Normal histology.—It cannot be too strongly impressed on the beginner that a thorough mastery of the normal appearances of tissues and organs is absolutely necessary before attempting to make an accurate study of morbid changes in them. He should not be satisfied with examining one specimen of an organ but as many as he conveniently can, in order to be fully acquainted with the many deviations from normal which may exist without actual disease. He should therefore obtain several animals, such as small dogs, cats, rabbits, frogs, &c., and remove their organs with all care, and harden them in the various appropriate fluids. He should also obtain specimens of normal human organs from the post-mortem room. Many normal tissues (skin, muscle, tendon, bone, &c.), can also be prepared from a limb amputated for an accident to a healthy patient. By preparing specimens in this way he will not only become the possessor of a set of slides illustrating normal histology, but will find also that he has acquired that proficiency in hardening and staining the specimens which practice alone can give.

The following account of the method of preparing different tissues is merely intended to indicate the lines on which the beginner should proceed. After some practice he will be quite able to select the modes of hardening and staining which special circumstances or cases may seem to demand.

The first part of these directions will refer to the preparation of normal tissues, the second part to morbid histology.

Blood.—For special methods of examination see Chapter VII.

Blood crystals—HÆmoglobin crystals, obtained from the blood of an animal, or enough may be collected at any operation. A little water or a little ether is added to the blood which is allowed to stand for half-an-hour after which a drop is allowed to evaporate slowly on a clean slide.

HÆmatin crystals.—The student should make himself thoroughly familiar with these, as their presence affords positive proof of the existence of blood colouring matter in a stain.

To obtain them a drop of blood should be allowed to dry on a slide. The dried blood is scraped into a little heap with a small piece of clean glass, and a drop of glacial acetic acid added. As it evaporates minute reddish-brown acicular crystals will appear.

HÆmatoidin crystals.—Obtained from the site of a bruise, or an old hÆmorrhage, e.g., a cerebral apoplexy or a hÆmatocele.

Simple squamous epithelium.—(Endothelium). Carefully strip off the lining of the parietal pericardium or parietal pleura, of a recently killed animal, or spread out its omentum on a piece of cork, and (1) stain the intercellular cement with nitrate of silver (p.82) so as to reveal the outlines of the cells. (2) Stain other specimens with hÆmatoxyline or alum carmine to reveal the nuclei.

Stratified squamous epithelium.—Specimens from skin of various parts, finger, groin, lip, tongue should be prepared. Harden in MÜller’s fluid.

Transitional epithelium.—Occurs in the pelvis of the kidney, ureter and bladder. It is very readily detached, especially if not hardened immediately after death. Remove as early as possible. If the bladder is taken it should be cut open and pinned out as flat as possible. Harden in osmic acid, or MÜller’s fluid and spirit. Embed preferably in celloidin.

Simple columnar epithelium.—Occurs in many parts. It may be studied in the salivary ducts, the intestine, kidney, &c., of any mammal.

Goblet-cells.—Seen abundantly among the columnar cells of the intestinal glands, and in the mucous glands of the mouth and of the cervix uteri.

Stratified columnar epithelium.—Occurs only in the urethra. Harden the penis of a cat in MÜller’s fluid, and cut transverse sections.

Ciliated epithelium.—Harden the trachea of a recently killed cat in osmic acid or MÜller’s fluid. Beautiful specimens may also be obtained from an ordinary nasal polypus, which should be put into hardening fluid immediately after removal.

Stain all sections of epithelium in picrocarmine, and in eosine and hÆmatoxyline.

Ordinary areolar tissue.—Difficult to obtain free from fat. It may be studied in the subcutaneous tissue of the section of the cat’s penis already made. A fragment of the tissue should also be removed and carefully teased in a drop of picrocarmine. Areolar tissue may also be studied in sections of skin, and in the capsules of the different internal organs.

Elastic tissue.—May also be studied in most sections of skin. If the ligamentum nuchÆ of a large quadruped (horse, bullock), &c., is available it yields the best specimens, or the human ligamenta subflava may be examined. Pin a piece out on a piece of wood or wax. Harden in MÜller’s fluid. Stain in picrocarmine. Both sections and teased specimens should be prepared.

