(2)Carbonate of potassium201/2oz.Water10021/2oz.
Mix the two solutions. Allow them to stand for an hour and filter.
It is used chiefly for clearing vegetable sections but may also be used for sections containing a large quantity of pigment. It is particularly useful in decolourizing sections of “madura foot” due to the presence of a black fungus.
Embedding of sections.—Before sections are made the tissues require to be embedded in some fluid, which will permeate their interstices, and is capable of being rendered firm so as to support the most delicate parts when the knife passes through the tissue.
The most generally useful substancesare:—
(1) gum, (2) celloidin, (3) paraffin or wax.
Gum.—Pickedcolourless gum arabic 2 parts, cold water 3 parts.
Leave with frequent stirring until dissolved. Add ten drops of carbolic acid to each ounce of the mucilage.
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. If this have occurred the surface can be softened sufficiently by breathing gently on it.
After cutting in gum, the sections are gently removed from the knife into distilled water by a soft camel’s hair brush, and left there for an hour or two, until the medium is entirely dissolved out. They may then be stained and mounted, or they may be put away in spirit for an indefinite time, and then stained and mounted.
Celloidinis for many purposes almost an ideal embedding medium. (1) It has great penetrating power; (2) it can be made of an admirable consistence for cutting purposes; (3) after sections are made it allows them to be very freely manipulated without fear of injuring them: (4) and being perfectly transparent and homogeneous in thin sections, it does not require to be removed from a section before mounting. It is insoluble in water, and in weak spirit; slightly soluble in alcohol of more than 90 per cent. strength, andvery readily soluble in ether, or in a mixture of alcohol and ether. The last solvent is the one commonly employed.
The embedding solution is madethus:—
Take some pure celloidin (“Schering’s,” sold in boxes containing an ounce of shavings, is very good) and pour on it about eight times its volume of a mixture of equal parts of absolute alcohol and ether. Allow this to stand all night until the celloidin is dissolved. The solution should be made about the consistence of ordinary mucilage.
It is also convenient to have a thinner solution made by using double the proportion of alcohol and ether. Both solutions should be kept in wide mouthed stoppered bottles, and the stopper should be well greased with vaseline as an additional obstacle to the evaporation of the volatile ether.
Before embedding a specimen it is necessary to dehydrate it thoroughly for twelve to twenty-four hours in absolute alcohol. It should then be placed in a mixture containing alcohol and ether for an hour or two, and afterwards transferred to the thin solution of celloidin for twenty-four hours, and then to the thick solution for the same period.The celloidin penetrates slowly and in the case of nerve tissues and other delicate structures it is wise to give the full allowance of time for the different steps. When the tissue has been thoroughly permeated by the celloidin, it is gently removed from the celloidin and placed in position on a piece of cork of suitable size for clamping in the holder of the microtome. Celloidin is painted round the object so that it is supported on every side. It is then left exposed to the air until the surface has become firm, when the cork is placed, with the tissue downwards, in methylated spirit. The cork floats but the tissue and celloidin remain submerged. At the end of twenty-four hours the celloidin will have become semi-opaque and opalescent, and of the same consistence as hard boiled white of egg. When it is impossible to wait so long, rapid hardening of the celloidin may be secured by immersing it in methylated chloroform in place of spirit, but the slower method gives more uniformly satisfactory results.
Pieces of tissue embedded in celloidin may also be cut on a freezing microtome. Afterthe celloidin has become firm by immersion in methylated spirit, the tissue with the celloidin round it may be cut off the cork, washed in water to remove the alcohol, and then soaked for an hour or two in gum, placed on the plate of an ether spray microtome, frozen and cut in the usual way.
Subsequent staining operations are conducted in the same way as for sections cut by hand or in gum. As celloidin is only slightly stained by hæmatoxylin, alum carmine, borax carmine, &c., it is not necessary to remove it from the sections, but it exhibits so intense a staining reaction with aniline dyes that it is necessary to remove it by treatment with alcohol and ether either before or after the staining operation.
The sections after staining may be mounted in Farrant’s solution (p.59), or in Canada balsam (p.61). If the latter medium is employed, the section should be clarified, after dehydration in alcohol, by means of oil of bergamot, or oil of origanum, instead of oil of cloves, as the latter dissolves out the celloidin and causes the section to break up.
