Staining

“ Tissues may be stained in the body during life (intravital staining) , or immediately after removal from the body (supravital or survival staining) , either before or after sectioning. (See Page 217.) Fixed tissues may be stained in bulk or in sections. ”

“ 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. ”

“ A better plan, perhaps, is to stain the section with carmine or logwood, and mount in balsam by the ordinary process. The best course to follow, however, especially in the case of transverse sections, is the double staining method. [18] For this purpose the sections in the first place must be subjected to the action of a solution of chloride of lime (1/4 oz. to a pint of water) until they become thoroughly bleached. ”

“ This sulphuring is simply the result of opacity, obtained by heating the glass to too high a temperature. If the staining is to be performed in the same firing as that by which the painting is to be fixed, it is quite clear that the outlines of the part to be stained must be painted in, with tar-oil, or with some such substance which is not affected by the moisture of the stain. ”

“ When the masses stained are large, or the whole gland is subjected to staining, of course the outer parts are more deeply stained than the inner ones, but it is at times convenient to have material of different depths of staining. After the process has continued long enough—the nuclei at least should be very distinctly colored—the material is transferred into a fluid containing glycerine five parts, water and alcohol each two parts, acetic acid one part. ”

“ Various methods have been advised for the intravital staining of cell-granules. Intravenous or intraperitoneal injections of methylene blue, alum carmine, neutral red and other stains will produce intracellular granule-staining in various organs of experimental animals. In the study of low forms of animal life staining solutions may be injected, or the animal or its parts may be examined in staining fluids. Human material can be examined by this method immediately after being removed from the body by operation or within 1-2 hours after death. ”

“ Body-staining is usual with both men and women, who do it for themselves, or get others to help them. There is no ceremony in connection with it. The colours generally adopted are red, greyish-yellow and black. The red stain is procured from an earth, which is obtained from the low countries; but they themselves also have an earth which is used, and produces a more bronzy red. ”

“ They are therefore usually prepared by treating them with some staining reagent, not merely to render them less transparent, but also to “differentiate” the elements of the section, by staining one part more deeply than another, or of a different colour. Thus hæmatoxyline stains the nuclei and rapidly growing parts of the tissue, leaving the formed material, as a rule, much more lightly tinted. Methyl violet again stains healthy tissues blue, and parts affected with waxy degeneration a red-violet colour. By combining stains also much differentiation of the tissue elements may be obtained. ”

“ DIAGRAM SHOWING DIAGONAL POSITION OF KNIFE IN COMMENCING TO MAKE A SECTION.
13. Staining Agents.—Before proceeding to mount the sections which have just been cut, it will be very advisable that they should be submitted to the action 32of some staining fluid, in order to render more clear and distinct their minute structure. Organic substances possess the property of being able to absorb various colouring matters from their solution, and to incorporate such colour into their own texture. ”

“ This method is not often used in pathologic work. The fixed and hardened tissue is cut into small pieces, placed in the staining solution for several days, washed thoroughly, dehydrated in alcohol, imbedded, cut, and mounted without further staining. Alcoholic solutions penetrate best; hæmatoxylin, hæmalum, carmine and alcoholic solutions of the aniline stains may be used. Metallic impregnation (gold or silver salts) of fresh or fixed tissues is but little used in pathology. (See Staining of Nervous System, and Spirochætes.) ”

“ Ehrlich method for tubercle bacilli.—Sections are stained for six to twenty-four hours in a one per cent. solution of gentian violet, methyl violet, methyl blue or fuchsine. They will stain more rapidly if the staining fluid be kept in an incubator at the body temperature. They should be removed from the staining fluid, and washed in distilled water, and then transferred (preferably on a glass section lifter) to a ten per cent. solution of nitric acid in distilled water until they are nearly decolourised. ”

“ As in most of these dark varieties it is necessary to first stain the wood a general color, the pigments and fluids most serviceable for plain staining purposes may be considered from the painter's and grainer's point of view, not from the polisher's. ”

“ In oil staining ordinary house woodwork and cheap panellings, apply the size before the stain. When the former is dry, it will be found that the oil stain, which now is graining color, also can be spread much better and more regularly, and that those sappy places which would otherwise have absorbed much stain are scarcely noticeable. In sizing white or stained wood, poor work often results from the quirks and mitres of mouldings receiving too much of the froth of the warm size. ”

“ In using this reagent it should be filtered into a watch glass, and the sections placed in it for at least an hour. There is no fear of overstaining, and they may be left all night. After they have been stained they must be thoroughly washed in water to remove the alum, otherwise numerous crystals of it will be seen in the field when the section is mounted. Sections may be mounted in Farrant’s solution or in Canada balsam. The staining effect improves very much after the section has been kept a few days. ”

“ Fixing and Staining.—A drop of the watery extract of the stain may be placed on a microscope slide and allowed to dry in the air, covered to protect it from dust, and then fixed by heating the slide over a spirit or Bunsen flame or by the use of absolute alcohol. When fixed, the preparation can be stained with eosin and methylene blue, methylene violet, or other nuclear dye. The film may also be fixed and stained with Leishman‘s solution in one procedure, or fixed by heat first and then stained with Leishman. ”

