3. Stain in Unna's alkaline methylene-blue solution (see page 246), diluted 1-10 with water, for one half to several hours.

4. Wash in water.

5. Differentiate and dehydrate in absolute alcohol until the pink color returns in the section.

6. Xylol.

7. Xylol balsam.

For celloidin sections use 95 per cent. alcohol, clear in origanum oil, and wash out with xylol.

It is important to get a deep stain with eosin, because the methylene-blue washes it out to a considerable extent. The eosin must be used first, because methylene-blue is readily soluble in an aqueous solution of eosin, and therefore is quickly extracted if the eosin is used after it, while on the other hand eosin is very slightly soluble in an aqueous solution of methylene-blue which is precipitated by any excess of eosin.

Diffuse or contrast-stains are useful to make prominent certain of the tissue-elements left uncolored by the nuclear stain. A greater richness of detail is obtained with diffuse stains if, after rather deep staining, the sections be washed out for some time in alcohol, because certain structures possess a greater affinity than others for certain diffuse stains, and by holding them are brought out sharply.

Of the diffuse stains, eosin, picric acid, and acid-fuchsin in Van Gieson's mixture are the ones most frequently employed.

Eosin is most frequently used as a contrast to alum-hematoxylin stains, but is often serviceable with alum-cochineal, methylene-blue, gentian-violet, etc. It brings out particularly well red blood-globules and smooth and striated musclefibers. The strength of the solutions used after hematoxylin varies from toper cent. according to the tissue and the fixative used. Zenker's preparations stain intensely in eosin, so that for them a very dilute solution is advisable. When desired as a contrast-stain to basic aniline dyes, eosin should be used first in a 5 per cent. or even saturated solu

tion, because otherwise it is likely to be washed out by the nuclear stain.

Picric acid is used for contrast with the carmine stains, more rarely with alum-hematoxylin. Striated muscle-fibers and cornified epithelium are rendered especially prominent by it. To stain with picric acid it is only necessary to add a few drops of a saturated aqueous solution to a dish of water, or of a saturated alcoholic solution to a little alcohol, and allow sections to remain in the solution for a few seconds.

Van Gieson's stain (see p. 249), a mixture of picric acid and acid fuchsin, is excellent as a contrast-stain to alumhematoxylin, especially when it is desirable to render prominent connective-tissue fibrillæ or certain pathological products. The nuclear stain with alum-hematoxylin must be rather deep, because the picric acid to some extent extracts or overpowers it.

1. Stain deeply in alum-hematoxylin.

2. Wash in water.

3. Stain in Van Gieson's solution three to five minutes.

4. Wash in water and dehydrate directly in

5. Alcohol, 95 per cent.

6. Oleum origani cretici.

7. Canada balsam.

Neutral Carmine (see page 245).—Neutral carmine is a diffuse stain, and is employed more especially for the central nervous system and for bone.

Filter one or two drops of the solution into 20 c.c. of distilled water, and leave the sections in the dilute solution over night. It is advisable to place a piece of filter-paper on the bottom of the dish for the sections to rest on, otherwise they may be stained on the upper side only. In double stains. with hematoxylin and carmine the sections should be stained first in the hematoxylin and then thoroughly washed in water for six to twelve hours before they are stained in the carmine. After the carmine they are again to be thoroughly washed in

water.

Combination Stains.-Biondi-Heidenhain Stain (see p. 250).-Tissues must be hardened in corrosive sublimate.

1. Stain paraffin sections six to twenty-four hours with the dilute solution.

2. Wash out a little in 90 per cent. alcohol.

3. Dehydrate in absolute alcohol.

4. Xylol.

5. Canada balsam.

It is important to place the sections directly from the staining fluid into the alcohol, because water washes out the methyl-green almost instantly.

Staining in Mass.-The staining of tissues in mass is a procedure much less employed in pathological than in normal histology, but still occasionally useful. For pathological tissues a variety of stains is generally necessary. It is therefore much better to make a series after one of the methods described, and then to stain the sections in whatever way seems best.