Celloidin is most useful for cutting sections of the coats of the eye, of the internal ear, and of bone marrow. It should always be used for the Weigert-Pal hæmatoxyline method of staining the nervous centres, as it protects the section from being injured by the transference from one fluid to another which is repeatedly required during the process. The stain is completely discharged from the celloidin by the decolourising solution used (p.90).
Paraffin.—Paraffin is a very convenient embedding medium for delicate structures, as very thin sections can be obtained and the paraffin need not be removed from the section until the latter is safely on the slide. It is unsuitable for large sections. Staining operations are not easily carried out after cutting in paraffin, and it is better to stain the blocks of tissue in bulk before embedding. The best stains for penetrating are borax carmine (p.75), alum carmine (p.76), and Kleinenberg’s hæmatoxyline (p.70). The tissue must be left in them for four to ten days.
Various kinds of paraffin are employed. It is usual to keep two kinds, one “soft,” melting at110° F., and another “hard,” melting at 140° F. A mixture of two parts of the hard and one of the soft will be found most generally useful. In winter a large proportion of the soft variety and in hot weather a larger proportion of the hard may be required. A paraffin mass which is always available has been suggested recently by Dr. F. E. Batten, who employs an ordinary white candle, composed of paraffin and wax. If the mass is found to be too hard, it can easily be made of a suitable consistence by adding a little paraffin with a low melting point.
To prepare a piece of tissue for embedding in paraffin, it should be stained, washed in distilled water, and as much moisture as possible removed by blotting paper. The block is then dehydrated, first in methylated spirit for several hours, finally in absolute alcohol. It is taken carefully by means of forceps from the alcohol and placed in xylol for an hour or two according to size. Superfluous xylol is removed from the surface, and the tissue placed in the melted paraffin. This will set round the cold tissue at once, but soon melts again and must be kept at a temperature justabove melting point for one to four hours, according to size. The tissue is then transferred to a mould (which can be easily made of paper), about half an inch cube, and melted paraffin poured round it until the mould is full. The mould may be made by folding a piece of paper to form a box about half an inch cube, or a small pill box may be used. Another convenient method is to place two L-shaped pieces of lead in contact with each other so as to enclose a space of suitable size as in the diagram (fig.3). The tissue is now hermetically sealed, and can be kept indefinitely if it is not convenient to cut it at the time. To prepare it for cutting, all superfluous paraffin is trimmed away with a warm knife, and the block is fixed on a piece of wood, cut so as to suit the clamp of the microtome, by melting the lower end of theparaffin block with a hot needle or wire and pressing it down on the wood.
Fig. 3.
Fig. 3.
Fig. 3.
When sections are cut they may be transferred singly to the slide (which should be lightly smeared beforehand with a saturated solution of celloidin in oil of cloves), or they may be cut so that the back of one section of the paraffin block adheres to the front of the next, and in this way a continuous delicate ribbon of serial sections is obtained. The ribbon is broken up into lengths of about two and a half inches and transferred to the slide, on which several ribbons may be placed side by side, and so a large number of sections kept in the order in which they are cut. A mark should be made on the slide to indicate where the series begins, and each slide should be numbered, so that the exact position of each section in the series can be recognised at once.
Before mounting, the paraffin must be removed from the sections. This is easily done on the slide in the case of single sections and of ribbons. If the sections are curled, a little warmth will make them unbend and lie flat. The slide is warmed over a spirit lamp until the paraffin justmelts. The sections will keep their places owing to the celloidin beneath. Xylol is then allowed to flow over the slide from a pipette, until the paraffin has been completely dissolved, which can be ascertained by glancing at the sections under the low power of the microscope. The slide is placed in an almost vertical position to let the xylol drain off, excess is wiped off from the edge of the slide with blotting paper, a drop of Canada balsam solution (p.61) is run on the slide, and a cover-glass of suitable size is applied.
Microtomes.—After a large amount of practice, persons with a fair amount of manual dexterity may acquire sufficient skill to be able to cut very satisfactory sections of specimens embedded in paraffin, &c., by hand. In the Pathological Laboratory of a large German University, until quite recently the use of a microtome was prohibited by the Professor, who is himself a most distinguished histologist. The amount of time expended before one acquires the necessary skill, and the cheapness and great convenience of the modern microtome have combined to throw hand cutting into the background, and some form of microtome is now almost universally adopted.
