Staining Animal Structures.
Specific stains are chiefly employed to assist the eye in distinguishing one elementary tissue from another. It is therefore necessary to stain all structures, as certain parts are seen to have a special affinity for one colouring agent rather than another, whereby they become more deeply stained, and consequently more clearly differentiated. For staining animal structures, borax, carmine, and hæmatoxylin are more frequently employed than others. The formulæ for each will be found in the Appendix “Formulæ and Methods.”
Staining Process.—Place the section in distilled water to wash away the alcohol; place a little of the carmine in a watch glass, and immerse the section in it for four or five minutes; then remove it to a solution composed of methylated spirit five parts, hydrochloric acid one part; shake well together. This solution should be kept ready for use. Immerse the section in this solution and leave it to soak for about five or ten minutes if over-stained, until the desired tint has been obtained. Sections of skin and fibrous tissue may be left until nearly all colour is removed, the glands and hair follicles will then be brought out more clearly. The section must be transferred to methylated spirit to remove all traces of acid, then to oil of cloves contained in a watch glass, lift the section from the methylated spirit by one of the lifters ([Fig. 250]), and carefully float it on the oil, in which it should be allowed to remain for about five minutes. This is the clearing process, the object of which is to remove the spirit and prepare the section for mounting in Canada balsam. First, however, place the section in filtered turpentine to wash away the oil of cloves; this is found to answer better than another plan adopted, that of removing the section from the oil of cloves and mounting it in balsam direct. The oil, however, has a tendency to darken the balsam.
Logwood or Hæmatoxylin Stains ([see Appendix] for the several formulæ). Staining by this agent is effected as follows:—
After the specimen has been hardened in any of the chromic acid solutions in use, transfer it to a seven per cent. watery solution of bicarbonate of soda for about five minutes, then wash well in distilled water. Spirit prepared preparations do not require to be transferred to the soda solution, but all sections must be washed before they are transferred to the logwood stain. To a watch glass nearly full of distilled water add ten or twenty drops of the logwood stain, in which it should remain for twenty or thirty minutes. Wash well with the ordinary tap water, which will fix the dye and cause it to become blue. Dehydrate in methylated spirit, clear in clove oil, and mount in dammar or Canada balsam.
Double-staining with Hæmatoxylin and Rosin.—Stain the section as directed above, then place it in an alcoholic solution of rosin, about one gramme of rosin to an ounce of methylated spirit, and let it soak for a few minutes; wash well in methylated spirit, clear in oil of cloves, and mount in balsam.
Canada balsam should be prepared for use as follows:—One ounce of dried balsam to one fluid ounce of pure benzole; dissolve, and keep in an outside stoppered bottle. Clear the section in clove oil, and place it in turpentine, clean a cover-glass and slide, place a few drops of balsam on the centre of the latter, take the section from the turpentine on a lifter, allow the excess of turpentine to drain away, and with a needle-point lift the section on to the balsam slide. Now take up the cover-glass with a pair of forceps ([Fig. 236]), and bring its edge in contact with the balsam, ease it down carefully as shown in [Fig. 237], so that no air bubbles are enclosed, and with the needle point press the surface of the cover until the section lies quite smoothly and flat, and the excess of balsam is pressed out. The slide should now be transferred to the warm-chamber, and there allowed to remain for a day or two, or until set and hardened.
Fig. 236.—Forceps for Mounting.
Any exuded balsam may be washed away with benzole and a soft camel’s hair brush; then dry the slide with an old piece of linen cloth, and apply a ring of cement or Japanner’s gold size. Other methods for staining and mounting will be found to answer quite as well—that of Beneke’s is a useful one for staining connective tissue.
Fig. 237.—Mode of placing Glass Cover on Object.
For staining connective tissue a modification of Weigert’s method of staining fibrine is resorted to. Portions of tissue that have been fixed in alcohol having been embedded in paraffin and cut, the sections are detached and placed on glass slides, and stained for ten or twenty minutes with gentian violet, ten parts, well shaken with water 100 parts; filter, and add five to ten parts of a concentrated alcoholic gentian violet solution. Afterwards treat for one minute with lugol solution, of a port wine tint, dry with filter paper and decolourise with aniline xylol (aniline oil two parts and xylol three parts). Decolourisation having been stopped at the right point (judged from experience) mount the sections in xylol balsam. The fibres of the connective tissue should appear stained of various shades of violet.