Tendon.—Readily obtained from an amputated limb. Harden in MÜller’s fluid. Make transverse and longitudinal sections. Stain with eosine and hÆmatoxyline.

A preparation should also be made by teasing a little of the fresh tendon in normal salt solution, and staining with picrocarmine.

Retiform or lymphadenoid tissue.—Seen in lymphatic glands and in the lymphoid follicles scattered along the sub-mucous coat of the alimentary canal.

Prepare sections in the ordinary way. Stain in eosine and hÆmatoxyline or in picrocarmine.

Some sections should also be prepared by pencilling (i.e., dabbing with a camel’s hair brush) or by shaking sections up in a test tube with water or normal salt solution. By this means the leucocytes are removed, and the structure of the adenoid tissue itself becomes more evident.

Fat.—Best studied in sections of skin and subcutaneous tissue, or in the mesentery of the cat. One specimen should be stained with osmic acid and picrocarmine and mounted in Farrant’s medium, and another in eosine and hÆmatoxyline and mounted in Canada balsam.

Pigment cells.—Branched cells are best studied in the living foot of the frog, where amoeboid movements may be seen in them when the light falling on the retina is made to vary in intensity. Permanent preparations are most conveniently made from the pallium of the common snail. The shell is removed, and the pallium snipped out with the scissors. It is then pinned out flat, hardened for a day in methylated spirit, and mounted unstained in Farrant’s medium. They are also well seen in sections of the choroid coat of the eye.

Hyaline cartilage.—Specimens may be obtained from any joint, from the costal cartilages of young animals, or from the thyroid cartilage and tracheal rings. It may be hardened in spirit. Stain with picrocarmine, eosine and hÆmatoxyline, and with methyl violet.

Elastic cartilage.—Prepared from the epiglottis, or from the cartilages of the ear, e.g., of a cat. Harden in spirit. Stain in picrocarmine or in dilute fuchsin.

White fibro-cartilage.—Obtained from intervertebral disc. Prepare and stain as for hyaline cartilage.

Bone:—

Unsoftened Bone.—Cut as thin a section as possible with a fine saw. Rub the section with the hand on a dry oil stone until it is as thin as possible. Then cement it by Canada balsam (liquefied by warming) to a piece of plate glass and continue the rubbing process with this, examining it now and then with the low power to see if it is thin enough. As soon as it is thin enough it is washed off the slide with methylated spirit, and washed to get rid of the fine bone dust. It should then be transferred to turpentine and may be mounted in balsam.

Softened bone.—Specimens may be obtained from an amputated limb or from the femur of a cat.

Specimens should be decalcified in chromic and nitric fluid, and the hardening completed in spirit. In studying the process of ossification, e.g., in the head of the humerus of a kitten, it is best to embed the specimen in celloidin before cutting sections, as the trabeculÆ of bone are very delicate, and easily detached.

Very beautiful double staining effects may be obtained with either picrocarmine, or eosine and hÆmatoxyline, and with eosine and methyl violet.

Bone marrow.—To obtain good sections of red bone marrow, take a piece of the clavicle or a rib, or of one of the carpal or tarsal bones. Decalcify in chromic and nitric fluid. Embed in celloidin. Stain with eosine and logwood, eosine and alum carmine, or alum carmine and picric acid. Mount in Canada balsam. The various cells present in bone marrow may also be studied by squeezing some fresh marrow from a rib, and making a cover-glass film, and preparing in exactly the same way as is directed in the case of blood films on page 116.

Tooth.—Best cut in situ from the jaw of a cat. Decalcify in chromic and nitric fluid, and cut both vertical and transverse sections. Stain in picrocarmine, or eosine and hÆmatoxyline.

Developing tooth.—Extremely good specimens may be obtained from the jaw of a newly-born kitten or puppy. Sections can easily be made shewing a milk tooth and a developing permanent tooth by its side.

The enamel is dissolved by decalcifying fluids. To study it a specimen of unsoftened tooth should be made, according to the directions given for bone.

Striped muscle.—Should be studied in various animals.