Fig. 4.—Cathcart’s Ether Spray Microtome.A, B. Wooden frame and supports. C. Glass runners. G. Screw for raising the zinc plate H. J. Ether bottle. L. Tube from air bellows.
Fig. 4.—Cathcart’s Ether Spray Microtome.A, B. Wooden frame and supports. C. Glass runners. G. Screw for raising the zinc plate H. J. Ether bottle. L. Tube from air bellows.
Fig. 4.—Cathcart’s Ether Spray Microtome.
A, B. Wooden frame and supports. C. Glass runners. G. Screw for raising the zinc plate H. J. Ether bottle. L. Tube from air bellows.
Of these there are a very large number in the market, each having special advantages, and often special drawbacks. A few of the more generally useful only will be described. We have microtomes for cutting in gum frozen by ether spray or ice, and those intended for cutting in paraffin or celloidin.
Cathcart’sether spray microtome(fig. 4).—This, or its more recent modifications (seelater), is perhaps the most useful and economical microtome for the purposes of the student. Its prime cost is low, it is small and portable as well as being clean and inexpensive to work with.
It consists of an oak frame which can be firmly clamped on to a table. On this frame are two narrow parallel supports about two inches high, which are covered by strips of plate glass, and serve as smooth rests along which the razor may glide in making sections. Between them is a brass well and in this a zinc plate firmly fixed in the horizontal position, which is almost at the level of the glass runners. It is capable of being raised or lowered through about3/8inch by means of a screw with a very fine and accurate thread. This screw is turned by a large milled wheel beneath the microtome. Just beneath the zinc plate are two small tubes, one connected with an india-rubber bellows, the other with a bottle at the side which contains ether. As the air issues from the first tube, it passes over the open end of the second, and thus draws the ether out and makes it play on the zinc plate, and at the sametime causes it rapidly to evaporate, and so reduces the temperature of the zinc plate.
In cutting sections with this microtome the tissue is taken out of the gum and placed on the zinc plate. The bellows are then worked until the gum on the zinc plate is completely frozen. The plate should be lowered by means of the screw until the surface of the piece of tissue is on a level with the glass runners. These and the razor should then be wetted with water. The razor being held firmly in the hand is pushed along the glass runners in a rather oblique direction. The plate should then be raised by turning the screw below through a very small arc, another section taken off and so on. Sections are carefully removed from the razor to a vessel of water by means of a soft wet camel’s hair brush. The needle should never be used for this purpose.
If the specimen is very delicate, and likely to be spoiled by being curled up on the knife, the latter should be kept cold by frequently dipping it in a vessel containing lumps of ice in water. The gum will then remain frozen after cutting, and support the tissue better. Each section should be at oncetransferred to a glass slide from the knife, washing it off with a stream of ice water from a pipette.
The knife that is used may be an ordinary razor, with the edge ground straight. It requires to be held steadily with both hands. As this is rather inconvenient, Dr. Sheridan Délépine suggested the employment of an ordinary plane iron such as is used in a carpenter’s plane. This only requires one hand, and the other can be kept on the head of the screw beneath to raise the plate at once after each stroke of the knife. Its disadvantages are that it is rather heavy for prolonged working, and that it is less easy to “set” than a razor.
A. Frazer has recently introduced a valuable improvement in the Cathcart microtome (fig.5).
In this the brass frame carrying the zinc plate and ether spray tubes is surrounded by a brass cylinder, in which it fits accurately, and is pushed up as desired by turning the screw beneath the instrument. This brass frame and with it the zinc plate, &c., can be easily drawn altogether out of the outer tube, and replaced by a second brass well, which exactly fits its place and can be raisedby the screw as desired. In this is a small toothed clamp which can be screwed up so as to hold a piece of wood carrying a piece of tissue embedded in paraffin. Sections can also be cut in celloidin with this instrument, but as oblique strokes with the knife cannot be made, it is impossible to get very thin sections. The combined microtome can be obtained for a guinea from Frazer, 22 Teviot Row, Edinburgh.
Fig. 5.—Frazer’s Modification of Cathcart’s Microtome.A. Microtome arranged for ether spray. B. Cylinder with clamp for holding object embedded in celloidin, &c. to replace ether spray apparatus.