Double Staining nucleated blood corpuscles. Two kinds of staining agents are required. Stain A: dissolve five grammes of rosin in half an ounce of distilled water, and add half an ounce of rectified alcohol. Stain B: dissolve five grammes of methyl green in an ounce of distilled water. Place a drop of frog’s blood on a glass slide, and with the edge of another slide spread it evenly over the centre of the slip, and put it away to dry; when quite dry flood the slide with stain A for three minutes, and wash with water, now flood the slide with Stain B for five minutes, wash again with water, and allow the slide to dry. Apply a drop of the prepared Canada balsam and a cover-glass.
Fig. 238.—Shadbolt’s Turn-table.
The blood of such mammals as are non-nucleated should be treated in a slightly different way. Spread a drop or two of blood on a slide and dry it quickly; then put the slide on Shadbolt’s turn-table ([Fig. 238]) and run a ring of cement around it; allow this time to dry, and then apply a second coating, and before this becomes quite dry place on it a clean glass cover, and press it down gently with one of the fine needles ([Fig. 229]), until firmly adherent.
Epithelium.—Remove from the mouth of a frog by scraping some squamous epithelium; the columnar must be taken from the stomach; place it in glycerine, or Farrant’s solution on the slide; apply a cover-glass, and with the point of the needle press it down until the epithelium cells are separated and spread evenly over the slide. Set this aside for a day or two, then wash away any of the medium which may have escaped; dry the slide, and run a ring of cement around the edges, on the turn-table. Portions of the intestine of a rabbit or other animal may be treated in the same way. If it is wished to make permanent specimens of such structures, the intestine must be hardened in a two per cent. solution of bichromate of potash for a couple of days, then washed until all colour is discharged, and removed to a solution of picro-carmine for twenty-four hours, after which allow the stain to drain away, when it will be ready for mounting.
By the aid of the handy little spring clip ([Fig. 239]), objects of delicacy when mounted may be left to dry and harden for any length of time.
Fig. 239.—Spring-clip for Mounting.
Striped muscular fibre, taken from the pig, must be teased out in a two per cent. solution of bichromate of potash, in which it should remain for two or three weeks, when it may be transferred to methylated spirit, and allowed to remain until required for mounting. Soak a piece in water to remove the spirit, place a small fragment on a slide in a few drops of water, and with a couple of needles tease the tissue up, so as to separate the fibres. Drain away the water, and apply a drop or two of Farrant’s medium and a cover-glass, which cement down as before directed.
Fibrous tissue may be served in the same way. Yellow elastic tissue must be first placed in a solution of chromic acid and spirit for ten days, and then treated as directed for muscular fibre.
Non-striated Muscle.—A piece of the intestine of a rabbit should be steeped in chromic acid and spirit for ten days, then washed in water; strip off a thin layer of the muscular coat, and stain in hæmatoxylin solution. Well wash in ordinary water until the colour changes to blue, when it will be fit for mounting. Place a fragment on a slide and a drop of water, and carefully separate the fibres with a pair of needles. Drain off the water, as it is now ready for mounting, place on slide, and add a drop or two of Farrant’s medium, and place on the cover-glass.
Nerve Tissue.—Dissect out the large sciatic nerve from a frog’s thigh, and stretch it on a small piece of wood, to which pin both ends of the nerve, and transfer it to a one per cent. solution of osmic acid for an hour or two. Wash in distilled water; tease up a small fragment on a slide (as shown in [Fig. 240]), and apply a drop or two of Farrant’s solution and cover-glass.
Tissues containing air should be soaked in water that has been boiled for ten minutes; this will displace the air. (For Farrant’s medium, [see Appendix.])
Glycerine Jelly.—Dissolve one ounce of French gelatine in six ounces of distilled water, and melt together in a hot-water bath. When quite dissolved, add four ounces of glycerine, and a few drops of creosote or carbolic acid. Filter through white filtering paper while warm, and keep in a capped bottle. This may be used instead of Farrant’s solution.
Fig. 240.—Method of Teasing out Tissue.
Nitrate of silver darkens by exposure; it is used in a half per cent. watery solution. Specimens to be acted upon should be washed in distilled water, to remove every trace of sodium chloride, and then steeped in the silver solution for some two or three minutes, after which they should be again washed until they cease to turn milky; then place them in glycerine and expose them to the action of light until they assume a dark brown colour, when they should be mounted in glycerine or glycerine jelly.
