Chapter XXVIII

THE STAINING OF SPECIAL ORGANS AND TISSUES.

I. BLOOD AND BLOOD-FORMING ORGANS.

The =blood= may be examined by means of _films_, _stained_ or _unstained_, or by _sections_, _celloidin_ or _paraffin_.

_A. FILMS._ The blood may be obtained from the pulp of the ring finger, from the skin over the knuckles, or from the posterior aspect of the lobe of the ear. The place selected should be carefully cleansed with water, soap and 1/1000 mercuric chloride solution, and finally with alcohol and ether. A puncture is made with a sterilized triangular needle or knife, or a stub-pen with one point broken off. The last-named makes a most useful and inexpensive instrument for this purpose. The puncture should be made by a quick and deep stab, so that sufficient blood can be obtained from one stab-wound. Pressure should not be employed to force blood from the wound. Bleeding may be encouraged by letting the arm hang down, or by applying pressure in the furrow of the terminal joint of the finger. The first drop of blood should be wiped away with a clean towel. When the second drop reaches the size of a pin-head touch it with the under side of a perfectly clean cover-glass, held by forceps, not by the fingers; place this cover-glass immediately upon another clean cover, so that the blood will spread out between the two covers in a thin film. The covers are then separated by sliding them apart without pressing or squeezing; place covers with film side upward, and dry in the air. The films should not be touched with the hands; forceps alone should be used to handle them. If the blood does not dry as quickly as it is spread the film will be too thick. Films may be made upon slides in the same way, or the drop of blood may be caught upon the edge of a clean cover, slide or “spreader” and then drawn rapidly across a slide. The dried film may be marked by scratching with a needle-point the number and date on the film itself. Blood-films may be fixed _without drying_ by exposure to the vapor of formol or osmic acid for several seconds and then dropping into absolute alcohol. Formol alcohol, saturated mercuric chloride solution or Flemming’s solution may also be used for the fixation of wet films, fixing for 5-10 minutes, and washing thoroughly after each of the last two solutions. The _dried film_ may be fixed by exposure to heat (110-115°C.) for 5-10 minutes for Ehrlich’s triple stain, and for 2 hours for the methylene-blue-eosin methods; 30-60 seconds at a temperature of 120°C. may suffice; the film should be brought at once into the required temperature. Heat-fixed films are improved by dipping them for a few minutes in mercuric chloride solution and then washing well before staining. _Acetone-free methyl alcohol_ (1-2 minutes), _absolute alcohol and ether in equal parts_ (½-12 hours), _formol-alcohol_ (1-2 minutes), _alcoholic mercuric chloride_ (absolute alcohol 25 cc., ether 25 cc., 5 drops of a 2 grms. mercuric chloride solution in 10 cc. of absolute alcohol) for 2-5 minutes, and _formol-vapor_ are the chief solutions used for fixing the air-dried film. For ordinary work methyl alcohol, formol alcohol, and the absolute-alcohol and ether mixture give good results; heat fixation brings out the granules well, and mercuric chloride is a good fixative for the leukocytes. The combination of fixation and staining, as in Leishman’s or Wright’s modification of the Romanowsky method, is also recommended for general work.

For the _staining of blood-films_ an almost endless variety of staining-methods can be found in the literature. Many of these represent slight deviations in the method of making the stain or in its application, such deviations marking stages of improvement in the development of the method. It is not necessary, therefore, to give all of these methods, but to consider only the latest modifications of value. In a general way _blood-stains_ may be divided into five classes:—

1. =HAEMATOXYLIN AND EOSIN.=

Fix in equal parts of absolute alcohol and ether for at least
30 minutes; stain with hæmalum and eosin, or Ehrlich’s acid
hæmatoxylin and eosin. By adding O.5 grm. of eosin to the formula
for Ehrlich’s acid hæmatoxylin a combination stain can be made that
is very good for blood-films fixed by heat or absolute alcohol and
ether. Stain 2-24 hours, wash, dry and mount in xylol balsam.

2. =EOSIN AND METHYLENE-BLUE.=

Fix by formol (dried film over 40 per cent formol for 1 minute);
absolute alcohol for 1 minute; stain 5 minutes in a 1 per cent
watery eosin; then without removing eosin place in watery
methylene-blue for 2 minutes; wash quickly; dry in air; balsam.

