are stained blue. The nuclei are red if the decolorization is carried far enough. It can easily be watched under the low power of the microscope. The method is not always successful, especially with tissues which are old. It is sometimes advisable to increase the proportion of xylol in the decolorizing solution, so that the extraction of the color may not be so rapid. With paraffin sections fastened to the slide with egg-albumin use alum carmine as the contraststain, because the acid used with the lithium-carmine method will dissolve the albumin and loosen the sections. Besides the fibrin, certain forms of hyaline are often stained by this method. Fibrin can be stained by the above procedure in sections of tissues hardened in Müller's fluid if the specimens are first placed for several hours in a 5 per cent. aqueous solution of oxalic acid to reduce the bichromate of potassium.

Mucin. The term "mucin" is applied to a proteid substance having certain chemical reactions, and also to certain other substances which give the same reactions, but do not belong to the proteids. These various substances of secretory and degenerative origin cannot be distinguished microscopically, and have been investigated but little chemically. The reactions in common are the following: they dissolve in water to form a slimy fluid; they are precipitated from slightly alkaline solutions by acetic acid; the fresh precipitate dissolves in alkalies and in neutral salt solutions. Acetic acid, usually employed for this purpose in a 1 or 2 per cent. solution, precipitates mucin in the form of threads or granules. This reaction with fresh tissues has long been the main test for mucin. The acetic acid is drawn under the cover-slip by means of filter-paper placed at the opposite edge. The preparation should be mounted in water, not in salt solution, which may hinder or entirely prevent the reaction from taking place. Of late certain color reactions have become prominent. Mucin is coagulated into threads by alcohol or corrosive sublimate, and in this form can be stained by a number of staining reagents. Alum-hematoxylin under certain conditions will stain mucin. Accord

ing to P. Mayer, these conditions depend on a certain degree of ripeness of the solution, on the presence of enough alum to keep the nuclei from staining deeply, and, most important of all, on the absence of any free acid. This is difficult to manage, unless the solution is carefully neutralized, on account of the acid properties of alum. Mayer therefore recommends staining the sections in muchematein (see page 243).

Various aniline dyes have been recommended for staining mucin: those most favorably spoken of are methylene-blue (Orth), Bismarck brown (P. Mayer), thionin (Hoyer), polychrome methylene-blue (Unna), and toluidin-blue. The drawback to most of the aniline stains is that they are quickly extracted by the alcohol used for dehydrating. On this account P. Mayer highly recommends Bismarck brown, because permanent mounts can be easily made with it. It is not extracted by alcohol, and it does not fade in Canada balsam like many of the others.

Hardening in corrosive sublimate and imbedding in paraffin are generally recommended as preferable to hardening in alcohol and imbedding in celloidin. Stain sections for five to fifteen minutes in a rather dilute aqueous solution of the dye chosen. Of Bismarck brown use a saturated aqueous solution, and stain, if necessary, twenty-four hours. With thionin, toluidin-blue, and polychrome methylene-blue metachromatic stains are obtained; the mucin is colored red, the rest of the tissue blue. Two special methods for staining mucin are given in detail :

Hoyer's Method with Thionin.-Mucin, red; everything else, blue. 1. Harden in corrosive sublimate, followed by

alcohol.

2. Paraffin sections are passed through xylol, chloroform, and 95 per cent. alcohol to free them from paraffin, and are then placed in a 5 per cent. aqueous solution of corrosive sublimate for three to five minutes.

3. Stain in a dilute solution of thionin for ten to fifteen minutes.

4. Alcohol.

5. Clear in the mixture of the oils of cloves and thyme. 6. Turpentine oil or oil of cedar.

7. Balsam.

Before the staining the sections must not be treated with iodin solution to get rid of the precipitate of mercury, because it spoils the staining.

Unna's Method with Polychrome Methylene-blue.—I. Stain paraffin or celloidin sections hardened in alcohol in polychrome methylene-blue five to ten minutes or longer. 2. Wash in acidulated water.

3. Fix in 10 per cent. solution of bichromate of potassium half a minute.

4. Wash in water.

5. Dry on slide with filter-paper.

6. Decolorize in aniline plus I per cent. hydrochloric acid (a few seconds only).

7. Wash off with oil of bergamot.

8. Balsam.

Pseudo-mucin dissolves in water to form a slimy material, and is precipitated from its solutions by alcohol in threadlike masses which are again soluble in water. It is not affected by acetic acid. Pseudo-mucin is found in certain ovarian and other tumors.

