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= (C_{16}H_{14}O_{6}) 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 (C_{16}H_{12}O_{6}), 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 (C_{2}H_{22}O_{12}). 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.