CHAPTER XXVIII.. The Staining of Special Organs and Tissues.

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