Staining Animal Structures.

Fig. 234.—The Cambridge Rocking Microtome.

Cambridge Rocking Microtome.—This new pattern Cambridge Rocking Microtome (Fig. 234) possesses advantages over other instruments in use for cutting flat sections, and not parts of a cylindrical surface. The tube containing the paraffin is 30 millimetres in internal diameter instead of 20 millimetres, as in the earlier forms. The forward movement is also increased, so that an object 12 millimetres long can be cut throughout its whole length. It is provided with a dividing arc for reading off the thickness of the section in thousandths of a millimetre. The razor may be fixed either with its edge at right angles to the direction of motion of the object, or diagonally, for giving a slicing cut. The object can also be raised and fixed in position clear of the razor.

This microtome has both steadiness and stiffness in its geometrical arrangement and bearings, while the simplicity and efficiency of itsmechanism for advancing the section between each stroke of the razor is remarkable. Although it may appear more complicated at first sight, it is found not to be so when brought into use.

Fig. 235.—Cathcart’s Microtome.Fig. 235a.—Section Cutting Holder for Microtome.

Fig. 235.—Cathcart’s Microtome.

Fig. 235a.—Section Cutting Holder for Microtome.

Cathcart’s Freezing Microtome.—This is a convenient and useful microtome for freezing purposes. Since its first introduction it has been much improved. The clamping arrangements give steadiness, and the principal screw is more effective; the freezing-plate is circular, and the arrangements made for preventing the ether from reaching the upper plate secures the object in view. This instrument can now be used for embedding as well as freezing. The directions for freezing are as follows:—

1. Place a few drops of mucilage (one part gum to three parts water) on the zinc plate.

2. Take a piece of the tissue to be cut, of about a quarter of an inch in thickness, and press it into the gum.

3. Fill the ether bottle with anhydrous methylated ether, and push the spray points into their socket. All spirit must of course have been previously removed by soaking for a night in water. The tissue should afterwards be soaked in gum for a like time before being cut.

Work the spray bellows briskly until the gum begins to freeze; after this work more gently. Be always careful to brush off the frozen vapour which, in a moist atmosphere, may collect below thezinc plate. If the ether should tend to collect in drops below the plate, work the bellows slower.

5. Raise the tissue by turning the milled head, and cut by sliding the knife along the glass plates.

6. After use, be careful to wipe the whole instrument clean.

7. Should the ether point become choked, clear by means of the fine wire provided for the purpose.

8. The instrument is intended for use with methylated sulphuric ether.

9. In clamping the instrument to a table, or other support, care should be taken that the zinc plate is in a horizontal position. If the plate be not horizontal, the gum will tend to run to one side.

The arrangement made for cutting and embedding sections consists of a cylindrical tube (Fig. 235a) fitting into the principal well of the microtome, within which is a hinged plate, upon which the screw acts, as in an ordinary vice. To bring this into use the freezing apparatus must be first removed, and the embedding tube placed in the well, and firmly pressed into place.

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 leftuntil 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 thelifters(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 Appendixfor 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 balsamshould be prepared for use as follows:—One ounce of dried balsam to one fluid ounce of pure benzole; dissolve, and keep in anoutsidestoppered 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 alifter, 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 inFig. 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 thewarm-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 Stainingnucleated 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 somesquamousepithelium; 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 iswished 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 tissuemay be served in the same way.Yellow elastic tissuemust 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 smallfragment on a slide (as shown inFig. 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 silverdarkens 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 anhour, 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.

Dr. W. Stirling46furnishes a brief but useful account of the methods he has employed with much success.

Osmic Acid and Picro-carmine.—Mix on a glass slide a drop of the blood of newt or frog and a drop of a one per cent. aqueous solution of osmic acid, and allow the slide to stand by. This will fix the corpuscles without altering their shape. At the end of five minutes remove any excess of acid with blotting-paper, add a drop of a solution of picro-carmine, and a trace of glycerine to prevent evaporation, and set aside for three or four hours to see that no overstaining takes place. At the end of this time the nucleus will be found to be stained red, and the perinuclear part yellow.

Picric Acid and Picro-carmine.—Place a drop of the blood of a frog or newt on a glass slide, and add a drop of a saturated solution of picric acid: put the slide aside and allow it to remain for five minutes; at the end of that time, when the acid has fixed the corpuscles (that is, coagulated their contents), any excess of acid should be removed as before. A drop of solution of picro-carmine should now be added, and a trace of glycerine, and the preparation set aside for an hour. At the end of that time remove the picro-carmine solution by means of a narrow slip of blotting-paper, and add a drop of Farrant’s solution of glycerine and apply glass-cover. The perinuclear part of the corpuscles will be seen to be highly granular and of a deep orange colour, whilst the nucleus is stained red. Some of the corpuscles will appear of a delicate yellow colour, and threads are seen extending from the nucleus to the envelopes. The preparation should be preserved and mounted in glycerine.