3. =MIXTURES OF EOSIN AND METHYLENE-BLUE.=

The numerous mixtures of methylene-blue and eosin are not very
stable, can be kept for a few days only, and give varying results.
_Jenner_ improved this method of staining greatly by collecting the
precipitate formed by the addition of eosin to methylene-blue, and
dissolving it in pure methyl alcohol, thus giving a solution that
fixes and stains at the same time. The _May-Grünwald_ method is
practically the same.

Jenner’s Method.

a. Water-soluble eosin, 1.25 grms.
Distilled water, 100 cc.
b. Medicinal methylene-blue, 1 grm.
Distilled water, 100 cc.

Mix equal parts of _a_ and _b_ in an open basin, stirring with
a glass rod. Let stand for 24 hours; filter; dry the residue at
50°C. Wash residue thoroughly with distilled water and again dry
thoroughly. Take 0.5 grm. of the dried powder and dissolve in 100
cc. of pure methyl alcohol. Filter. Solution keeps well.

1. Make blood-film. Dry in air. Do not fix.

2. Cover film with stain, keeping under watch-glass to prevent
evaporation Stain 2 minutes.

3. Wash in distilled water until the film has a pink color. Dry
in air. Mount in xylol-balsam.

Neutrophile granules are red, eosinophile rose red, basophile
granules violet, red blood cells and central portion of
blood-platelets are terra-cotta, leukocyte nuclei and granules in
red blood cells are blue, protoplasm of nuclei and outer portion of
platelets light blue.

4. =MODIFICATIONS OF THE ROMANOWSKY METHOD.=

A large group of stains has resulted from various applications
of the Romanowsky idea of uniting equimolecular proportions of
methylene-blue and eosin, and the solution of the dyes so obtained
in some suitable solvent. These dyes consist of mixtures of
methylene violet, methylene azure, eosinate of methylene blue,
etc., and can be obtained from Grübler and Co. under various names,
such as _Azur-blau_, _Bleu Borrel_, _Giemsa’s stain_, _Leishman’s
stain_, etc. _Hastings_, _Leishman_, _Wright_ and others have
combined the Romanowsky method with that of Jenner by dissolving
the new dyes obtained by their various modifications in pure methyl
alcohol, so as to form a solution that will fix and stain at the
same time. _Hastings’ stain_ is a modification of _Nocht’s stain_;
_Wright’s stain_ is a modification of the _Leishman-Romanowsky_
method. The revised directions given by _Wright_ for making and
using his stain are here given. Wright’s method and the Giemsa
stain possess all of the staining advantages afforded by the
variations of the Romanowsky method, and are alone given here. The
former is recommended for blood-work, the latter for the staining
of protozoa.

Wright’s Blood-stain.

To a 0.5 per cent aqueous solution of sodium bicarbonate add
methylene-blue (B.X or medicinal) in the proportion of 1 grm. of
the dye to each 100 cc. of the solution. Heat the mixture in a
steam sterilizer at 100°C. for one full hour, counting the time
after the sterilizer has become thoroughly heated. The mixture
should be placed in a flask of such size and shape that the
fluid will not be more than 6 cm. deep. After heating, allow the
mixture to cool, placing the flask in cold water if desired, and
then filter it to remove the precipitate. When cold the fluid
should have a deep purple-red color when viewed in a thin layer by
transmitted yellowish artificial light. It does not show this color
while warm.

To each 100 cc. of the filtered mixture add 500 cc. of a 0.1 per
cent aqueous solution of yellow water-soluble eosin, and mix
thoroughly. Collect on a filter the abundant precipitate which
immediately appears. When the precipitate is dry, dissolve it in
pure methyl alcohol (Merck’s) in the proportion of 0.1 grm. to 60
cc. of the alcohol. To facilitate solution the precipitate is to be
rubbed up in a porcelain dish or mortar with a spatula or pestle.
This alcoholic solution is the staining solution. It should be
kept in a tightly-stoppered bottle. Should it become concentrated
through evaporation methyl alcohol in proper quantity should be
added.

