CHAPTER XXV.
STAINS AND STAINING METHODS.—NUCLEAR
AND PROTOPLASMIC STAINS.
THEORIES OF STAINING. The exact nature of the process of staining has not yet been determined. Various theories have been advanced, explaining the affinity of the tissues for certain dyes, on the ground of a chemical, mechanical or chemicophysical action. The chemical theory assumes the formation of an insoluble compound through the chemical combination of tissue and stain; the physical theory is based upon the assumption that the process is purely physical or mechanical, while the chemicophysical theory holds that it is neither purely physical nor purely chemical. The process is not controlled by the molecular weights alone of the substances concerned, but does depend upon the conditions controlling the formation of solutions in general. Therefore, the theory most widely accepted at the present time is the solution-theory, which assumes that the staining-process is a solution of the dye in the tissue, and that the stained tissue-element is a fixed solution of the stain in its substance. This solution of the dye in the tissues may be a direct action between the two (direct or substantive stains); or it may be brought about only by the interaction of a third substance (indirect or adjective stains). The third substance is called a mordant, and the combination of the dye with the mordant is known as a lake. The mordant may be added to the stain or to the tissue, either before or at the time of staining. Many of the fixing-fluids are mordants, particularly those containing chromic acid or its salts. Alum, iron, and many of the metals are the most commonly used mordants. In a general way acid mordants are used for basic colors, and basic mordants with acid colors.
The stains most commonly used in pathologic work are:—
1. Natural Dyes:—Hæmatoxylin and Carmine.
2. Aniline Dyes:—a, Acid.—Eosin, erythrosin, acid fuchsin, orange G, picric acid, sudan III, and scarlet R (Fett-ponceau).
b, Basic.—Methylene blue, methylene violet, thionin, toluidin blue, kresyl-echt-violett, methyl violet, gentian violet, crystal violet, basic fuchsin, dahlia, aniline blue, methyl green, iodine green, safranin, Bismarck brown, and vesuvin.
In a general way it may be said that basic stains are nuclear stains, and acid stains are protoplasmic. Neutral stains are usually diffuse stains; when formed by the combination of acid and basic dyes they usually act as selective stains for some especial tissue-element or cell-constituent. Metachromasia, in its narrowest sense, is the term applied to that staining-phenomenon, in which a single-chemical entity gives different colors to different tissue-elements. In this sense iodine, in its action upon glycogen and amyloid, is a true metachromatic substance. The majority of the so-called metachromatic stains, however, do not possess true metachromasia, since their metachromatic powers are dependent upon a mixture of two or more dye-stuffs in the one compound. The most important stains of this class are gentian violet, methyl violet, crystal violet, dahlia, thionin, toluidin-blue, polychrome methylene blue, methylene azure, and kresyl-echt-violett. The chief chromotropic substances are amyloid, mucin, mast-cell granules and cartilage. Metachromatic reactions are at their best usually when examined in water; they are affected by alcohol and usually destroyed by carbol-xylol. Sections stained by metachromatic stains should be quickly dehydrated by absolute alcohol and blotting, and cleared in xylol, when mounted in balsam.
I. NUCLEAR STAINS.
1. Haematoxylin (C16H14O6) is an ether extract of the wood of Hæmatoxylon campechianum, a tree found in the West Indies and Central America. In itself not a dye, it becomes one of the most valuable when oxidized to hæmatein (C16H12O6), and combined with alum, iron or other mordants to form a lake. It then stains nuclei a deep violet-blue or black color that is practically permanent. Mucin, lime-salts, bacteria, and colonies of actinomyces are also stained varying shades of blue. If the staining process is prolonged the entire tissue, as well as celloidin, becomes more or less heavily stained blue. A pure nuclear stain is obtained by interrupting the stain at the right time (examine in water); or if the sections are over-stained they may be differentiated in acid alcohol (1 per cent hydrochloric acid in 70 per cent alcohol). Hæmatoxylin stains well after all fixing-solutions except osmic acid; some of its staining-formulæ stain slowly after fixation in Zenker’s fluid. On the whole, it is by far the best general nuclear stain for laboratory and diagnostic work. It is employed in numerous staining formulæ, the most useful of which are given here. These formulæ differ chiefly in the time of staining, “ripening” of the stain (oxidation of hæmatein), intensity of stain, necessity of differentiation, etc.
a. Böhmer’s Alum-haematoxylin.