Picro-carmine and Aniline Dye.—For glandular tissue, none of the aniline dyes answer so well as iodine green, used in the form of a one per cent. watery solution. Stain the tissue in picro-carmine, wash it in distilled water acidulated with acetic acid, and stain it in a solution of iodine green. As it acts rapidly, care must be taken not to overstain. Wash the section in water, and then transfer it to alcohol; finally clear with oil of cloves. The washing should be done rapidly, as the spirit dissolves out the green dye. All preparations stained with iodine green must be mounted in dammar.

Picro-carmine and Iodine Green.—Stain a section of the cancellated head of a very young bone (fœtal bone) in picro-carmine, wash it in distilled water, and stain it with iodine green, and mount in dammar. All newly-formed bone is stained red; that in the centre of the osseous trabeculæ, the residue of the calcified cartilage in which the bone is deposited, is stained green. Many of the bone corpuscles are also stained green.

Ossifying cartilage, the back part of the tongue, Peyer’s Patches, solitary-glands, trachea, and bronchus, may all be treated in the same way. In preparing the skin, take a vertical section from the sole of the foot of a fœtus. The cuticle and superficial layers of the epithelium are dyed yellow, the rete Malpighii green; and the continuation of these cells can be traced into the ducts of the sweat-glands, which are green, and form a marked contrast to the red stained connective tissue of the cutis vera, through which they have to ascend to reach the surface. The outer layer of the grey matter of the cerebellum with Purkinge’s cells is, when double stained, red, while the inner or granular layer is green. Logwood and iodine green stains the mucous glands of the tongue green, and the serous glands, lilac logwood stain.

Eosin and Iodine Green.—Eosin is used as the ground colour. Stain the tissue in an alcoholic solution of eosin, which will colour it very rapidly, usually in a few seconds. Wash the section thoroughly in water acidulated with acetic or hydrochloric acid, a one per cent. solution, and stain with iodine green. This will double stain bone and cerebellum; but if logwood is substituted for the latter, the cerebrum and general substance become stained by the eosin, while the logwood colours the nerve-cells a lilac.

Gold Chloride and Aniline Dyes.—The tissue must be impregnated with chloride of gold, and then stained with either aniline blue, iodine green, or rosin. The tail of a young rat, containing as it does so many different structures, is an excellent material for experimenting upon. Remove the skin from the tail, and place pieces half an inch long into the juice of a fresh lemon for five minutes, wash it to get rid of the acid. The fine tendons swell up under the action of the lemon acid, and permit of the more ready action of the chloride of gold solution. Place the piece for an hour or more in a one per cent. solution of gold, remove it and wash it thoroughly, and thenplace it in a twenty-five per cent. solution of formic acid for twenty-four hours. This reduces the gold. During the process of reduction the preparation must be kept in the dark. The osseous portion has then to be decalcified in the ordinary way, with a mixture of chromic and nitric acid. After decalcification preserve the whole in alcohol. Transverse sections of the decalcified tail are made, and may be stained with a red dye, as rosin, and afterwards with a watery solution of iodine green. Mount in dammar.

Fig. 241.—Injecting Syringe.Fig. 242.—Water Bath and Melting Vessels.

Fig. 241.—Injecting Syringe.

Fig. 242.—Water Bath and Melting Vessels.

The injection of animal bodies practised by the older anatomists, to render the vascular system more apparent, has not been superseded by the more modern methods of staining. The method of injecting even small bodies requires some skill, and a few pieces of apparatus made expressly for the purpose. First, a special form of brass syringe of such a size that it may be grasped with the right hand, the thumb at the same time covering the button at the top of the piston-rod when drawn out to the full. InFig. 241the piston rod is seen withdrawn,ais the body, with a screw at the top for firmly screwing down the cover,b, after the piston,c, is replaced;eis a stop-cock, to the end of which either of the smaller cannulæ,g, isaffixed. The transverse wires are for securing them tightly with thread to the vessels into which they are to be inserted. In addition to the syringe, two or three tinned vessels are required to contain size, injecting fluid, and hot water.

The size must be kept hot by the aid of a water bath; if a naked fire be used there is danger of burning it. A convenient form of apparatus for melting the size, and afterwards keeping it at a proper temperature, isFig. 242.

Fig. 243.—Artery Needle.