Colloid and Hyaline.-The terms colloid and hyaline. are not yet sharply limited to definite chemical substances. The term colloid was originally applied to the homogeneous substance found in the thyroid gland, but has been broadened to include various substances of a similar appearance. The term hyaline is still more indefinite, but its use may be said to be applied most generally to those homogeneous substances which stain deeply with various stains, in contradistinction to those which like colloid show no marked affinity for staining reagents after ordinary fixatives.

Unquestionably, numerous substances of different chemical composition and of varying origin have been grouped under these two titles because of their physical and optical characteristics-namely, that they occur as glassy, refractive, homogeneous, occasionally colored gelatinous or firm masses.

Chemically, very little that is definite is known about them, and they possess no peculiar chemical reactions. Several attempts have been made to classify them in accordance with their reactions to various staining reagents.

Von Recklinghausen applied the term colloid to all the homogeneous, transparent-looking substances, including mucin, amyloid, etc., and reserved the term hyaline for a special group, which, according to him, is characterized by the following peculiarities: it resembles amyloid in physical characteristics, but does not react to iodin; it stains deeply with acid dyes, such as eosin and acid fuchsin.

Ernst has recently endeavored to differentiate two groups of hyaline substances, colloid and hyaline, by means of their reaction to Van Gieson's picro-acid fuchsin solution. According to him, true hyaline stains with acid fuchsin alone, and appears of a deep-red color, while colloid, of which the typical example is found in the thyroid gland, stains with both picric acid and acid fuchsin, so that it appears of an orange or yellowish-brown color. He has also tried to prove that all colloid is derived from epithelial cells, while all hyaline comes from connective tissue or from blood-vessels.

According to Von Kahlden, these differential staining reactions with Van Gieson's mixture claimed by Ernst for colloid and hyaline are by no means justifiable, because true colloid often stains a deep red. Furthermore, Unna has shown that in the skin connective-tissue cells can give rise to the so-called true hyaline, of which part is acidophilic and part basophilic, while the intercellular substance gives rise to colloid.

The last attempt to classify the various homogeneous substances on the basis of their reactions to dyes, apparently the only method possible at present, has been made by Pianese as a result of his studies of the various degenerative processes occurring in cancer-cells. He used a special fixative (see p. 225) and five different staining methods (see p. 250, methods III. A and B, IV., V., and VI.). Of these methods, III. B is the best, because it gives a characteristic color to each substance-hyaline, brick-red; colloid, bright

green; mucin, clear sky-blue; and a substance resembling amyloid, a dark reddish-violet. Besides these distinct reactions for colloid, hyaline, mucin, and a substance resembling amyloid, he found others less definite; one of these he calls pseudo-mucin and another pseudo-colloid. As a basis for his studies he took the reactions of amyloid, mucin (intestine), colloid (thyroid gland), and hyaline (hyaline remains of ovarian follicles, hyaline degeneration of renal glomeruli), with the same stains after fixation in his own hardening mixture.

The above brief historical statement is considered necessary to show the present views in regard to these various, more or less indefinite, homogeneous, transparent substances. For demonstrating them after the usual hardening reagents, of which alcohol and corrosive sublimate are perhaps the best, a double stain with alum-hematoxylin and eosin is very useful. Certain of the homogeneous substances stain deeply with eosin; others, like the transparent drops and masses occasionally found in the walls of the blood-vessels of the brain, stain with hematoxylin. Sometimes good results can be obtained with Weigert's fibrin stain or with carbol-fuchsin. The most generally useful stain, aside from alum-hematoxylin and eosin, is probably Van Gieson's mixture.

1. Stain deeply in alum-hematoxylin.

2. Wash in water.

3. Stain three to five minutes in a saturated aqueous solution of picric acid, to which is added enough of a saturated aqueous solution of acid fuchsin to give it a deep-red color. The effect of various proportions is sometimes useful. 4. Wash in water.

5. Alcohol.

6. Oleum origani cretici. 7. Balsam.

The transparent homogeneous substances usually stain. from orange to deep red in color; connective tissue, red.

Unna's Method for Hyaline and Colloid Material.-A. Harden in alcohol. 1. Acid fuchsin (2 per cent. aqueous solution) five minutes.