Fig. 5.—Frazer’s Modification of Cathcart’s Microtome.A. Microtome arranged for ether spray. B. Cylinder with clamp for holding object embedded in celloidin, &c. to replace ether spray apparatus.
Fig. 5.—Frazer’s Modification of Cathcart’s Microtome.
A. Microtome arranged for ether spray. B. Cylinder with clamp for holding object embedded in celloidin, &c. to replace ether spray apparatus.
There is another modification which is more generally useful, and at the same time more expensive than the original model. In this, instead of glass runners to support the knife, there is a flat glass plate about eight inches square sufficiently large to allow of “Swift’s plough” (fig.6) being used for the purpose of cutting sections. This instrument consists of a triangular brass frame, supported on three legs, each of which is a screw, tipped with ivory. There is one screw in front and two behind. Beneath the plate, and held in position by the posterior screws in front, and a little clamp behind, is a razor with the edge directed forwards. The edge can be raised or depressed by turning the anterior screw, on whichthe frame is supported. Before sections are cut the edge of the razor should be brought down to the level of the tissue, taking care that all the legs are equal in length. The plough should then be firmly grasped with both hands, (the index finger of one hand being left free to turn the anterior screw) and pushed rather obliquely through the tissue. The edge of the razor is then slightly lowered by turning the screw through a very small angle, and another section made, and so on. With a little practice very thin uniform sections may be made with great rapidity.
Another useful ether spray microtome is that made by Jung of Heidelburg. The knife swings round a pivot, and there is an ingenious ratchet arrangement which works synchronously with each swing of the knife, to raise the tissue automatically the requisite distance for the next section to be made. The exact thickness of the sections can be graduated with great nicety by a simple contrivance. The instrument can be obtained in this country for about £2. It works satisfactorily, but, with practice, the student will get equally good results with the cheaper“Cathcart.”
Williams’ ice freezing microtome(fig. 6).
This consists of a round mahogany water-tight box provided with an exit tube below, and covered with a strong plate glass lid. Firmly fixed in the centre of the floor of the box is a stout brass pillar surmounted by a brass disc which fits into a hole in the centre of the glass lid, so that its surface is on a level with that of the lid.
Fig. 6.—Williams’ Ice Freezing Microtome, with Swift’s plough.
Fig. 6.—Williams’ Ice Freezing Microtome, with Swift’s plough.
Fig. 6.—Williams’ Ice Freezing Microtome, with Swift’s plough.
To use it, the box is filled with alternate layersof pounded ice and salt; the lid is then put on and fixed by means of a lateral screw. The tissue to be frozen is gently removed from the gum and placed on the brass disc and plenty of gum painted round it. It should then be covered with a tin cap for a few minutes until frozen. Sections are made with a Swift’s Plough (p.44).
Fig. 7.—Schanze Microtome (see text).
Fig. 7.—Schanze Microtome (see text).
Fig. 7.—Schanze Microtome (see text).
Schanze microtome(fig. 7) is the pattern used in the Leipsic laboratories. It consists of aheavy iron frame with a large base. The knife is carried in a clamp which slides along the full length of the instrument, gliding upon two smooth plates of iron which are arranged at an angle to one another. The knife must be moved very steadily and gently, as when using a long blade vibrations are easily set up which prevent good sections being obtained. The surfaces of contact must be kept scrupulously free from dust, and lubricated with equal parts of olive oil and castor oil. There are several object holders, which can be removed and interchanged, one connected with an ether spray apparatus, another suitable for holding an object embedded in paraffin, and a third for grasping an object embedded in celloidin. When celloidin is employed, a specially long knife must be used, and it must be fixed very obliquely in the clamp. The object holder is raised by a fine screw worked by a large brass toothed wheel. There is a ratchet arrangement, by which the object may be raised automatically any desired distance, after each stroke of the knife. It gives most satisfactory results with celloidin and paraffin.(Messrs. R. and J. Beck are the agents). Its cost is about £5.
Becker’s microtomeis made on exactly the same principles as the Schanze. The modifications are that the carrier glides on glass plates instead of iron ones, and that instead of the whole surface of the carrier being in contact with the plates, there are a few smooth ivory buttons only. Friction is thus reduced to a minimum, and very uniform sections can be obtained. The price is the same as that of the Schanze.
Frazer has introduced a “student’s sliding microtome” on the same principle as the Schanze which costs about £3.