A pair of strong forceps for seizing the vessel, and a small needle (Fig. 243) is also necessary for passing the thread round the vessel into which the injection pipe has been inserted. These complete the list of apparatus. To prepare the material for opaque injections, take one pound of the finest and most transparent glue, break it into small pieces, put it into an earthen pot, and pour on it three pints of cold water; let it stand twenty-four hours, stirring it now and then with a stick; set it over a slow fire for half an hour, or until all the pieces are perfectly dissolved, skim off the froth from the surface, and strain through a flannel for use. Isinglass and cuttings of parchment make an excellent size, and are preferable for particular injections. If gelatine be employed an ounce to a pint of water will be sufficiently strong, but in very hot weather it is necessary to add a little more gelatine. It must be first soaked in part of the cold water until it swells up and becomes soft, when the rest of the water, made hot, is to be added. The size thus prepared may be fixed with finely levigated vermilion, chrome-yellow, blue salts, or flake white.

To prepare the subject, the principal points to be attained are: to dissolve the fluids and completely empty the vessels; relax the solids; and prevent the injection from coagulating too soon. For this purpose it is necessary to place the animal, or part to be injected, in warm water, as hot as the operator’s hand will bear. This should be kept at nearly the same temperature for some time by occasionally adding hot water. The length of time required is in proportion to the size of the part and the amount of its rigidity.

Injecting the systems of Vessels with different colours: Carmine and Gelatine Injection.—Carmine 30 grains, strong liquid ammonia 60 drops, glacial acetic acid 43 drops, gelatine solution (one ounce in six ounces of water) two ounces, water one ounce: dissolve the carmine in the ammonia and water in a test tube, and mix it with one half of the warm gelatine, add the acid to the remaining half of gelatine, and drop it little by little into the carmine mixture, stirring it well with a glass rod during the mixing; filter through flannel, and add a few drops of carbolic acid to make it keep. It is very important that the stain should be quiteneutral, the test of which is the colour and smell of the fluid. It should be a bright red, and all trace of smell of ammonia must be removed.

Prussian or Berlin Blue and Gelatine.—Take 1½ ounces of gelatine, place it in a vessel and cover it with water; allow it to stand until all the water is absorbed and the gelatine is quite soft, then dissolve in hot water. Dissolve one drachm (60 grains) of Prussian or Berlin blue in six ounces of water, and gradually mix it with the gelatine solution, stirring well with a glass rod during the mixing; then filter as before.

Watery Solution of Berlin Blue.—Dissolve 2½ drachms of the blue in 18 ounces of distilled water, and filter. This staining fluid is used for injecting the lymphatic system.

Directions for Injecting.—The animal to be injected must be first killed by chloroform, and injected while still warm; to secure this place the body in a water bath, at a temperature of 104° Fahrenheit. Expose the main artery of the parts to be injected, clear a small portion of it from the surrounding tissues, and place a ligature of thin tissue or silk round it, by means of the small artery needle (Fig. 243). With a pair of sharp-pointed scissors make an oblique slit in the wall of the vessel, insert the cannula, and tie the ligature firmly over the artery behind the point of the cannula, into which put the stop-cock. Fill the syringe with injection fluid, which must not be too warm, and take care not to draw up any air-bubbles; insert the nozzle of the syringe into the stop-cock and force in a little fluid; remove the syringe so that the air may escape, re-insert the syringe, repeat the process until no air-bubbles escape, and then proceed slowly with the injection. Half an hour will be required to complete the process in an animal the size of a rabbit. To judge of thecompleteness of the injection, examine the vascular parts of the lips, tongues and eyes; if satisfactory, tie the ligature round the artery and withdraw the syringe; place the animal in cold water for an hour to consolidate the injection fluid. When cold dissect out the organs, cut them up, and place them in methylated spirit to harden. Change the spirit every twenty-four hours for the first three days. The hardening process will be complete in ten days.

To inject lymphatics by the puncture process, a small-sized subcutaneous syringe should be used, filled with a watery solution of the prepared stains. Thrust the nozzle into the pad of the foot, (or tongue), and then rub the limb to cause the injection fluid to flow along the lymphatic vessels into the glands.

When the blue stain is used add a few drops of acetic acid to the spirit while the hardening process is going on.

Of Injecting Different Systems of Vessels with Different Colours.—It is often desirable to inject different systems of vessels distributed to a part with different colours, in order to ascertain the arrangement of each set of vessels and their relation to each other. A portion of the gall-bladder in which the veins have been injected with white lead, and the arteries with vermilion, forms an attractive preparation. Each artery, even to its smallest branches, is seen to be accompanied by two small veins, one lying on either side of it. By this method four different sets of tubes have been injected—the artery with vermilion, the portal vein with white lead, the duct with Prussian blue, and the hepatic vein with lake. There are also opaque colouring matters which may be employed for double injections.