Dissolve 5 grms. of hæmatoxylin crystals in 50 cc. of absolute alcohol; add this drop by drop, while stirring, to 1,000 cc. of a 1 per cent solution of potassium alum. Expose in open vessel to air and light for 1-2 weeks. Filter before using.
b. Hansen’s Haematoxylin.
To 200 cc. of alum-hæmatoxylin solution brought to the boiling-point add 2 cc. of a concentrated solution of potassium permanganate. Cool quickly; filter when cold. Can be used at once without further ripening. It tends to stain diffusely.
c. Delafield’s Haematoxylin.
To 400 cc. of a saturated solution of ammonia alum add a solution of 4 grms. of hæmatoxylin in 25 cc. of absolute alcohol. Expose mixture to air and light for 3-4 days; filter; then add 100 cc. of glycerin and 100 cc. of 95 per cent alcohol, and filter. Expose to light until solution is dark enough, then keep in tightly-stoppered bottle. It is a strong stain, and may be diluted with distilled water when desired. The solution keeps well.
d. Ehrlich’s Acid-haematoxylin.
Dissolve 2 grms. of hæmatoxylin in 100 cc. of absolute alcohol. Add this to a saturated solution of potassium alum in water 100 cc., glycerin 100 cc., and glacial acetic acid 10 cc. Allow mixture to stand for a week exposed to air and light; then filter. Keep in well-stoppered brown bottles. The solution stains best when it is six months old, and may be kept for several years. It does not overstain, and on the whole is more useful than Böhmer’s, Hansen’s or Delafield’s.
e. Mayer’s Haemalum.
Dissolve 1 grm. of hæmatein in 50 cc. of 90 per cent alcohol and warm. Add this solution to a solution of 50 grms. of potassium alum in 1,000 cc. of distilled water dissolved by heating. Mix warm, cool, and filter. With hæmatein no ripening is required, and the solution can be used at once. Hæmalum is a more precise nuclear stain, but stains more slowly than the formulæ given above.
It may be prepared directly from hæmatoxylin crystals by dissolving 1 grm. of hæmatoxylin in boiling water; add water up to 1 litre, and cool. Add 0.2 grm. sodium iodate and 50 grms. potassium alum, dissolving at room temperature; 50 grms. chloral hydrate and 1 grm. citric acid may be added to make solution keep better.
f. Mayer’s Acid-haemalum.
Add 2 cc. glacial acetic acid to 100 cc. of hæmalum solution. It stains the nuclei more precisely than hæmalum.
g. Weigert’s Iron-haematoxylin.
Dissolve 1 grm. of hæmatoxylin in 100 cc. of 96 per cent alcohol. Allow to ripen several days, but this solution should not be kept longer than six months. Make a second solution of 4 cc. of liq. ferri. sesquichlor. (German Pharmak. IV, sp. gr. 1,124), 1 cc. of concentrated hydrochloric acid and 100 cc. of water. Mix equal parts of each solution just before staining. The mixture stains well for 5-8 days, so that a quantity of stain sufficient for this time only should be made up. The two stock solutions are easily made and keep well. The nuclei stain quickly and deeply, and differentiation and long washing are unnecessary when hydrochloric acid is included in the second solution, as given above. After-staining with eosin, picric acid or the Van Gieson’s mixture gives better results with Wiegert’s iron-hæmatoxylin than with any other hæmatoxylin.
Method of Staining with Haematoxylin.
1. Stain 1-15 minutes, controlling progress of stain by examination of section in water, on slide, using low-power.
2. If sections are over-stained, differentiate in ½-1 per cent potassium-alum or in acid alcohol.
3. Wash thoroughly in tap water, until a good blue is obtained. Exposure to ammonia vapor or washing for a few seconds in lithium-carbonate solution will hasten the development of the blue color. If these reagents are used the section should afterwards be thoroughly washed in water.
(Stain with a plasma stain, if contrast is desired.)