The leg of an insect such as a cockroach may be hardened in osmic acid. One leg should be hardened in a straight position so as to fix the fibrils in the fully extended position, another should be bent up so as to get specimens of relaxed fibrils.

Portions of muscle should be removed, and teased on a glass slide in some staining fluid such as picrocarmine, a tenth per cent. solution of eosine or quarter per cent. of safranine.

Sections of amphibian and mammalian muscle should be prepared to show their differences in structure. The most convenient part to select is the tongue, as a view of the fibres is obtained both in longitudinal and transverse sections. Sections should be stained in eosine and hÆmatoxyline which gives a beautiful effect. For special stains for intra-muscular nerve endings see page92.

Heart muscle.—A portion should be teased fresh in picrocarmine or eosine, another portion hardened in MÜller’s fluid, and sections made and stained with eosine and hÆmatoxyline.

Unstriped muscle may be obtained by teasing a fresh portion of the muscular coat of the small intestine of an animal, or by sections of the hardened intestine, bladder or uterus. Stain in picrocarmine or preferably eosine and hÆmatoxyline.

Nerves.—The special methods for staining nerve tissues are detailed in Chapter VI. The student must remember that the ordinary staining methods are also applicable to nervous tissues.

Nerve terminations:—

Meissner’s corpuscles.—Take the tip of an index finger immediately after amputation. Place part of it at once in chloride of gold solution, and the rest in MÜller’s fluid until it is hardened.

Sections stained with chloride of gold should be mounted in Farrant’s medium. The other sections may be stained in picrocarmine or eosine and hÆmatoxyline.

Pacini’s corpuscles.—May be dissected out on the smaller branches of the digital nerves, or may be found in the mesentery of the cat. The latter should be spread out on wood, hardened in MÜller’s fluid, stained in hÆmatoxyline, and mounted in balsam.

Other forms of tactile corpuscles may be studied in the tongues of frogs, ducks, or geese. A network of nervous fibrils should be studied in the cornea. Take the cornea of a newly killed frog or cat and stain with chloride of gold (p.82).

The end plates in which the nerves terminate in muscle may be studied by placing specimens of living muscle of some cold blooded animal into chloride of gold solution, and staining rather deeply.

Arteries.—Take a piece of the aorta, a piece of some medium artery, as the renal or radial, and harden in MÜller’s fluid. Stain in picrocarmine and always in eosine and hÆmatoxyline. Arterioles are best studied in sections of the various organs. Thus they are seen in each Malpighian body of the spleen, in the boundary zone of the kidney, and so on. A longitudinal surface view can also be obtained by staining and examining the pia mater.

Veins.—Remove, harden, and stain in the same way.

Capillaries.—May be very well seen in the foot of the frog.

Stun a frog by striking its head, or by chloroforming it. Fix it on a piece of card with a V shaped notch at one end. Tie one of the hind feet by means of threads attached to its toes so that the web of the foot is gently stretched over the V. The foot can then be readily examined under a 1/2inch objective. The foot must be brushed from time to time with normal salt solution to keep it moist. The movement of blood in the capillaries, &c., can then be studied for an hour or two. After death the mesentery should be spread out on a piece of wood, and hardened for a few days in MÜller’s fluid.

Stain with eosine and hÆmatoxyline.

Lymphatics.—Commencement of lymphatics in serous membrane. Stain a piece of cat’s omentum in nitrate of silver (p.82) for some minutes. After washing keep in glycerine for about a week and then stain in hÆmatoxyline and mount in Farrant’s medium.

Lymphatic glands.—The lymphatic glands of the neck of the cat may be used. Harden in MÜller’s fluid. Stain in picrocarmine, eosine and hÆmatoxyline.

Skin and sweat glands.—Sections should be made from pieces taken (a) from the sole, (b) from the skin of the body, (c) from the axilla of an adult to study the pigment. Harden in MÜller’s fluid. Stain in picrocarmine or eosine and hÆmatoxyline.

Hairs and sebaceous glands.—Take a portion of the scalp, or of the skin of a puppy. Harden in MÜller’s fluid. Stain in eosine and hÆmatoxyline, and mount others unstained.