Injecting the Lower Animals.—The vessels of fishes are exceedingly tender, and require great caution in filling them. It is often difficult or quite impossible to tie the pipe in the vessel of a fish, and it will generally be found a much easier process to cut off the tail of the fish, and put the pipe into the divided vessel which lies immediately beneath the spinal column. In this simple manner beautiful injections of fish may be made.

Mollusca(slug, snail, oyster, &c.).—The tenuity of the vessels of the mollusc often renders it impossible to tie the pipe in the usual manner. The capillaries are, however, usually very large, so that the injection runs very readily. In different parts of the bodies of these animals are numerous lacunæ or spaces, which communicatedirectly with the vessels. Now, if an opening be made through the integument of the muscular foot of the animal, a pipe may be inserted, and thus the vessels may be injected from these lacunæ with comparative facility.

Insects.—Injections of insects may be made by forcing the injection into the general abdominal cavity, when it passes into the dorsal vessel and is afterwards distributed throughout the system. The superfluous injection is then washed away, and such parts of the body as may be required removed for examination.

Natural injection of Medusæ may be effected without injuring the vessels, with an opening at the side remote from it. The medusa must be placed in a glass vessel, with the bell downwards, and a bell-jar ending in a narrow tube above is placed over it and made air-tight; the medusa is then covered with the injection-mass, the air in the glass is exhausted, and as the sea-water runs out by slits in the lower side of the annular canal, the coloured fluid runs in. In the case of leeches and large species of earthworms, the natural injection is made from the ventral sinus. In all cases a glass tube is used, with a finely drawn-out point. The injection is complete when the injection issues from the counter-opening. Besides the animals mentioned, large caterpillars, beetles, and larvæ of various kinds are favourable objects for injection; the glass cannula being introduced into the posterior end of the dorsal vessel, and the counter-opening made in the ventral vessel, andvice versâ.

Staining Living Protoplasm with Bismarck Brown.—Henneguy having treatedParamœcium aureliawith an aqueous solution of aniline brown (known as “Bismarck Brown”), found that they assumed an intense yellow-brown colour. The colour first appears in the vacuoles of the protoplasm, and then in the protoplasm itself, the nucleus generally remaining colourless, and becoming more visible than in the normal state. If a yellow-tinted paramœcium be compressed so as to cause a small quantity of the protoplasm to exude, it is seen that it really is the protoplasmic substance which becomes coloured. All the Infusoria may be stained with Bismarck brown, but no other aniline colour employed exhibits the same property—they merely stain the Infusoria after death, and are in fact poisonous. Living protoplasm does not as a rule absorb colouring matters, and as Infusoria are chiefly composed of protoplasm,attempts have been made to ascertain whether protoplasm in general, of animal or vegetable origin, behaved in the same way in the presence of aniline brown. A tolerably strong solution of Bismarck brown was therefore injected under the skin of the back of several frogs. After some hours the tissues became uniformly tinted a deep yellow; the muscular substance especially had a very marked yellow tint. The frogs did not appear in the least incommoded. Small fry of trout placed in a solution stained rapidly and continued to swim about. Finally, a guinea-pig, under whose skin some powder of Bismarck brown had been introduced, soon presented a yellow staining of the buccal and anal mucous membranes and of the skin. Seeds of cress sown on cotton soaked with a concentrated solution of the Bismarck brown sprouted, and the young plants were strongly stained brown; but on crushing the tissues and examining them under the microscope, it was ascertained that the protoplasm of the cells was very feebly coloured: the vessels, on the contrary, showed a deep brown stain up to their termination of the leaf. The mycelium of a mould developed in a solution of Bismarck brown was clearly stained after having been washed in water, whilst it is known that the mycelium, which frequently forms in coloured solutions, picro-carmine, hæmatoxylin, &c., remained perfectly colourless. Other aniline colours injected under the skin of frogs stained the connective tissue as deeply as did the Bismarck brown; but the striæ of muscle remained colourless. We may conclude, then, that Bismarck brown possesses the quality of colouring living protoplasm both in plants and in animals.