4. Dehydrate in 80 and 95 per cent alcohols.
5. Clear in carbol-xylol.
6. Mount in balsam.
Over-ripened hæmatoxylins may stain reddish or even brownish, and too diffusely. In such cases the celloidin will be deeply stained. The addition of alum-water to the stain may counteract the fault. Alum-hæmatoxylins must always be filtered before using, as precipitates are constantly formed as the result of oxidation.
2. Carmine. Carmine is the coloring matter of cochineal, the dried bodies of the female coccus cacti, and is obtained chiefly from Honduras. The coloring principle is carminic acid (C2H22O12). When combined with alum, borax, lithium, etc., carmine gives a good, permanent nuclear stain, varying from reddish violet to deep scarlet. It is used chiefly in pathology to give a contrasting nuclear stain to the various pigments, and when specific blue stains have been used for fibrin, mucin, bacteria, elastic tissue, etc., or when a blue injection-mass has been used. Alum-carmine is the most precise nuclear stain. Differentiation with acid-alcohol is necessary after staining with borax- or lithium-carmine. Lithium-carmine is on the whole the best of the three for use as a contrast-color to the various pigments.
a. Alum Carmine.
Carmine ½-1 grm., 1-5 per cent alum solution 100 cc.; boil 20 minutes; cool; filter. Add crystal of thymol as preservative.
1. Stain 15 minutes to several hours.
2. Wash thoroughly in distilled water.
3. Dehydrate in 80 and 95 per cent alcohols; clear in carbol-xylol: mount in balsam. Nuclei are a light reddish violet; the plasma is slightly stained (muscle) or not at all.
b. Lithium Carmine.
2-5 grms. of carmine to 100 cc. of a cold saturated water solution of lithium carbonate; filter.
1. Stain 1-3 minutes; transfer directly to acid alcohol (1 cc. of hydrochloric acid to 100 cc. of 70 per cent alcohol) without putting section into water; differentiate ¼-6 hours, until nuclei alone retain the color. Control differentiation by examination on the slide in acid alcohol.
2. Wash thoroughly in water.
(Use plasma stain, as picric acid, if desired.)
3. Deyhdrate in 80 and 95 per cent alcohols; clear in carbol-xylol; mount in balsam.
c. Borax Carmine.
Dissolve by boiling 0.5-0.75 grm. carmine and 1-2 grms. of borax in 100 cc. of water. To the hot solution add 5 cc. of a O.5 per cent acetic acid until solution is deep red. After 24 hours filter and add crystal of thymol.
Stain and differentiate as with lithium-carmine, but leave the sections somewhat longer in the stain (15 minutes).
3. Basic Aniline Stains. The basic aniline stains are used as general stains for bacteria in sections, and at the same time stain the nuclei. Methylene blue and fuchsin are employed especially for this purpose. The metachromatic dyes (thionin, kresyl-echt-violett, etc.) are also used as nuclear stains in combination with their metachromatic reactions with mucin, amyloid, mast-cells, etc. Methylene blue is used in the study of the blood-forming organs, cell-inclusions, parasites in the tissues, etc. Safranin and fuchsin are used for staining mitotic figures. (See Staining of Mitoses.) Bismarck brown is employed sometimes in preparing sections for microphotography. Methyl green is an intense chromatin stain and is used in various combinations. Since these dyes are not very permanent, and are easily washed out in dehydrating and clearing fluids, as well as “running” in balsam mounts, they are rarely employed as pure nuclear stains in pathologic work.
When the basic-aniline stains are used a saturated water or concentrated alcoholic solution (1½-2 per cent in 40 per cent alcohol) may be employed as stock-solution and diluted 1:5 or 1:10, as desired. The sections are stained 3-5 minutes, then differentiated in absolute alcohol, cleared in xylol, and mounted in balsam. Methylene-blue is used by some workers as a nuclear stain contrasted with eosin for tissues fixed in Zenker’s solution, giving better effects with this fixation than the hæmatoxylins. Unna’s alkaline methylene-blue formula is employed (methylene-blue 1 grm., carbonate of potassium 1 grm., water 100 cc.). Dilute 1:10 or 1:5 for staining. Stain 10-15 minutes, wash quickly in water, differentiate in 95 per cent alcohol; dehydrate in absolute, clear in xylol, mount in balsam. When celloidin sections are used, 95 per cent alcohol may be used for dehydrating, blotting on the slide with several changes of xylol.