1. Cover film with a given quantity of staining fluid by means of
a medicine dropper.

2. After 1 minute add to the staining fluid on the film the same
quantity of distilled water by means of the medicine dropper,
and allow the mixture to remain for 2-3 minutes according to the
intensity of the stain desired. A longer period of staining may
produce a precipitate. Eosinophile granules show best after short
staining. The quantity of diluted stain on the preparation should
not be so great that some of it runs off.

3. Wash the preparation in water for 30 seconds or until the
thinner portions of the film become yellow or pink in color.

4. Dry, and mount in balsam.

Films more than a few hours old do not stain as well as fresh ones.

The _red cells_ are orange or pink in color. Polychromatophilia
and punctate basophilia or granular degeneration are well shown.
Nucleated reds have deep-blue nuclei, and their cytoplasm is
usually bluish. The _lymphocytes_ have dark purplish-blue nuclei
and cytoplasm of a robin’s-egg blue, in which a few dark-blue
or purplish granules are sometimes present. The nuclei of the
_polynuclear neutrophilic leukocytes_ are dark-blue or dark
lilac-colored, the granules reddish-lilac. The _eosinophiles_ have
blue or dark lilac nuclei, a blue cytoplasm and eosin-red granules.
The _large mononuclear leukocytes_ have a dark lilac or blue
nucleus, cytoplasm pale blue or blue with dark-lilac or deep purple
granules. Mast-cells have purplish or dark-blue nuclei, bluish
protoplasm and coarse dark purple or black granules. _Myelocytes_
have dark blue or lilac nuclei, blue cytoplasm, and dark-lilac
or reddish-lilac granules. _Blood platelets_ are blue with small
violet or purplish granules in their central portions. _Malarial
parasites_ have a blue body and lilac or red chromatin. _Spirochæte
pallida_ is pale blue.

Giemsa’s Method.

_a_, One per cent water solution of azur-blau; _b_, one per cent
watery solution of eosin. For staining take 1 cc. of _b_, add 10
cc. of water, and then 1 cc. of the azur-blau solution. Stain 10
minutes to 1 hour.

Giemsa’s Old Method.

Azur II—Eosin 3.0 grm.
Azur II 0.8 grm.
Glycerin (Merck’s pure) 250.0 cc.
Methyl-alcohol (Kahlbaum I) 250.0 cc.

To 1 cc. of distilled water in a small, perfectly clean graduate
add 1 drop of the stain, shaking very gently. Make very thin
film; dry in air; fix 15-20 minutes in absolute alcohol. Cover
preparation with a thin layer of the freshly diluted stain for
10-15 minutes, renewing stain at end of 10 minutes. Wash in a
stream of water. Differentiate over-stained preparations in
distilled water. Dry with absorbent paper; mount in balsam. Stains
the _spirochæte pallida_ and _malarial organisms_. The Giemsa
solution may be obtained from Grübler. A more intense staining can
be obtained by adding to the water used for diluting the stain 1-2
drops of a 0.1 per cent solution of potassium carbonate.

5. SPECIAL ELECTIVE STAINS FOR THE BLOOD.

1. =Ehrlich’s Triple Stain.=

Saturated watery solution of Orange G 120 cc.
Saturated watery solution of acid fuchsin 80 cc.
Saturated watery solution of methyl green 100 cc.
Glycerin 50 cc.
Distilled water 300 cc.
Absolute alcohol 180 cc.

Mix gradually; allow to stand for several months; do not shake or
filter. Remove stain with pipette. Fix by heat, or pure methyl
alcohol for 5 minutes. Stain 5-10 minutes; wash thoroughly, dry
and mount in balsam. Neutrophile granules violet; eosinophile,
a bright red; nuclei of the neutrophilic and eosinophilic cells
greenish-blue; nuclei of the lymphocytes deep-blue; nuclei of the
large mononuclears pale blue; those of red cells intense blue; red
cells copper red. The Aronson-Philipp modification is more variable
and less satisfactory.

Pappenheim’s Stain for Lymphocytes.

3-4 parts of polychrome methylene-blue or methyl green to 1-2 parts
of pyronin. Fix in absolute alcohol. Nuclei blue-green; protoplasm
bright red.