Take the femur of cat, or rabbit, remove as much of the muscle as possible and macerate it in water until quite clean; on removal hang it up to dry. With a fine saw make transverse and longitudinal sections. File the section down until flat, and smooth. Take some Canada balsam, place a piece on a square of glass and warm gently over a lamp until the balsam is plastic enough to allow of the section being pressed into it, and set it aside to consolidate. Take a hone (“Water-of-Ayr” stone), moisten it with water, and rub one side of the section upon it until quite smooth, then place the glass slip, with the section stillattached, into methylated spirit, and in a very short time the section will be separated; wash it and remount it on the reverse side, and proceed to rub it down on the hone until it appears to be thin enough for mounting. Polish both sides on a polishing strop with Tripoli powder, and mount in Canada balsam.

Fig. 244.—Small Lathe for cutting and polishing Sections of Teeth.

Teeth.—The enamel of the teeth is a much harder structure than that of bone, consequently it is found necessary to have recourse to a cutting machine. Hand machines have been introduced for this purpose, but the small lathe described in the earlier editions of my book has in no way been superseded by later cutting machines.Fig. 244represents the small lathe used for cutting and polishing every kind of hard substance. With regard to the teeth, two sections should be made perpendicular to one another through the middle of the crown and fang of the tooth from before backwards, and from right to left, which will show the peculiar structure of the enamel. The section must be cemented to the carrier of the stock of the lathe, or to the metal platea, and kept in position by the steel holderb; the wheel being set in motion by the first treadle. The embedding materials in use are either gum-shellac or Canada balsam. The former is more generally employed by the lapidary and grinder of lenses than the latter. As the enamel is liable to fracture under the saw, it will be necessary to lessen the friction by dripping water on the saw as it is made to revolve. Thick sections can be quickly ground down against the corrondum wheel. The final polishing of the section may be done on the lathe, or by rubbing the flattened surface with water upon a “Water-of-Ayr” stone, and ultimately set up in Canada balsam, which must not be too fluid, or it will penetrate thelacunæandcanaliculi, fill up the interspaces, and cause them to become quite invisible. As the flatness of the polishing surfaces is a matter of importance, the stones themselves should be tested from time to time, and when found to present an uneven surface must be rubbed downon a granite stone with fine sand, or on a lead plate with emery powder. If it is decided to use Canada balsam as the embedding material, this must be prepared in the following manner:—The section of tooth or bone must be attached to a slip of well-annealed glass by hardened Canada balsam, and its adhesion effectually secured by placing the slide on the cover of a water bath, or in the hot-chamber (Fig. 256), when the balsam, a thick drop of which should be used, will spread out by liquefaction. The slide should then be removed and allowed to cool in order that the hardness of the balsam may be tested. If too soft, as indicated by its readily yielding to the pressure of the thumb-nail, the heating process must be repeated, care being taken not to cause it to boil and form bubbles; if too hard, which will be shown by its chipping, it must be remelted and diluted with fluid balsam, and then set aside as before. When it is found to be of the right consistence, the section must be laid upon its surface with the polished side downwards; the slip of glass is next to be gradually warmed until the balsam is softened, care being taken to avoid the formation of bubbles, then press the section gently down with a needle upon the liquefied balsam, the pressure being just applied on one side rather than over the whole surface, so as to drive the superfluous balsam towards the opposite side; finally, an equable pressure over the whole will secure a perfect attachment of the section without air bubbles. If, however, these should present themselves in drying, and they cannot otherwise be expelled by pressure, it will be found better to take the section off and relay it as before. The thickness of the layer of balsam may be reduced by rubbing it down before applying the glass-cover.

Rock Sections.—Small pieces of rock may be ground down by the aid of the lathe, or on a zinc plate, with emery powder and water, until one side is rendered smooth and flat. Then fasten the polished side of the section to a square of glass on the metal holder of the lathe, with dried Canada balsam, as directed for bone, and allow it time to become consolidated. When moderately thin take a piece of plate-glass and some fine emery or putty-powder and rub the section down as thin as possible. When found to be thin enough wash it well in water, and put it aside to dry, or warm it over a spirit-lamp, and with a needle push the section off the glass into a watch-glass of benzole or turpentine, and allow it to soak until all the balsam isdissolved out. Wash again in turpentine, and mount in Canada balsam, with or without a cover-glass. Sections of echinus spines, shells, stones of fruits, &c., are prepared in the same way as those of bones and teeth; but when the grinding is finished, the sections must be passed through alcohol into oil of cloves, after which they should be mounted in Canada balsam. If tolerably thin, sections of these substances can be cut in the lathe; in the first instance, there will be no actual occasion to attach them to glass at all, except for the purpose of obtaining a hold upon the specimen for polishing, but the surface thus attached must afterwards be completely removed in order to bring into view a stratum which the Canada balsam may not have penetrated.