The nucleolus has an affinity for the acid stains. With the modifications of the Romanowsky methylene-blue-eosin methods the nucleolus stains red, the nucleus blue.
II. DIFFUSE OR PLASMA STAINS.
The most commonly used diffuse stains are eosin, erythrosin, acid fuchsin, orange G, and picric acid. They are practically never used alone, but are employed as contrast-stains to the nuclear stains. Eosin, orange G, acid fuchsin and picric acid may be used as counterstains for hæmatoxylin; picric acid is used as the best contrast to the carmines, while eosin and orange G are employed as counterstains for methylene blue. The combination of hæmatoxylin and eosin is by far the best general staining method for laboratory and diagnostic work, except for tissues fixed in Zenker’s solution; for these the combination of methylene blue and eosin is preferable. Ammonia carmine is sometimes used as a diffuse stain for bone and the central nervous system. The majority of the diffuse stains wash out easily in water, alcohol and xylol, hence sections thus stained should not be allowed to remain too long in these fluids.
a. Eosin. Two forms of eosin are obtainable, one soluble in water, the other in alcohol. Saturated solutions of both kinds should be kept as stock solutions and diluted as occasion demands. For use after hæmatoxylin a ½ per cent solution is advisable; with Zenker’s fixation a more dilute solution may be used, as tissues so fixed stain intensely in eosin. If used as a contrast-stain to methylene-blue, eosin is used first in a 5 or 10 per cent solution, as the basic nuclear stain takes out some of it. Some workers express a preference for the aqueous solution of eosin, others for the alcoholic; the alcoholic solution stains more uniformly and with less differentiation than the other. Eosin is particularly good as a contrast-stain for tissues containing red blood cells when fixed in formol, mercuric chloride or Zenker’s.
b. Orange G. Used in a 1 per cent water solution. Requires longer time for staining than eosin.
c. Acid Fuchsin. A saturated water solution is kept in stock and diluted as needed. Must be used in weaker solutions than eosin, as it more quickly overstains, and cannot be washed out so well.
d. Picric Acid. Keep in stock either a saturated water or saturated alcoholic solution, and dilute as needed. As it washes out more readily than eosin, the staining solution should be stronger than for the latter, and the sections somewhat over-stained to allow for some loss of stain. Picric acid gives a brownish tint to nuclei stained with hæmatoxylin or carmine, and will take out the stains completely, if allowed to act too long.
e. Ammonia Carmine. One grm. of carmine is dissolved, without heating, in 50 cc. of distilled water and 5 cc. of strong ammonia water. The fluid is then exposed in an open dish until the odor of ammonia is lost; it is then filtered. When ready for use dilute by filtering 1-2 drops into 20 cc. of distilled water.
III. COMBINED NUCLEAR AND DIFFUSE STAINS.
The diffuse or plasma stains may be combined with the nuclear in one staining solution, or used in succession. The latter method gives better results. The nuclei are stained first, the diffuse stain being used after the washing-out following the use of the nuclear stain, except in the methylene-blue and eosin method in which to obtain the best results it is necessary to stain with the eosin first. Nuclear hæmatoxylin may also be followed by a combination of plasma stains, as in the Van Gieson’s mixture of picric acid and acid fuchsin, Delépine’s mixture of rubin and orange, White’s erythrosin and picric acid mixture, etc. In these mixtures the different affinities of the plasma-stains give rise to differential or selective staining effects.
a. Haematoxylin and Eosin.
1. Stain in any one of the hæmatoxylins.
2. Wash thoroughly.
3. Stain in dilute water or alcoholic eosin until section is bright rose-red.
4. Differentiate eosin-staining, as desired, by rapid or slow washing in water.
5. Dehydrate quickly in 80 and 95 per cent alcohols.
6. Clear quickly in carbol-xylol. (If carbol-xylol takes out eosin too rapidly add some of the dry eosin stain to it. Hæmatoxylin-stained sections can be placed in such eosin-carbol-xylol and will take up the eosin beautifully.)
