Double staining is employed for the purpose of obtaining a contrast between the nuclei and the plasms and interstitial substance. The nuclear stain is employed first, as the contrast stain is weaker and colors the tissues more diffusely.

Neutral Carmin

Carmin powder........................... 5 gm.

Aq. ammon. fort.......................... 1 c.c.

These rubbed together, then add:

Distilled water............................ 200 c.c.

Boil until the ammonia is driven off. Allow the solution to stand uncorked for about a week, then filter. The solution works better as it becomes older.

To prepare the stain for immediate use add just enough ammonia to the carmin to make a paste. This should be thinly spread on the sides of the mortar and allowed to dry. Pulverize again, let it remain exposed to the air for twenty-four hours, then dissolve in cold water; it is then ready for use.

To stain sections: Add the stock solution to distilled water until a clear pale red color results. The sections remain in this until they become plainly red, up to twelve hours. The best results are obtained by staining for a long time in a weak solution. Strong solutions stain more rapidly. Wash thoroughly in water, dehydrate, clear, and mount.

The counterstain best used is hematoxylin, and it should be employed first.

Eosin

Either the form soluble in water, which is the better, or that in alcohol may be used. A few drops of a concentrated solution of either variety is added to a small dish of water and the sections stained until they are of a reddish color - one to three minutes.

Then washed in water.

Dehydrated in alcohol. Should be careful not to leave in the alcohol too long, as it gradually dissolves out the stain.

Cleared and mounted.

This method is preceded by staining in hematoxylin. In such cases the nuclei are blue. In specimens fixed in formalin or sublime solutions the red blood-cells stain a bright red or copper color and the blood-vessels are prominent. Eosino-phile cells show up plainly. The other tissues show a diffuse reddish tinge.

Picric acid is generally used in combination with some other stain, as in Van Gieaon's method. As the picric acid decolorizes the sections, they should be overstained in the hematoxylin. If iron hematoxylin is used instead of Delafield's, the decolorization does not occur to the same extent.

Van Gieson's method for nervous tissue:

Aqueous sol. acid fuchsin (1 per cent.)........ 15 c.c.

Sat. aq. sol. picric acid...................... 50 "

Water.................................... 50 " mallory's anilin blue stain 287

For connective tissue:

Aq. sol. acid fuchsin (1 per cent.)........... 5 c.c.

Sat. aq. sol. picric acid..................... 100 "

Sections are:

1. Overstained in Delafield's hematoxylin.

2. Washed thoroughly in water.

3. Stained in Van Gieson solution three to five minutes.

4. Washed in water one-half minute.

5. Dehydrated, cleared, and mounted.

Nuclei are stained brownish red; connective tissue, varying shades of light red; axis-cylinders, brownish red; myelin sheaths, yellow; neuroglia and sclerosed fibers, red; amyloid, rose or reddish brown; hyaline, red; colloid, orange or red.