Walter S. Colman

“ For hardening formal should be used as a two to five per cent. solution in distilled water. It may also be used as a ten per cent. solution for mounting museum preparations, but there is some tendency for a cloudy deposit to form on the glass after a time. It is the most suitable hardening agent at our disposal for eyes. It rapidly fixes the tissue elements, but does not cause much contraction. It may also be used for hardening the brain and spinal cord. Large quantities of fluid must be used for the latter purpose and it must be frequently changed. ”

“ In consequence of its not making the tissues shrink, it does not squeeze the blood out of the vessels and where the organ has been congested before death, we may, by using Müller’s fluid, preserve a natural injection of the capillaries.
3. There is comparatively little danger of over-hardening the tissue and rendering it brittle.
4. Sections of organs hardened in Müller’s fluid are usually firm and easy to manipulate. They do not tend to curl up or adhere to one another as much as those hardened in spirit. ”

“ Nissl’s method stains the protoplasm without greatly reducing its transparency and allows us to study details of cell structure. Small portions of tissue, removed as soon as possible after death, are hardened in alcohol. Sections are then cut, preferably in gum, as celloidin is inconvenient owing to its staining so deeply with aniline dyes.
Sections are transferred from alcohol to a one-half per cent. aqueous solution of methylene blue, which is heated in a watch glass till it steams freely, but short of the boiling point. Stain for about a quarter of an hour and allow to cool. ”

“ 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. ”

“ Specimens are thoroughly freed from all trace of the hardening fluid by washing in water, and the tissue is then placed in the gum solution for at least twelve hours, or if enough carbolic acid be added, it may be left there for an indefinite time.
When frozen, gum forms a firm non-crystalline mass, which supports the tissue on all sides. It must not be frozen too deeply, or it becomes hard and rather brittle and is apt to injure the razor. ”

“ The specimens should never be impaled with needles, or unsightly holes, which may even be mistaken for pathological appearances, will appear when a section is examined under the microscope.
It requires some practice to know when the tissue is sufficiently hardened. The object aimed at is to make them not really hard but tough. It is almost unnecessary to add that in testing this with the fingers the utmost gentleness must be observed, or serious damage may be done to the tissue.
When the tissue is sufficiently hardened the hardening fluid must be thoroughly dissolved out. ”

“ An incision must be made into the left ventricle, and a cannula passed up into the aorta and firmly secured by a ligature passed round the aorta with the assistance of forceps or an aneurism needle. Any blood is cleaned away and the animal is then placed in the water bath for about ten minutes. The tube from the bottle containing the injection fluid is then filled by gentle pressure on the syringe, and clamped when full. Its end is then placed on the cannula and secured there by a ligature. The pressure should be raised by squeezing the syringe until the manometer registers one inch. ”

“ The solution may be kept at the temperature of the body in an incubator. The organisms will often stain more readily if the section be passed through a 1 in 2000 solution of corrosive sublimate before putting it into the staining fluid. After staining the section is washed in distilled water and then in methylated spirit until it appears almost decolourised. Some prefer to decolourise the tissues by washing in a half per cent. solution of acetic acid instead of methylated spirit. Practice is required before the correct time for decolourising is accurately estimated. ”

“ It is not practicable to dehydrate by means of alcohol as it would cause sudden shrinking of the tissues. When the section is dry a drop of Canada balsam is applied and it is covered with alcohol in the usual way. The nerve cells and their processes are stained a deep slate colour, as are the nuclei of the connective tissue cells, while the ground work of the neuroglia is faintly stained and of a neutral grey tint. This method gives beautiful results both with normal and morbid specimens. ”

“ 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. ”

“ Before buying a microscope the student should obtain an illustrated price list from any of the firms mentioned above, and, having selected an instrument, he should test it very carefully, or better get some experienced friend to test it for him, before deciding to purchase it. Delicate test objects such as diatoms, scales of butterfly’s wing, or a stained specimen of micro-organisms should be employed. The coarse and fine adjustments should be tried. They should work freely and smoothly and without any delay. ”

