regards succedaneous staining, the rule should be followed to stain first with the nuclear stain and secondarily with the acid dye, an excess of which latter can be extracted with alcohol, 70-95%. Often the acid dye will act as an agent of differentiation on the basic, so that the after-treatment with alcohol will extract more of the latter dye.

Picric Acid.

We have already been acquainted with trinitrophenol as a fixing agent; it plays a much more important part as a stain. Picric acid is an excellent plasma stain; its main use is for double staining after carmine and hæmatoxylin. We must, however, always bear in mind, that being a monobasic acid, it will strongly attract the nuclear stains of our specimen, similar to hydrochloric and acetic acid. It is necessary, therefore, to overstain in the nuclear dye, if a good double staining is desired. It is used in a 1% watery solution, staining cell bodies, connective tissue, muscle, red blood cells intensively yellow in a few minutes.

Acid Fuchsin

is the alkaline salt of rosaniline-trisulpho-acid, the latter being prepared by treating fuchsin with fuming sulphuric acid. It is a dark red powder, easily soluble in water, not quite so readily in alcohol. The watery solution is instantly decolorized by an alkali.

Acid fuchsin has attained great importance in histology, since it forms a component of many important staining mixtures. It is also very serviceable for staining after nuclear staining has been accomplished, a 0.05–0.1% solution in water being very efficient for this purpose. A possible overstaining can be corrected by washing in hydrant water. It plays an especially important part in the demonstration of Altmann's bioblasts.

Bleu de Lyon

(Lyon blue) is made also by sulphurizing a basic dye and occurs as a dark blue powder, readily soluble in water, more difficultly in alcohol.

It is well adapted for afterstaining of carmine preparations, for which purpose it is used in a 0.1% solution. Staining is continued until the sections begin to assume a blue color, when we transfer them into 70% alcohol. In well fixed specimens we can demonstrate nerves and differentiate them from the surrounding connective tissue by this method.

Light Green.

Light green is a dark green powder, readily soluble in both alcohol and water. It is used in the same manner as the preceding.

Orange G.

Orange G. is a representative of that large group of dyestuffs known as azo colors, which are of great importance in commercial dyeing. It forms small yellowish red crystal plates, which are soluble in water and to some extent in alcohol. Orange G. is one of our best plasma stains. It is especially used after hæmalum staining, in a 1% watery solution; it stains in a few minutes. The washing is done in 70% alcohol.

Bordeaux R.,

also an azo dye, is a reddish brown powder, readily soluble in water.

It is made use of in a 0.2% watery solution, which must be applied before the nuclear staining takes place; it stains in about five minutes, after which specimens are washed briefly in water and, as previously stated (see p. 57), are subjected to hæmatoxylin-iron alum. The more the differentiation advances the more will the red color due to the Bordeaux reappear.

Eosin.

Eosin is the sodium salt of a color acid, tetrabromfluorescin. In commerce it is found as a red powder, easily soluble in water. When a mineral acid is added to the watery solution, the insoluble color acid will be precipitated.

Eosin is extensively used as a plasma stain, especially following hæmatoxylin. The simplest manner of using it is to make a concentrated solution of the preparation called "eosin yellowish," and diluting the same with ten to twenty times its volume of water. The thinner the solution, 'the longer the staining will last. After staining we wash in 70% alcohol. Eosin is of great value in the staining of blood and blood parasites.

Indifferent Stains.

Indifferent Stains

Such is the name applied to a small group of dyes, the most important properties of which are their relative solubilities. They are totally insoluble in water, dissolve with difficulty in alcohol, but are very readily soluble in fats. Chemically these dyes are indifferent, i.e., they possess no salt-forming groups. Under this group belong, for instance, sudan III and scarlet R. (Scharlach R.), both of which are azo dyes. They are reddish brown or red powders, difficultly soluble in alcohol.

Sudan III and Scarlet R.

for Fat Staining.

A concentrated solution is made in 70% alcohol, the frozen sections are taken from water and placed in 50% alcohol for a few minutes, then into the staining fluid for fifteen to thirty minutes, after which they are washed in water. Fat will appear intensively red, likewise the sheaths of peripheral and central nerves. This process may be combined with a nuclear stain, e.g., hæmalum, as well as with a plasma stain, e.g., picric acid. In such a case we first stain with hæmalum, wash in water, transfer into 50% alcohol and thence into sudan; the specimen is then washed again in water, stained in a watery picric acid solution and finally rinsed with water. In the after-treatment we must of course avoid strong alcohol and all solvents of fat.