With regard to smaller bodies, these can scarcely be treated in any other way than by attaching a number of them to slips of glass at once, and in such a way as to make them mutually support each other. Thus in making horizontal and vertical sections offoraminifera, it would be impossible to slice them through unless they were laid close together in a bed of hardened Canada balsam, and first grinding away one side and then turning and rubbing down the other. My friend, Dr. Wallich, many years ago communicated to me the ingenious plan adopted by himself when mounting and turning a number of these minute objects together. The specimens being cemented with Canada balsam, in the first instance, to a thin film of mica, and then attached to a glass slide by the same means, when ground down to the thinness desired, the slide must be gradually heated just sufficiently to allow of the detachment of the mica-film and the specimen it carries; a clean slide with a thin layer of hardened balsam having been prepared, the mica-film is transferred to it with the ground surface downward. Its adhesion by drying having been complete, the grinding and polishing should be proceeded with; and as the mica-film will yield to the stone without any difficulty, the specimen now reversed in position may be further reduced to the requisite thickness for mounting as a permanent object.

Staining and Mounting Vegetable Tissues.—Bacteria I propose to treat of in a separate section. Vegetable tissues generally will first receive attention, and their differentiation is based on the employment of delicate gradations of colour stains. The more strikingresults are obtained byMultiple Staining, while the cell contents are rendered more palpable. On this account colouring media have been divided intonuclear,plasmic, andspecific. The first are chiefly valued in proportion as they prove to have a selective affinity for the nuclei of cells, and leaving the protoplasm comparatively unstained. Such stains are needful for fresh and young tissues. On the other hand,plasmicstains colour tissue uniformly, and are used to give a ground colour by way of contrast; andspecificstains are chiefly employed to distinguish certain elementary structures from the mass of cellulose which forms the basis of vegetable tissue, and which is also met with to a slight extent in animal membranes.

Cellulose, as it occurs in plant life, presents a variety of physical properties: sometimes it is soft, as in young plants, and again quite dense in older structures. This fact accounts for the varying results obtained when cellulose is subjected to the action of staining fluids, and whether the cellulose occurs in a nearly pure form, as in cotton fibre, or in the modified form of lignine or woody-fibre. Stains which readily attack young tissue have little or no effect upon it in its maturer form. It is of much importance, then, in the staining of fibres, as well as sections for the microscope, that the cellulose should take the stain uniformly.

The staining of tissues may be effected in four ways. First, when the stain has sufficient affinity for the tissue to be retained by it without the intervention of any outside agent. Second, when the stain and mordant are mixed and applied to the tissue in one solution. These two are the simplest and easiest methods of staining. Third, when the tissue is first immersed in the staining liquid and then transferred to some other liquid which shall fix the colour upon the tissue. Fourth, when the tissue is first impregnated with the mordant, or fixing agent, and then immersed in the stain. The last method is the one usually followed in commercial works, and it is to be recommended in the staining of microscopical preparations which do not readily take the stain.

Nuclear Stains.—As in both vegetable and animal sections it is generally the nuclei which form the landmarks of the structure, so the most important class of reagents which are used in any of the branches of microscopical work are the “nuclear stains.” There are several of these stains, the most important of which is thehæmatoxylin, and when proper solutions are used the results are very satisfactory. Many formulæ have been given, but there are three only reliable, Delafield’s, Kleinenberg’s, and Ehrlich’s, in all of which alum is present as an ingredient; the idea being that the alumina forms with the colouring matter an insoluble lake, and so acts as a mordant.

InDelafield’ssolutions a large proportion of alum to hæmatoxylin is used, and methylic alcohol (wood-spirit in the place of rectified spirit).

ForKleinenberg’ssolution many different formulæ exist. Squire’s improved formulæ for both stains is given in the Appendix, “Formulæ and Methods.”

Hæmatoxylin solutions stain the nuclei violet, and in order to change this into blue, the sections should be transferred to water taken from the house supply, not distilled water; but as the alkalinity of the water varies in different localities, a better and more uniform result is obtained by using a weak solution of bicarbonate of sodium (half a grain to the ounce).

Carmineis also much in vogue as a nuclear stain, and the two solutions more generally employed are Greenacher’s alcoholic borax carmine, and Orth’s lithium carmine. Under ordinary circumstances they act as general stains, affecting the ground tissue as well as the nuclei. By subsequent treatment with acidulated alcohol or acidulated glycerine the colour is discharged from the ground tissue without seriously affecting the nuclei. Used in this way, carmine becomes a good nuclear stain. It should be remembered that the sections must not be washed in pure water, as the colour will to a great extent be discharged; nor in acidulated water, as the carmine will be precipitated.