7. Mount in balsam.
Hæmatoxylin and eosin can also be combined in one stain, but the results are not as good as those obtained by successive staining.
b. Haematoxylin and Picric Acid.
1. Stain with Weigert’s iron-hæmatoxylin, or overstain with any other hæmatoxylin.
2. Wash thoroughly.
3. Stain in saturated water solution of picric acid, diluted one-half, until sections are a bright yellow. If left too long in the stain, the hæmatoxylin will become brown or may be wholly lost.
4. Wash, dehydrate and clear quickly, as for eosin. (Dry picric acid may be added to the carbol-xylol.)
5. Mount in balsam.
c. Haematoxylin and Acid Fuchsin.
Stain with hæmatoxylin, and after washing use a 1 per cent water solution of acid fuchsin, until section is sufficiently red; wash; dehydrate; clear; mount.
d. Haematoxylin and Orange G.
Stain with hæmatoxylin, and after washing use a 1 per cent water solution of orange G, staining ¼-3 hours. Treat otherwise as for eosin-staining.
e. Carmine and Picric Acid.
Stain with borax- or lithium-carmine; differentiate in acid alcohol and wash thoroughly. Then counterstain with picric acid, as for hæmatoxylin and picric acid. Carmine and picric acid may also be combined in one stain, as picro-carmine, but this is rarely used at the present.
f. Eosin and Methylene-blue.
(For tissues fixed in mercuric chloride or Zenker’s.)
1. Stain in a 5-10 per cent aqueous eosin for 20 minutes or longer, until a deep eosin-stain is obtained.
2. Wash out excess of eosin in water.
3. Stain in Unna’s alkaline methylene-blue, diluted 1-4 or 5 with water, 10-15 minutes.
4. Wash in water.
5. Differentiate in 95 per cent alcohol, keeping the section in constant motion to obtain a uniform decolorization. Control process under microscope. Wolbach advises the use of a 0.75-1.5 per cent solution of colophonium in methyl alcohol as a differentiating medium instead of 95 per cent alcohol. For tissues fixed in formol or alcohol a 10 per cent solution should be used.
6. When the background is pink, dehydrate quickly with absolute alcohol, or in 95 per cent by blotting on slide with xylol, until clear.
7. Clear in xylol.
8. Mount in balsam.
g. Van Gieson’s Method.
1. Stain in Weigert’s iron-hæmatoxylin, or overstain if any other hæmatoxylin is used. (Weigert’s gives the best results, as it does not decolorize so readily.)
2. Wash thoroughly.
3. Stain in Van Gieson’s mixture (acid fuchsin 1.5 grms., saturated water solution of picric acid [0.6 per cent] 150 cc. This mixture keeps well. Add 1 cc. of this stock solution to 10 cc. of saturated water solution of picric acid. Stain in this for 10 seconds).
4. Wash quickly; dehydrate in alcohol; clear in xylol or carbol-xylol; mount in balsam.
I have obtained the best results by making the Van Gieson’s mixture by taking an ordinary small staining-dish nearly full of saturated water solution of picric acid, and adding to this, drop by drop, sufficient saturated water solution of acid fuchsin to make the solution just dark enough so that the finger cannot be seen through the staining-dish. The hæmatoxylin-stained section is put into this mixture for a few seconds, until it appears to become lighter. The section is then washed in 95 per cent alcohol, dehydrated, cleared and mounted.
The Van Gieson method is extremely valuable in pathologic work, because of its varied differential reactions. The nuclei are brown or black, protoplasm is ochre-yellow, connective-tissue light red, voluntary and involuntary muscle yellow, axis-cylinders red, connective-tissue hyalin deep rose-red, epithelial hyalin yellow, orange or brownish, amyloid yellow or brownish pink, mucin yellow or brownish, fibrin yellow or brown, necrotic areas yellow or brownish, lime-salts brown to brownish blue or violet.
Combinations of rubin and orange (Delépine) and erythrosin and picric acid (Powell White) are also advised as differential combination stains, but are not so useful as Van Gieson’s. Other combination methods are to be found in the various modifications of the Ehrlich triple stain.