being less in old, well-ripened stains than in others freshly prepared. This depends also upon the proper hardening and fixation of the tissue and also upon its age. Fresh tissues will stain more deeply and more quickly than old ones.

4. The staining of refractory tissues may be assisted by:

(a) Concentration of the stain.

(b) Staining for a longer time, up to twenty-four hours.

(c) Heating up to 370 C, delafield's hematoxylin 281

(d) Adding mordants, as acids and alkalis, anilin oil, etc.

5. The sections should be carefully washed in water to remove all traces of the decolorizing agents used.

6. Sections should be thoroughly dehydrated before being mounted; otherwise those areas containing water will not be transparent and will contain what appear to be oval pigment particles.

Method of staining and mounting sections.

1. Stain.

2. Wash, usually in distilled water.

3. Alcohol (80 per cent.) two to three minutes.

4. Alcohol (95 per cent.) three to five minutes.

5. Alsolute alcohol two to three minutes.

6. Clearing fluid until the specimen sinks below the surface, two to three minutes.

7. Place section on slide, blot off the excess of clearing fluid, and mount in balsam, using a cover-glass.