“ Staining is something on which opinions differ greatly. Some artists claim that only the natural colour of the wood should be used, but a great deal of staining is done, and we must leave artistic arguments to others.
The extent to which staining is carried may be illustrated by the following finishes used on one kind of wood—oak:
The writer believes that staining to make imitations is wrong, such as staining cherry or birch to give the impression of mahogany. ”

“ I have been staining with this method for years, yet I cannot say why the results differ so much. Some time ago I stained a frog for the nerve-endings in muscle and obtained the most beautiful results, but in the number of stainings I have made since (trying to follow the same method) I have not been by far as successful as then. In breaking up the gland I do not always separate all the pieces, but try to remove the connective tissue holding together the small lobules with the dull end of a needle, and then throw the coherent mass into the stain. ”

“ 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. ”

“ For staining in sections mercuric chloride or Zenker’s fixation followed by staining with polychrome methylene-blue, Giemsa’s or the modifications of the Romanowsky method may be employed.
3. Plasmodium Malariae. For films make medium smears (not too thin) ; fix with equal parts of absolute alcohol and ether for 1⁄2-1 hour; or fix and stain in the same solution (Leishman-Romanowsky, Wright’s stain, etc.) . For single staining methylene-blue, carbol-thionin, etc., may be employed; for double staining eosin and methylene-blue. ”

“ The best vessels in which to stain sections are small jars of white porcelain, capable of holding about two fluid ounces, and furnished with lids—they are much preferable to beakers or watch glasses, for owing to the white background which they afford it is very easy to watch how the staining is proceeding. The carmine solution which we have just described is both too strong and of too great density to be used in its pure state. ”

“ 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. ”

“ When staining pine dark, it is preferable to use the stain before sizing, if the grain is desired to be very prominent, a full coat should be spread, and then shortly afterwards all the stain lying on the surface may be rubbed off with old cloth or rag free from fluffiness. ”

“ Staining of Spores in the Rod.—Prepare the films as usual. Cover with carbol-fuchsin, using plenty of stain so that it will not dry on the slide; heat until vapor arises, not to boiling; cool until the stain becomes cloudy and heat again until the stain clears, and repeat once more; wash in tap water and then wash in 1 per cent. H2SO4 three times, dropping on plenty of acid, tilting and running this over the slide three times and then pour off and use fresh acid and repeat this once. Wash thoroughly in distilled water, then stain with Löffler’s blue one to three minutes. ”

“ I have found it necessary to use an objective at least as high as one-fifth 118or one-sixth, with central illumination without diaphragm, as cases will frequently occur where the staining is so faint, that with a lower power they will escape observation, though a good, wide angle, four-tenths inch, will show them well when strongly stained. ”

“ If it is scratched and spotted it can be scrubbed clean and given a new coat of Jackson’s varnish stain and be in a moment as good as new if not better. Just one word en parenthése, as it were, about stains. Do not let anything anyone can say induce you to attempt the beautiful green staining we all so much admire at home, for if you do, it can be nothing but a most ghastly failure. True, the particular piece of furniture will be green, but such a green! ”

“ The main use of the microscope in bacteriology is in the study of stained preparations of the organisms. Staining makes bacteria opaque and hence more easily seen than the transparent unstained forms. Some methods of staining also show morphological structures which are either imperfectly recognized in the unstained cell, spores, or are not visible at all—capsules, metachromatic granules, flagella. ”

“ It is much used in staining embryonic specimens in bulk before embedding in paraffin, and was strongly recommended for that purpose by Foster and Maitland Balfour.
Various formulæ have been given from time to time. That advised by Squire (Methods and Formulæ, p. 25) can be accurately made up without much difficulty.
Mix and add
Allow it to stand and any excess of calcium sulphate, &c., to separate. Filter and add
A little thymol should be added as a preservative. ”

“ Stain is, however, used of giving an intended and perhaps pleasing color to wood, glass, etc., by an application of coloring-matter which enters the substance a little below the surface, in distinction from painting, in which coloring-matter is spread upon the surface; dyeing is generally said of wool, yarn, cloth, or similar materials which are dipped into the coloring liquid. Figuratively, a standard or a garment may be dyed with blood in honorable warfare; an assassin's weapon is stained with the blood of his victim. ”

“ This change of colour depends upon the free access of air and the presence or absence of chemical substances in the air, so that it is almost impossible to infer the age of a blood-stain by its colour. On dark-coloured materials the stains are rendered more visible by the aid of artificial light, such as candle-light; on light-coloured materials, on leather, wood, iron, and stone, they are more visible in good daylight. By reason of the coagulation and the albuminous composition of blood, dry stains stiffen the fabric when thin, and on thicker woollen materials the fine fibres become matted. ”

“ A decorator if asked to imitate in stain on white wood a piece of parquetry or inlaid wood, might reply that such a thing was impossible, alleging as a reason that by employing liquid stain in the same way as a distemper—that is to say, by the aid of a stencil to reproduce the pattern—the stain, as soon as it became absorbed would be found to “run,” and so giving to the pattern imitated an indistinct or blurred edge. ”