For staining in bulk only a limited number of solutions are available-either those, like alum-carmine and alumcochineal, which do not stain beyond a certain point, or those, like lithium and borax-carmine and Heidenhain's hematoxylin, which may be decolorized so as to leave only the nuclei stained, The process of staining differs from that for sections only in the length of time required for each step. Tissues cm. thick will need from one to two days in the staining solution.

MITOSIS.

For the study of karyomitosis it is important that the tissue be perfectly fresh-that is, just removed from a living animal or from one just dead—and that it be fixed in a suitable reagent as quickly as possible. The best results cannot be obtained with tissues put into a hardening fluid over half an hour after removal from a living animal. On the other hand, mitotic figures can be demonstrated in tissues which have been dead for some time (twenty-four hours or more) before being put into a fixing reagent, but the details of the figures are not so perfect as those in perfectly fresh tissues, and the figures are not so numerous, because some

of them have completed their changes and can no longer be recognized. It is therefore evident that mitosis can be studied much better in tissues from the lower animals, or in tissues removed by operation from the human body, than in the organs and tissues removed at post-mortem examinations.

The choice of fixing reagents for the study of mitotic figures is important. The figures can often be demonstrated after hardening in alcohol or even in Müller's fluid, but for their careful study quicker and more perfect fixing reagents must be used. Nearly all of the reagents employed penetrate slowly, so that it is absolutely necessary for the best results that the tissue to be hardened be cut into very thin slices, rarely over 4 mm. in thickness and preferably not over 2 mm. The amount of fixing reagent used should always be at least ten to fifteen times as great as the volume of the tissue, and should be changed if it becomes cloudy. The most important fixing reagents are―

1. Flemming's solution.

2. Hermann's solution.

3. Pianese's solution.

4. Zenker's fluid.

5. Corrosive sublimate.

6. Orth's fluid.

The first three solutions penetrate with much difficulty, so that tissues placed in them should be especially thin. The most generally useful stain for mitosis is probably safranin. The time of staining varies with the solution used. Babes' is the quickest. The mitotic figures should be stained deeply then, when treated with alcohol slightly acidulated with hydrochloric acid, they will retain the color, while the resting nuclei will yield up most of theirs and become very pale or even colorless. In consequence of this intense stain mitotic figures can then be very readily found.

Directions for Staining Karyomitotic Figures with Safranin.-1. Stain paraffin sections five minutes to twentyfour hours, according to solution used.

2. Wash in water.

3. Wash in 95 per cent. alcohol to which are added a few drops of acid alcohol.

4. Wash in pure 95 per cent. alcohol, followed by absolute alcohol.

5. Xylol.

6. Xylol balsam.

For celloidin sections, clear in oil of bergamot or oleum origani cretici after the 95 per cent. alcohol, wash out in xylol, and mount in xylol balsam. Safranin can be used after any of the above fixing reagents.

Other useful stains are carbol-fuchsin and aniline-gentianviolet, used in the same way as the safranin. The GramWeigert method gives good results after Flemming's solution.

After fixing in corrosive sublimate mitotic figures can be demonstrated by the Biondi-Heidenhain solution, which stains resting nuclei blue-violet and mitotic figures green. After Pianese's solution his special staining mixtures should be used (see page 250). His methods are said to give beautiful results.

THE STAINING OF BACTERIA IN TISSUES.

Bacteria are demonstrated in sections of tissues almost entirely by means of the aniline dyes, of which three have thus far proven themselves to be particularly valuable— namely, methylene-blue, gentian-violet, and fuchsin. These dyes are employed in aqueous or dilute alcoholic solutions, of which the effective staining power is greatly increased by means of heat and by the addition to the solutions of certain chemical substances.

The effect of moderate heat is obtained by placing the sections in the incubator for several hours, or greater heat for a short time is utilized by warming the staining solution on the slide over a small flame for a few seconds or minutes, keeping the fluid steaming, but not allowing it to boil.

Of the various methods employed to increase the staining power of aniline dyes by means of chemical substances, the most successful have been the use of caustic potash with