STAINING MIXTURES

Staining Mixtures, Homogeneous

and Heterogeneous.

As we have previously observed, multiple staining (excepting metachro matic staining) can be accomplished by either using the various dyes in succes

sion or by staining with a mixture of the same. Three possibilities must be considered in the making of such a mixture: The component dyes may be basic, they may be acid, or they may partly be basic and partly acid. The first two varieties are easily prepared; such homogeneous mixtures are compounded of numerous basic and likewise a number of acid dyes. In the third variety, the heterogeneous mixtures, we are confronted with certain difficulties. If we mix the watery solutions of a basic and an acid dye, a reaction will take place, the color acid of the acid and the color base of the basic dye uniting to form a new dye, which we name neutral. These neutral dyes are generally insoluble in water, therefore a precipitate will be thrown down. We thus must expect a precipitate when mixing solutions of both acid and basic dyes. In order to bring such precipitate into solution we must employ other solvents, such as ethyl and methyl alcohol, acetone or methylal. Another way of procedure is to use an excess of the acid dye or to add an extra acid dye. What the nature of the solution is in the latter case cannot be stated with certainty, the probability, however, is that aside from the neutral dye it will also contain both the acid and the basic dyes in their original form.

Of the homogeneous mixtures only those of acid dyes are of interest to us; they are extensively used for afterstaining following nuclear dyes, in which case they will furnish an excellent differentiation between connective tissue and muscle. Sections stained with a heterogeneous mixture will show the chromatin of the nuclei, the basic substance of cartilage, mucus and the granules of mast cells in the basic colors, while all other elements will take up the acid dye or dyes, as the case may be.

Picrofuchsin.

Here we deal with a mixture of picric acid and acid fuchsin; it is prepared by mixing 45 cm3 of concentrated watery picric acid solution with 5 cm3 of a 2% solution of acid fuchsin. It is used after nuclear staining with hæmalum or iron-hæmatoxylin; it acts in five to ten minutes, the excess dye being extracted with 70% alcohol. Connective tissue will take on a bright red stain, while cell protoplasm and muscle will appear yellow.

Picro-Indigcarmine,

a solution of indigcarmine in picric acid, is prepared by dissolving 1 gm of the former in 300 cm3 of a concentrated watery solution of the latter. This mixture is suitable for the after-staining of carmalum, paracarmine and safranine specimens. In five to ten minutes it will impart a brilliant blue color to connective tissue, cell protoplasm, and muscle taking on a grass-green hue. After staining, the specimens are washed in 70% alcohol.

Picrocarmine.

Its preparation necessitates magnesia water, which we can make by adding well water to magnesia usta, leaving this mixture stand for eight days, vigorously shaking it from time to time, so that we can always be sure to have an excess of magnesia present. Pulverized carmine (0.2 gm) is boiled in 100 cm3 of magnesia water for half an hour in a flask loosely plugged with cotton. After cooling we filter and add a few drops of formalin. 100 cm3 of a 0.5% watery solution of picric acid with 0.15 gm of magnesia carbonate are also

heated to boiling, cooled and filtered. The two solutions combined will give us the stain ready for use. Five to ten minutes will suffice to have nuclei stained red, all other structures taking a yellow stain.

Azureosin Solution.

This stain, also known as "Giemsa solution," contains 3 gms of eosin and 0.8 gm of methylene-azure dissolved in 250 gms of glycerine and 250 gms of methyl alcohol. One drop of the solution in 1 cm3 of water will give a ready stain; its main use is for blood slides.

Biondi Solution.