The Cambridge Rocking microtome.—This instrument, as made by the Cambridge Scientific Instrument Company, or the slightly modified form made by Messrs. Swift (fig.8), is the best instrument for cutting sections of small objects embedded in paraffin. Ribbons of serial sections can be obtained from it with greater ease and certainty than with other microtomes. This microtome differs from those which have been previously described in that the knife is fixed,while the object is moveable. The microtome consists of an oblong heavy metal stand. A long bar is arranged so that it rides in see-saw fashion on two strong vertical pillars arising from the frame. One end of this bar is hollow, and receives the piece of wood carrying the tissue embedded in paraffin, which is firmly clamped in position. This end is depressed by means of a strong spiral spring. In order to raise it there is an arrangement by which the other end of the bar is depressed by a cord which revolves round a pulley. When the handle is turned, the tissue is raised, and when the cord is relaxed, the spring pulls the tissue firmly and steadily down. The razor, which must have a straight edge, is fixed firmly by screws, with its edge upwards at the end of the microtome. The object is then adjusted so that in its descent a thin slice is taken off by the razor. There is an ingenious arrangement by which the depression of the bar to raise the section pushes it a little further in the direction of the razor. The distance can be graduated from1/500to1/3000inch. The actual working of the machine is therefore very simple.The position of the block containing the tissue to be cut having been adjusted so that the razor just cuts it, the free end is depressed by means of the pulley. This also pushes the section a little beyond the razor. The strong spring then draws the tissue steadily past the edge of the razor, and a thin section is left on the blade. This may be at once transferred to a slide, or if the paraffin be of the proper consistence, another cut may be made, when the two sections should adhere by their edges, and so by repeating the movement a continuous ribbon may be obtained. If there is difficulty in obtaining a good ribbon, it will usually be got over by taking a little soft paraffin and attaching it by means of a hot needle to the lower end of the paraffin block. The cost of the instrument is about £5.
Fig. 8.—Swift’s Modification of the Cambridge Rocking Microtome.
Fig. 8.—Swift’s Modification of the Cambridge Rocking Microtome.
Fig. 8.—Swift’s Modification of the Cambridge Rocking Microtome.
Fresh sections.—Although these are not so satisfactory as hardened specimens for accurate histological work, it is often very useful to make them both in the post-mortem room where an immediate opinion of the nature of the tumour or diseased organ is desired, and also in the operating theatre. With a little practice sectionsmay be cut, stained, and mounted, within ten minutes of the removal of the specimen from the body. In this way important information may be afforded to the operating surgeon, and in not a few cases it has caused the proposed treatment to be entirely altered. Thus, in one case, a supposed chronic periostitis was shown to be a sarcoma, and the limb was amputated. In another, a supposed sarcoma of the thigh was found to be a gumma, when a portion was removed and microscopically examined.
A portion of the specimen should be placed without any preparation on the zinc plate of the freezing microtome, and some gum painted round it. It is then frozen. The serum in the tissues is not in sufficient mass to injure the knife when it is frozen. The knife should be wetted with, and sections transferred to, either pericardial serum, or3/4per cent. solution (70 grains to the pint), of common salt, neither of which causes the cells to swell up as plain water does. They should be carefully floated out on a glass slide, an operation which requires much more patience than in the case of hardened sections, as fresh sections areless coherent and also more sticky, so that the edges tend to curl up on the knife, &c. They should then be examined, one unstained, simply mounted in salt solution; another stained with picrocarmine and examined in the saline solution; and a third stained in picrocarmine, mounted in Farrant’s solution, and preserved. The last usually gives the best results, the picrocarmine staining becoming quite brilliant after a week. The glycerine, however, is apt to make the sections shrink a good deal, and the weight of the cover-glass tends to break up the unhardened section.
1. Byflotation.
In this method the section whether stained or unstained is placed in a bowl of water, or normal salt solution (p.53). A clean slide is then introduced into the water at an angle of about 60°, a little more than half of its length being submerged. The section is then brought up by the needle and floated as far as possible into position on the slide. One corner is then fixed by the needle, and on gently withdrawing the slide the section should lie flat. If any folds are left no attempt should be made to smoothen them out with a needle, but the slide should be re-immersed until the folded part of the section is under water. It should then be gently withdrawn, when the fold will disappear. This manœuvre must be repeated in different directions until the section lies quite smoothlyon the slide. Stained and unstained sections are floated out in this way before being mounted in Farrant’s medium, and unstained sections previous to staining in picrocarmine.