Staining of Blood-platelets.

The blood-platelets may be examined in the fresh state by coating
a cover-slip with Deetjen’s agar-solution (boil 5 grms. agar-agar
in 500 cc. distilled water, filter hot, and to each 100 cc. of the
filtrate add 0.6 grm. sodium chloride, 6-8 cc. of a 10 per cent
solution of sodium phosphate and 5 cc. of a 10 per cent solution
of sodium diphosphate). Place drop of blood on this coating and
examine on warm stage. For permanent stained preparations bleed
into a fixing and staining fluid (equal parts alcohol and ether and
Romanowsky’s stain) or use Wright’s stain.

Bremer’s Diabetic Reaction.

Take a clean cover-glass, smear one-half with normal blood, the
other half with diabetic blood. Fix for 2 hours at 120°C., or in
equal parts of absolute alcohol and ether at 60°C. for 4 minutes.
Stain in a 10 per cent watery methylene-blue for 2 minutes, wash
off the stain in water, and stain for 10 seconds in a ⅛ per cent
watery eosin. Wash, dry and mount in balsam. In diabetic blood the
red cells are green; in normal blood red. While this reaction is
constant in diabetic blood it also occurs in leukæmia, Hodgkin’s
disease, exophthalmic goitre and multiple neuritis. A 1 per cent
solution of _Biebrich scarlet_ stains diabetic blood intensely,
normal blood but slightly. On the other hand, a _1 per cent
methylene-blue_ and a _1 per cent Congo red_ stain normal blood
intensely and diabetic blood slightly.

Staining of Glycogen in Leukocytes.

To a solution of Lugol’s (100:3:1) add sufficient gum arabic to
make a syrupy mixture. Keep tightly corked. Place a drop of this
solution upon an air-dried film. After 1 minute dry with blotting
paper. Examine with oil immersion. A positive reaction is shown
by the presence of a diffuse brown or reddish-brown coloration or
granules in the cell-body of the polymorphonuclear leukocytes.

Staining of Fat in Blood.

Stain in solutions of scharlach R or sudan III in 70 per cent
alcohol.

Staining of Blood-parasites.

The _malarial parasites_, _trypanosomes_, _Leishman-Donovan
bodies_, _sporidia_, _piroplasma bigeminum_, _spirilla_ and
_spirochætes_ and the _filaria_ may all be stained with Wright’s
or Giemsa’s modification of the Romanowsky method, or by any of
the modifications of this method. (See also Staining of Animal
Parasites.)

_B. SECTIONS._ The blood is allowed to drop directly into
Flemming’s solution and allowed to stand for 24 hours. It is then
washed in water by repeated decanting, or the coagulum may be
placed in a bottle covered with muslin, and then exposed to running
water. It is after-hardened in alcohol and imbedded in paraffin.
Safranin should be used to stain the sections. This method is
especially good for the demonstration of mitoses in the blood-cells.

Bone-marrow.

Prepare films and fix and stain, as for blood films. For sections,
fix the marrow in formol-Müller’s, mercuric chloride, Zenker’s,
etc.; imbed in paraffin; cut very thin sections; stain with
Ehrlich’s triple stain or Wright’s modification of Leishman’s
stain. To distinguish the young forms of erythrocytes and
leukocytes _Trambusi_ fixes in Flemming’s, stains the sections
in a 1 per cent thionin solution in aniline-water (4:100),
differentiates in acid-alcohol, and then brings the sections into
a watery eosin and finally an alcoholic eosin, and mounts in
xylol-balsam.

Spleen and Lymphnodes.

Fresh material may be obtained by means of a trocar, and may be
examined in the fresh state, or films may be prepared, fixed
and stained, as for blood-smears. Sections of fixed tissues may
be obtained by the use of the same fixing and staining methods
employed in the study of the blood or bone-marrow. For the study
of the reticulum Mallory’s reticulum stain or the digestion-method
may be used. In ordinary work formol-fixation followed by
eosin-staining is of great value in distinguishing hæmolymphnodes
and lymphatic glands.

II. BONE.

For ordinary work decalcification is necessary except for those
pathologic conditions in which the lime-salts have been lost (see