This is the name given to a mixture containing one basic color, methyl green, and two acid dyes, acid fuchsin and Orange G. Its efficiency depends on the care taken in its preparation, which is as follows: The colors to be used should be ordered from the "Aktiengesellschaft für Anilinfabrikation" at Berlin, the following names being asked for: Methyl green N.M.P., acid fuchsin S.M.P., and Orange G.M.P. Of the former we take 3.4 gms, of the second 4.2 gms and of the third 3.0 gms; all three are placed in a small porcelain mortar and carefully triturated, so that a uniform powder results, which is dissolved in 100 cm3 of distilled water. If a permanent solution is desired, the container must be made of the very best Jena glass. The best plan is to use an Erlenmayer flask, place in it the dry powder mixture, add the prescribed amount of water, cork well and, shaking it repeatedly, let the mixture stand on the paraffin oven for two to three days. After this period complete solution will have taken place, resulting in a dark brown fluid with a slight red tinge. This stock solution, when properly taken care of, will keep for a long time, and before using must be acidified and diluted. The following procedure, though approximate only, is to be recommended as practically very efficient for acidulation. Place 1 cm3 of acetic acid in a 100 cm3 graduated tube and add distilled water up to the 100 cm3 mark, mix well and empty the glass; the traces of acid adhering to the wall will suffice for the acidulation. In a second smaller graduate 5 cm3 of the stock solution are placed, and rinsing thoroughly with distilled water the contents are transferred to the acidulated tube, adding enough rinsing water to bring the solution up to 50, and now our stain is ready for use; it will keep well for a few days. As regards the dilution, one need not literally cling to the stated proportion. As far as the material is concerned, we must differentiate between frozen and paraffin sections. In the former case nearly all fixatives will yield good results, e.g., formalin, the various corrosive sublimate mixtures, nitric acid, also chrom-osmium-acetic acid, if the specimens have not been subjected to its action for too long a period. Of the paraffin sections only those fixed in sublimate or its mixtures are to be recommended as suitable. Celloidin preparations are only then admissable, if the celloidin has been extracted prior to the staining. The duration of the staining has little influence on the results; ten minutes will suffice; again we may stain for twentyfour hours with impunity. The staining is followed by a short wash with 0.5% acetic and the specimens are transferred into 70% alcohol, where thick color clouds will be seen to emerge. The use of the alcohol must not be protracted for very long, but as soon as the section takes on a red color, it should be put

through absolute alcohol and thence transferred to xylol. The results obtained are most excellent. A bluish green is imparted to nuclear chromatin, basic substance of cartilage and mucus; a bluish violet to the mast-cell granules; a red color appears in the nucleoli, the homogeneous juice of the nuclei, the centrioles, spheres and achromatic spindles, the cell protoplasm, collagenous and elastic tissues, contractile substances, oxyphilic cell granules, basic substance of bone, uncalcified dentine. The hæmoglobin of the erythrocytes will appear orange-colored. The shading of the red is so fine as to differentiate all various elements among themselves. In facility and simplicity of execution and variety of applicability this method is not excelled by any other. One disadvantage of Biondi solution stands out against its many valuable properties, the fact that specimens prepared in such manner will not keep indefinitely. This hampering objection, however, may be remedied by strictly observing the rules for finishing and mounting microscopic specimens, as laid down on pages 76-79.

APPENDIX TO METHODS OF STAINING

THE IMPREGNATION METHODS

Essentials of Impregnation.

Metal impregnation is the term applied to a process in which we aim to saturate a block or a section of our specimen with a solution of a metal salt, the latter undergoing such reduction in certain parts of the tissue, which enables it to form colored compounds or to be changed to the metal itself, respectively. Thus the metal might be precipitated in these places as fine granules, or these certain tissue elements may appear distinctly colored, without any precipitate being formed. In the latter case a differentiation between impregnation and staining is impossible.

Metals Concerned.

Of the metals used for this purpose only the noble metals and of these only silver and gold will interest us. Another impregnation, that with osmic acid, has been discussed among the fixation methods and impregnation in that case, when applied to fresh tissues, naturally serves as a fixation as well, since the salts of the noble metals will all coagulate albumin.

The metal salt solution may be allowed to act on either the fresh specimen or the fixed specimen or finally on sections made from the latter. Each of these three methods will answer the purpose.

The Reduction

of the Metal Salt.

Reduction, the most important phase in the process of impregnation, may be induced without our aid by the tissue itself; ordinarily, however, we augment the process by the use of some aiding factors. One such factor is light— diffuse daylight or the direct rays of the sun. Under its influence the changes will take place much more rapidly and more intensely. Another such factor is found in acidulation; the reduction does not take place in distilled water, but