Alum carmine and alum cochineal are useful nuclear stains, not requiring after-treatment.

Picro-carminesare also largely used. The following formulæ will be found the most useful:—

Ammonia Picro-carmine.—Carmine, one gramme; strong solution of ammonia, three cc.; distilled water, five cc. Dissolve the carmine in the ammonia and water with a gentle heat, then add saturated aqueous solution of picric acid, 200 cc.; heat to boiling, and filter.

Picro-Lithium Carmine.—The following is generally preferred for use—Lithium carmine solution, 100 cc.; saturated solution of picric acid, 270 cc.

There are several aniline dyes which are used for nuclear staining: methylene blue, methyl green, safranine, gentian violet, vesuvine, fuchsine, and Hoffmann’s blue.

The usual process is to stain in ¼ or ½ per cent. aqueous solutions and wash in methylated spirit. Methylene blue and methyl green have the reputation of being so readily washed out in the methylated spirit as to be worthless. This is obviated by washing the sections (when removed from the stain) in distilled water, previous to the differentiation in methylated spirit. Treated in this manner, the nuclear staining is very beautiful. This also applies to Hoffmann’s blue and partly to vesuvine; with the latter, however, it is not a necessity. Safranine and gentian violet worked better by transferring the sections directly from the stain into 90 per cent. alcohol.

Contrast Stains.—Very frequently other stains are used to dye the ground a colour which is in contrast to that employed for the nuclei. Brown, orange, or pink are used after nuclear blue or green. Carmine is generally counterstained yellow or indigo-blue; and fuchsine red, as in tubercle bacilli, is counterstained with nuclear blue. It is important that the ground stain should be made weaker than the principal stain, so that the whole tissue may be shown without detracting from the nuclei. The following colours are used as counterstains for animal sections, but they prove less useful for vegetable sections: benzo-purpurine, eosin, erythrosine, orange, acid rubin, and picric acid.

Examples ofspecificstains are fuchsine, methylene blue, and gentian violet for bacteria; osmic acid for fatty elements; victoria blue and rose bengale, for demonstrating elastic tissue; methyl violet, iodine, and safranine, for amyloid degeneration. Methylene blue is one of the most useful of aniline dyes, and one of the most variable in composition.

Iodine green, or methyl green, has long been in use as a reagent for amyloid, starchy matters, in ignorance of the fact that the reaction is due to the methyl violet, contained as an impurity in the iodine green. It is exceedingly difficult to obtain a green quite free fromviolet. As nuclear stains they are identical, and the amyloid reaction, being dependent wholly upon the contained violet, varies, not with the formula of the green, but with the extent to which it has been purified.

Cellulose reactions.—After the nuclear stains, the most important reagents to the botanist are those which affect cellulose and its several modifications. Pure cellulose is coloured yellow by iodine, the colour being changed to a blue on the addition of slightly dilute sulphuric acid, or a strong solution of zinc. Solutions containing iodine, iodide of potassium, and chloride of zinc, give a violet reaction with unaltered cellulose, and yellow with lignine.

Schulze’s zinc re-agent must be used with a certain amount of caution, as the chloride of zinc and potassium undergo decomposition. The formula now in use is as follows: Take of zinc chloride solution (sp. gr. 1·85) 70 cc., potassium iodide 10 grammes, iodine 0·1 gramme; but this solution can only be employed as a re-agent and not as a dye, and structures stained with it cannot be mounted in any of the ordinary media, and the only fluid for ringing them down is caoutchouc cement.

Cellulose can be stained permanently by carmine, hæmatoxylin, nigrosine, methylene blue, safranine, and fuchsine. The aniline dyes are used in dilute aqueous solutions containing one-eighth or one-fourth per cent. of dye. When the cellulose undergoes the change known as lignification its reactions are altered. It is coloured yellow by chloro-zinc iodine, red by phloroglucin, yellow by aniline chloride. The two latter are much assisted by hydrochloric acid. The results of these reactions also cannot be preserved in the usual mounting media.

Sections containing mixed tissue, partly unaltered cellulose and partly lignified, give striking results with aniline dyes, and with this additional advantage can be preserved for years.

Double Staining.—When a section is passed through methyl green solution and afterwards one of carmine, the lignified portion is coloured green and the unlignified red. Acid green may be used in the place of methyl green, with a similar result. When picric acid is used with carmine, ingrosine, or Hoffmann’s blue, the picric acid dyes the ligneous portion and the others colour the unlignified structure, red, black, and blue respectively.