Hairs from various parts of the body should also be soaked for some hours in liq. potassÆ and mounted unstained in Farrant’s medium. They may be bleached subsequently by treatment with eau de Javelle (p.27).

Brain and spinal cord.—Must be removed from the body with extreme care, all stretching or squeezing being avoided. Harden slowly in MÜller’s fluid to which a fourth of its bulk of water may be added.

The best staining reagents to employ are eosine and hÆmatoxyline, alum carmine or borax carmine, aniline blue-black, &c. Staining methods, see Chapter VI.

Eye.—Harden the eye of a recently killed bullock, cat, or other animal in formal (p.23), puncturing the sclerotic in places to allow the hardening fluid to penetrate. In about a week make a horizontal section through the eye. The anterior half (the lens having been removed) may be satisfactorily cut in gum. Sections of the crystalline lens are not very satisfactory. The best way to get specimens of the fibres is to tease a piece of the fresh lens of a fish (e.g., a cod) in a 1/40 per cent. aqueous solution of eosine. Wash the eosine off the slide with 1/2 per cent. acetic acid, and mount in Farrant’s solution.

The posterior half of the eye should be embedded in celloidin, as otherwise it is extremely difficult to get sections of the retina in its proper relation to the other coats.

Mount some specimens unstained. Stain others with the ordinary stains.

Internal ear.—Decalcifying the temporal bone of a cat, dog, guinea pig, &c., in chromic and nitric fluid. As soon as the bone is decalcified complete the hardening of the soft parts in methylated spirit, embed in celloidin, and cut sections in the longitudinal axis of the cochlea. Owing to the extreme hardness of the bone in adults it will be found best to use the petrous bone of newly born animals.

The semi-circular canals will be most readily studied in the temporal bone of fishes, or of birds, e.g., the common fowl. They also must be cut in celloidin, and stained in the ordinary way.

Nose and olfactory epithelium.—It is difficult to obtain specimens from the human subject, but very satisfactory preparations may be made from the dog, or more conveniently in a new born puppy where the bones are still cartilaginous. Harden the latter in MÜller’s fluid, decalcify adult specimens in chromic and nitric fluid. Specimens of ciliated epithelium, &c., will be obtained from the lower part, and of the special olfactory epithelium from the upper part. Stain in eosine and hÆmatoxyline.

Lungs.—Carefully remove the lungs of a cat without injuring the bronchi or trachea. Introduce a cannula into the trachea and gently inflate the trachea with air. Ligature the trachea and place the lung in MÜller’s fluid, a weight being attached to keep the organ submerged. Harden for about six weeks, and then make sections of the various parts.

To demonstrate the endothelium of the alveoli, inject instead of air, nitrate of silver. Allow it to remain in for half an hour, then remove it by washing, and harden in MÜller’s fluid.

Beautiful casts of the alveoli, &c., may be obtained by placing a cat’s or human lung under the receiver of an air-pump, and when the air is completely exhausted, injecting fusible metal into the bronchus. The lung tissue is then removed by corrosion or by maceration. Portions of the casts should be removed, fixed in a glass cell with a spot of Canada balsam, and examined by reflected light.

Thyroid gland.—Best obtained from a young subject either human or an animal.

Harden in MÜller’s fluid. Stain in picrocarmine or eosine and hÆmatoxyline. Also stain sections in safranine, which stains the colloid material, and also picks out any colloid formation in the cells themselves.

Thymus.—Remove from a foetus or a very young animal, and prepare in the usual way.

Tongue.—That of the cat or rabbit serves very well.

Ordinary transverse sections should be made, and also sections through the circumvallate papillÆ in order to study the “taste buds.”

Salivary glands.—Those of a cat or dog do very well.

Sections should be made from each of the three glands.

Stomach.—That of the cat or dog should be studied. The organ must be removed immediately after death before any post-mortem digestion of the coats has occurred. The stomach should be opened, washed gently and pinned out flat, with as little stretching as possible on a piece of wood, and hardened in MÜller’s fluid.

Sections should be made (a) longitudinally through the cardiac end to show the transition from the oesophageal to the gastric mucous membrane, (b) from a portion of the greater curvature, (c) longitudinally through the pyloric valve.

