Löwit's Formic-acid Method.-1. Place very small bits of fresh tissue in a mixture of formic acid I part, and water I to 2 parts, until they become transparent (a few seconds to several minutes).

2. Transfer to chlorid of gold, 1 to 1.5 parts to 100 of water, for fifteen minutes.

3. Formic acid, I part to water 3 parts, for twenty-four hours.

4. Concentrated formic acid twenty-four hours. Preserve in glycerin or balsam.

All the steps except the first should be performed in the dark.

Ranvier's Formic-acid Method.-1. Boil together 8 c.c. of a 1 per cent. solution of chlorid of gold and 2 c.c. of formic acid. When the solution is cold place very small bits of tissue in it for one hour, in the dark.

2. Wash quickly in water.

3. Expose to diffuse light in a mixture of formic acid 10. c.c. and water 40 c.c. Reduction takes place slowly (twentyfour to forty-eight hours).

4. Harden in 70 per cent., then 90 per cent., alcohol in the dark.

Osmic Acid (perosmic acid, osmium tetroxid) is used as a fixing reagent and for staining fat and myelin, by which it is reduced. As osmic acid is quickly reduced by organic substances, care must be taken in making up the solution. Remove the label from the sealed tube in which the acid comes, and place the tube, after cracking off one end, in a glass-stoppered bottle containing enough water to make a 2 per cent. solution. If desired, the tube can be broken after it is in the bottle by violent shaking. It should be borne in mind that osmic acid is very-irritating to the bronchial mucous membrane.

In a 1 or 2 per cent. solution osmic acid is used to stain fat in teased preparations or frozen sections of fresh tissues. In Marchi's method it is used to stain fat in tissues which have been hardened for some time in Müller's fluid. As a fixing reagent it is usually combined with other reagents, as in Flem

ming's solution, both for its property as a fixative and for the purpose of staining any fat present.

STAINING METHODS.

THE purpose of staining is to render prominent the different tissue-elements, so that they may be readily recognized and studied. The constant tendency now-a-days is toward selective or differential staining methods, by which but one. tissue-element will be colored to the exclusion of all others, or at least of any element that might be confused with it morphologically. These selective stains, which really are micro-chemical color reactions, enable us to differentiate from each other with ease and accuracy cellular and intercellular elements, or pathological products which otherwise look alike.

The list given on page 263 does not pretend to be either complete or perfect in arrangement, but will give some idea of the various elements which we wish to stain. Those for which we now possess more or less perfect differential stains are printed in italics.

The simplest selective stain is, of course, that for nuclei, and it can be obtained with a great variety of staining reagents. The most difficult element to stain differentially, although it can be done under certain conditions with a fair amount of success, is probably the axis-cylinder and its terminal processes.

Tissue-elements and pathological products differ from each other, not only in form and consistency, but also in chemical properties. While perfect preservation of form is sufficient to distinguish certain cells or elements from each other-as, for instance, polynuclear leucocytes from lymphoid cells-differentiation based on micro-chemical tests is always to be preferred when possible. A few of the tests employed are colorless, like the precipitation of mucin by acetic acid. Certain tests, like the methylene-blue or gold stain for axis-cylinders, can be applied to fresh tissues only.

Others, like the various amyloid reactions, can be obtained with fresh or hardened tissues. Most of the micro-chemical reactions, however, can be employed only with tissues which have been properly preserved. It is exceedingly important, therefore, that a tissue-element be so fixed and hardened that its peculiar chemical properties be preserved intact, otherwise a differential stain for it is impossible. Each tissue-element is a law unto itself. For example, the peculiar chemical properties of red blood-globules depend on the presence in them of hemoglobin. As a differential stain of the red blood-globules depends on fixing this substance in them, it is necessary to find out the chemical properties of hemoglobin, such as the fact that it is soluble in water or dilute alcohol, but not in salt solution, and that it is fixed in the red blood-globules by heat, absolute alcohol and ether equal parts, corrosive sublimate, formaldehyde, bichromate of potassium, etc.

While differential stains depend in part on the chemical properties of the tissue-elements, they also depend to a certain extent on the chemical properties of the staining reagents and the decolorizers used.

Some of the tissue-elements can be stained differentially in a number of ways, sometimes after one fixing agent, sometimes after another. The simplest differential stains are those where certain tissue-elements stain directly in a given solution after they have been properly fixed. Good examples are-Ehrlich's triple stain for certain protoplasmic granules in leucocytes, and the direct stain for elastic fibers with an acid alcoholic solution of orcein.

Other differential stains depend on the property of certain elements to hold colors they have once taken up when treated with decolorizers. The best example of this is the tubercle bacillus, which holds certain stains through various acids, or aniline hydrochlorate, followed by alcohol, and, if necessary, by a contrast-stain.

Still another varied group of elements (certain bacteria, fibrin, neuroglia-fibers, etc.) depends for a differential stain in part on changes produced in gentian- or methyl-violet by

iodin, in part on the decolorizer employed for extracting the coloring reagent.

Although the steps of the various staining methods differ considerably, they may be roughly arranged in the following order:

I. Staining.
2. Differentiating.
3. Decolorizing.
4. Dehydrating.
5. Clearing.

6. Mounting.

Very often two or more of the steps are combined in one, as when aniline oil is used for decolorizing, dehydrating, and clearing sections stained for certain bacteria. Sometimes the staining process occupies more than one step, as in Weigert's myelin-sheath stain. In alum-hematoxylin the differentiating reagent, the excess of alum, is combined with the stain; in Gram's method the differentiating reagent, iodin, forms a step by itself.

NUCLEAR STAINS.

For general histological work few stains are more valuable or can be more highly recommended than alum-hematoxylin, either alone or in contrast with eosin. Properly made and used, the solution stains the nuclei sharply and of varying degrees of intensity, depending on the character of the cells. Besides the nuclei, however, it stains. other tissue-elements in delicate shades of blue, so that they are readily visible, and thus more or less differentiated from those structures which fail to stain.

Of the carmine stains, lithium carmine, followed by picric acid, will be found the most brilliant, generally useful, and permanent.

Safranin gives, perhaps, the most permanent stain of any of the basic aniline dyes, and confines itself very sharply to the nuclei. It is much used after certain fixing reagents, such as Flemming's and Hermann's solutions. Eosin, followed by methylene-blue, gives beautiful results, especially when