By means of this stain the endothelial cells of the lymphatics, blood vessels, &c., and the nodes of Ranvier, constrictions in medullary nerves, are rendered visible. Sections of any of these may subsequently be stained by logwood or carmine.
Several methods have been adopted for staining with gold chloride. Dr. Klein’s and Professor Schäfer’s are among the best.
Dr. Klein’s method of showing the nerves of the cornea is as follows:—Remove the cornea within fifteen minutes of death; place it in a half per cent. chloride of gold solution for half an hour, or an hour; wash in distilled water, and expose to the light for a few days; in the meantime occasionally change the water. Then immerse it in glycerine and distilled water, in the proportion of one to two; lastly, place it in water, and brush gently with a sable pencil to remove any precipitate, when it will be fit for mounting in glycerine. The colour of the cornea should be grey-violet.
Schäfer adopts another method—a double chloride of gold and potassium solution.
Osmic acid, first used by Schultze, is useful for the demonstration of fatty matters, all of which it colours black; it is also valuable for certain nerve preparations. Specimens should be allowed to remain in a one or two per cent. aqueous solution of the acid from a quarter to twenty-four hours, when the staining will be completed; but if it is desired to harden specimens at the same time, they should remain in it for some few days. Osmic acid does not penetrate very deeply, therefore small portions should be selected for immersion. This is a useful stain for infusorial animals.
Chloride of palladium, another of Schultze’s staining fluids, is used to stain and harden the retina, crystalline lens, and other tissues of the eye, the cornified fat and connective tissues remaining uncoloured. The solution should be used very weak:—Chloride of palladium, one part; distilled water, 1,000 parts. Specimens should be mounted in glycerine at once, or further stained with carmine.
Dr. Schäfer employs a silver nitrate and gelatine solution for demonstrating lung epithelium; this is made as follows:—Take of gelatine ten grammes, soak in cold water, dissolve, and add warm water to 100 cc. Dissolve a decigramme of nitrate of silver in a little distilled water, and add to the gelatine solution. Inject this with a glass syringe into the lung until distension is pretty complete. Leave it to rest in a cool place until the gelatine has set; then cut sections as thin as possible, place them on a slide with glycerine, and expose to light till ready for mounting.
Of the double stains Mr. Groves prefers only those where the double colour is produced by a single process—or stains in which one colour is first employed, and then another. Single stains are picro-carmine, carmine and indigo carmine, aniline blue and aniline red.
Picro-carmine is specially useful for staining sections hardened in picric acid. It is prepared in several ways:—
1. Add to a saturated solution of picric acid in water a strong solution of carmine in ammonia to saturation.
2. Evaporate the mixture to one-fifth its bulk over a water bath, allow it to cool, filter from deposit, and evaporate to dryness, when picro-carmine is left as a crystalline powder of red-ochre colour.
Sections can be stained in a one per cent. aqueous solution, requiring only ten minutes for the process; wash well in distilled water, and transfer them to methylated alcohol, then to absolute alcohol, after which they are rendered transparent by immersing in oil of cloves or benzole, before mounting in balsam or dammar.
To summarise Mr. Groves’ recommendations:—
1. Let the material be quite fresh.
2. (a) Take care that the hardening or softening fluid is not too strong. (b) Use a large bulk of fluid in proportion to the material. (c) Change the fluid frequently. (d) If freezing be employed, take care that the specimen is thoroughly frozen.
3. (a) Always use a sharp razor. (b) Take it with one diagonal sweep through the material. (c) Make the sections as thin as possible; and (d) Remove each one as soon as cut, for if sections accumulate on the knife or razor they are sure to get torn.
4. (a) Do not be in a hurry to stain, but (b) Remember that a weak colouring solution permeates the section better, and produces the best results; and (c) That the thinner the section the better it will take the stains.
5. (a) Always use glass slips and covers free from scratches and bubbles, and chemically clean. (b) Never use any but extra thin circular covers, so that the specimens may be used with high powers. (c) Always use cold preservatives, except in the case of glycerine jelly, and never use warmth to hasten the drying of balsam or dammar, but run a ring of cement round the cover.
6. Label specimens correctly; keep them in a flat tray, and in the dark.