Eosine and hÆmatoxyline form the best stain for the alimentary canal.

Intestine.—Prepare in the same way as the stomach. Make sections from (a) the upper part of the duodenum to show Brunner’s glands, (b) the ileum, (c) a Peyer’s patch, (d) the vermiform appendix, (e) the colon.

Liver.—Make an injection of one specimen with carmine and gelatin (p.120). Harden in methylated spirit. Others should be hardened in MÜller’s fluid and stained in the usual way.

Kidney, supra-renal, and pancreas.—Same preparation as for liver.

Spleen.—Harden in MÜller’s fluid.

Mount one section unstained. Shake another up with water in a test tube to shew the structure of the pulp. Stain others in eosine and hÆmatoxyline.

Bladder.—Must be removed and pinned out immediately after death, as otherwise the epithelium will be macerated off. Consequently it must be taken from an animal, as a cat. Harden in osmic acid. Cut in celloidin as the coats are very apt to become detached.

Penis and testis.—Readily obtained from dog, cat, or rat.

Stain with eosine and hÆmatoxyline.

Uterus, ovaries, and Fallopian tubes.—May be obtained from the post-mortem room or from the lower animals. Harden in MÜller’s fluid, and make sections from the cervix, the body of the uterus, the Fallopian tube, and the ovary.

Stain with eosine and hÆmatoxyline.

Embryological specimens.—For systematic work special manuals should be consulted.

Specimens should be hardened in osmic acid or in MÜller’s fluid, and cut in celloidin, or paraffin.

Cloudy swelling.—Specimens are obtained from organs of subjects who have died in the early stage of some fever. They should be always hardened in MÜller’s fluid, as the appearances alter if the tissue is kept in spirit for any length of time.

Fatty degeneration.—Prepare from patients who have died of exhausting diseases, phosphorus poisoning, &c.

Stain in osmic acid. Mount in Farrant’s medium and keep in the dark.

Mucoid degeneration.—Study in goblet cells of normal intestine or of ovarian cysts. There are no satisfactory selective stains for mucin.

Colloid degeneration.—Occurs in the thyroid gland, in the tubules of the kidney in many diseases, and the prostate of the old.

Stain in safranine.

Waxy or lardaceous degeneration.—Best studied in liver, spleen, or kidneys. It should be searched for in persons who have died from a long illness, accompanied by suppuration, e.g., phthisis or bone disease. Mount one section unstained, stain another in methyl violet, a third in a weak solution of iodine, and examine the latter at once both by transmitted and reflected light. The iodine stain is not permanent. Another section should be stained in osmic acid, followed by methyl violet, as waxy and fatty degeneration frequently co-exist.

Hyaline degeneration.—Seen in the arterioles of the spleen in some cases of typhoid and diphtheria. The ordinary staining methods must be used.

Calcareous degeneration.—Occurs after fatty degeneration in gummata and in atheromatous arteries. It also occurs in the matrix of the costal cartilages after middle life. Mount one section unstained and examine if possible with the polariscope. Stain others in safranine.

Pigmentary degeneration.—May be studied in brown atrophy of heart, nutmeg liver, &c. It is also seen well in spinal and cerebral nerve cells of the aged. Harden in MÜller’s fluid and mount sections unstained.

It will be unnecessary to recapitulate the methods for hardening the various diseased organs as the directions for the normal organs hold good. If the presence of micro-organisms be suspected, harden in methylated spirit or absolute alcohol, but as a rule both for diseased organs and tumours MÜller’s fluid will be found the most satisfactory reagent for general use.

It sometimes happens, however, that it is inconvenient to wait several weeks, until the MÜller’s fluid has hardened the specimen sufficiently, before making sections. In this case the best plan is to make fresh sections, or else to cut a slice about one-eighth of an inch thick, and harden for about three days in plenty of methylated spirit, or in formal (p.23).

Tumours.—MÜller’s fluid should be employed, unless a more rapid agent is required.

Methylated spirit may be used in the case of epithelioma, adenoma, &c., but for sarcoma, myxoma, tumours containing cysts or much blood, MÜller’s fluid yields by far the best results.