Eosin stainis the most useful for sieve-tubes and plates. Make a strong solution of eosin in equal parts of water and alcohol, and stain the section for five or ten minutes. Wash well in methylated spirit, dehydrate, clean in oil of cloves, and mount in Canada balsam.

Bleaching Process.—The bleaching and clearing of vegetable structures before staining is a very necessary process, especially so if starch be present in any quantity. Clearing agents are of two kinds—those which act by virtue of their property of strongly refracting light, and those which disintegrate and dissolve the amyloid cell contents. To the first class belong the essential oils, as oil of cloves, Canada balsam, glycerine, and other similar bodies; to the second class, solutions of potash, phenol, and chloral hydrate. The actual value of some of these agents is questionable. The process usually preferred is as follows: Place the sections in a fresh clear solution of chlorinated lime, allowing them to remain until quite bleached, say from two to four or five minutes; then gently warm in a test-tube for a few seconds, and quickly replace the solution with distilled water and boil for two or three minutes; repeat the treatment with boiling water three times; wash with a one per cent. solution of acetic acid, and finally with distilled water. The sections are then quite ready for staining operations.

When the stem is hard and brown, a solution of chloride of lime should be used—a quarter of an ounce of chloride dissolved in a pint of water, well shaken and stood by to settle down, then pour off the clear fluid for use. For hard tissues this solution answers well, but it is not suitable for leaves, as they require not only bleaching, but the cell contents should be dissolved out to render them transparent. A solution of chlorinated soda answers well for both stems and leaves. It is prepared as follows:—

To one pint of water add two ounces of fresh chloride of lime, shake or stir it well two or three times, then allow it to stand till the lime has settled. Prepare meanwhile a saturated solution of carbonate of soda—common washing soda. Now pour off the clear supernatant fluid from the chloride of lime, and add to it, by degrees, the soda solution, when a precipitate of carbonate of lime will be thrown down; continue to add the soda solution till no further precipitate is formed. Filter the solution, and keep it in a well-stoppered bottle in the dark, otherwise it speedily spoils.

Sections bleached in chlorinated soda must, when white enough, be washed in distilled water, and allowed to remain in it for twenty-four hours, changing the water four or five times, and adding a few drops of nitric acid, or at the rate of eight or ten drops to the half-pint, to the water employed before the final washing takes place. From water transfer them to alcohol, in which they must remain for an hour or more.

Although alkaline glycerine has been recommended for several purposes in micro-technique, it is not so well known as it should be how serviceable it is as an extempore mounting solution in vegetable histology. The best mixture for general use is composed of glycerine 2 ozs., distilled water 1½ oz., solution of potash, B.P., ½ oz. This combines the refringent property of the glycerine with the clearing action of the caustic potash, while the swelling action of the potash is considerably diminished.

Cutting Sections of Hard Woods.—The lathe and circular saw will be found as useful for cutting sections of the harder kinds of woods, as for bone structure. It may be necessary to subject the older and consequently harder pieces of wood to the action of steam for a few hours to soften them, and afterwards transfer them to methylated spirit, before making an attempt to cut sections. But the more open woods, of one, two, or three years’ growth, will show all that may be required, and these can be cut by hand, or with the microtome, as already described.

With a little practice the finest and thinnest possible slices may be cut by hand. It is usual to take off the first slice to give a smooth and even surface to the specimen. Then turn the screw to raise it a little, sprinkle the surface with spirit and water, and cut with a light hand. Remove the cut sections with a fine camel’s-hair brush or a section lifter (Fig. 250) to a small vessel containing water, when the thinnest will float on the surface, and remove to methylated spirit and water, where they should remain until they can be mounted. Sections of hard woods, and those containing gum-resins, or other insoluble material, must first be kept in methylated spirit or alcohol, and finally transferred to oil of cloves, to render them sufficiently transparent for mounting in Canada balsam.

If the structure of an exogenous wood is required to be examined,the sections should be made in at least three different ways: the transverse, the longitudinal, and the oblique, or, as they are sometimes called, the horizontal, vertical, and tangential, each of which will exhibit different appearances, as seen inFig. 245:bis a vertical section through the pith of a coniferous plant, and exhibits the medullary rays known to the cabinet-maker as the silver grain;eis a magnified view of a part of the same; the woody fibres are seen with their dotsl, and the horizontal lineskindicating the medullary rays cut lengthwise;cis a tangential section, andfa portion of the same; the medullary raysm m, and the woody fibres with vertical slices of the dots, are shown. Instructive preparations will be secured by cutting oblique sections of the stem. The sections seen are made from the pine. All exogenous stems, however, exhibit three different appearances, according to the direction in which the section is made.

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