“ Besides this, the tissue when placed in the fluid contained a good deal of water which will have diluted it and consequently an early change is desirable. Afterwards the fluid requires to be changed only as often as it becomes turbid, or any deposit occurs, usually about once a week.
While hardening, specimens should be kept in a cool place, as warmth favours changes in the cells, &c.
In manipulating the portions of organs, forceps should always be used and these with great gentleness. ”

“ Osmic acid is invaluable for staining fatty particles in the cells.
For ordinary use the one per cent. stock solution (p. 21) should be diluted with ten times its bulk of distilled water, and sections stained in it all night in a dark cupboard, or the watch glass containing them may be placed inside a small box.
The sections must be washed thoroughly in plenty of water. If desired they may be stained subsequently in picrocarmine or methyl violet if waxy degeneration also be present. Sections should be mounted in Farrant’s solution, as Canada balsam usually gives disappointing results. ”

“ Three methods (two of which are modifications of the first) are employed far more often than any others. By these methods the myelin coating is stained. Tissues must have been hardened previously for many weeks in Müller’s fluid, or some other bichromate solution. They are then overstained in a solution of hæmatoxyline, and the section treated with a suitable bleaching reagent, when the colour is discharged from all the tissue elements except the nerve fibres. ”

“ If the tissue is very delicate, and likely to be injured by changing from one vessel to another, or if it is larger than the section lifter will conveniently carry, it should be floated out on a glass slide, and, as much water as possible having been removed by blotting paper, should be dehydrated by adding a little alcohol from a pipette once or twice. Most of the alcohol should then be removed by tilting the slide, and before the remainder has evaporated, some oil of cloves or bergamot should be added from another pipette. ”

“ 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. ”

“ For delicate structures and for fresh sections the transference to spirit, and the subsequent flying out of the section when returned to water are risky, and the best method of treating these is to put the vessel containing them under the receiver of an air pump, if one is available, and slightly exhausting the air. ”

“ 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. ”

“ The liquefied gelatine is forced into the artery by a syringe or by air pressure. It is essential that the pressure be uniform and steady. This is so much more easily managed with air pressure that this method is strongly recommended to the beginner. But, whatever method be adopted, perfect results can only be obtained with certainty after long practice. Sometimes too high pressure will be employed and the vessels give way, at others the injection may not reach the capillaries at all. The most scrupulous attention to details is essential. ”

“ 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. ”

“ Both the hardening and the subsequent washing must be carried on in the dark.
Osmic acid is also a most valuable staining reagent (see p. 81) .
Carbolic acid (5 per cent.) .—May be used to harden almost any tissue, but is particularly useful for hardening nervous tissues such as brain or spinal cord which are afterwards to be preserved as museum specimens. It does not discharge the colour of a specimen so rapidly as spirit. ”

“ Dehydrate in alcohol, clear in xylol, and mount in balsam. Goodall advises a mixture of pyridine and xylol for clearing, and mounts in strong xylol-dammar solution, without a cover-glass.
Very careful attention to details and much practice are required before uniformly good results can be obtained. The results are extremely beautiful and well repay the labour expended on them. The cells and their processes appear black on a yellowish ground. ”

“ 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 skin must previously be washed with soap and water or ether, to remove any grease or epithelial scales. The puncture should be made firmly so that blood may escape freely. The finger or ear must not be squeezed. Specimens must be made rapidly before red corpuscles have run into rouleaux. The slides and coverslips employed must be scrupulously clean, or it is impossible to get really good films. ”

“ But those who do not desire to make a special study of bacteriology may often require to examine for the presence of organisms in sections, or in various excretions, and it is hoped that they may find the following short description of special methods sufficient for their purpose. ”

“ Much practice is required in using either of the methods before one can judge accurately how long to leave sections in the staining reagents or decolourising agents, and the beginner must not be discouraged if at first he is unable to obtain good results although he follows the book directions most minutely. ”