2. Bytransferencewith asection lifter.
This method is employed in mounting in Canada balsam in order to transfer the section from the clarifying agent (p.63) to the slide. The lifter is polished, and insinuated under the section. The section being held in position by the needle is now raised from the fluid, excess of which is removed by holding the section in position with a mounted needle, and tilting the lifter so as to allow it to drain off.
Removal of air bubbles from sections.—When sections contain many air bubbles, the best plan is to leave them in methylated spirit for a time. The bubbles then coalesce and escape from the section.
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 receiverof an air pump, if one is available, and slightly exhausting the air.
The most frequent cause of air bubbles in mounted specimens, however, is the employment of cover-glasses which have not been thoroughly cleansed. Proper cleansing is best effected by placing the covers when bought in a shallow wide mouthed stoppered bottle containing strong nitric acid, and leaving them in this fluid for twenty-four hours. The acid should then be drained off and water run through the vessel from a tap, until the washings no longer give an acid reaction with litmus paper. The water should then be drained off, and the glasses covered with absolute alcohol. They can be removed one by one and rapidly dried as required. With cover-glasses properly cleansed in this manner, not only will air bubbles be avoided, but the covers will be dried much more easily with the cloth, and fewer will be broken in the process.
Another very frequent cause is the transference of air bubbles with the mounting medium on the glass rod. This occurs especially if the rod be fused to the stopper. The proper bottles to use,both for Farrant’s medium and balsam are “balsam bottles” which have no stopper, but the mouth is closed by a glass cap which fits accurately (fig. 9). A short glass rod is attached to the cap, and is used to transfer the medium to the slide.
Fig. 9.—Balsam bottle.
Treatment of folded sections.—The folding may bedue:—
(1.) To the section having creased through being cut with a knife whose surface was not perfectly smooth. This is best remedied by placing the section in methylated spirit for a minute, and then transferring it to a bowl of clean water, when the section will rapidly rise tothe top, and spread itself out flat on the surface of the water, in consequence of the alcohol rapidly diffusing out at the edges into the surrounding water.
(2) To the section containing a large amount offat, as in those of the skin and subcutaneous tissue. The fat may be removed from the fat cells without materially altering the appearance of the section. This is done by dehydrating the section in alcohol, and then transferring to a watch glass containing ether or chloroform to extract the fat. The tissue should be washed free from ether in the alcohol and then transferred to the bowl of water, and allowed to float out. This process does not interfere with subsequent staining operations.
Farrant’s solution:—
Gum Arabic (picked, colourless)equal parts.GlycerineWater
In making this solution the best gum arabic must be used, and only the clearest pieces of this.“Powdered gum acacia” should be avoided, as though it looks white it often yields a brown mucilage, and besides is frequently adulterated with starch, &c.
The glycerine and water should be mixed and the gum arabic added. The mixture should be allowed to stand for some weeks, with frequent stirring until the whole of the gum is dissolved. Then allow it to stand for a week or two longer in order that the dirt may subside, and the bubbles rise to the top. The scum should be removed and the clear fluid decanted from the sediment into a “Balsam bottle” (p.58) containing a few drops of a saturated solution of arseniate of sodium and a small lump of camphor.
If properly made it is an extremely useful mounting reagent. It does not clarify the tissues too much, and in consequence of its containing gum it dries at the edges and cements the cover-glass more or less firmly in a week or two. If this is not the case the medium contains too much glycerine and more gum must be added to compensate for this. This drying at the edge prevents any further evaporation while the glycerine keeps the section permanently moist.
The camphor and arseniate of sodium prevent the formation of fungi. Sections preserve their original appearance in this medium for many years. After a long time they are apt to become a little cloudy and granular.
Unstained sections should always be mounted in Farrant’s medium, as the Canada balsam process renders them quite transparent. It is suitable for almost any tissue stained or unstained, but sections of the nervous centres require to be mounted in Canada balsam, owing to the opacity of myelin when mounted in glycerine.
Canada balsam solution:—The medium is madethus:—
The ordinary Canada balsam which is of a treacly consistence is heated gently in a water bath for some hours, to drive off turpentine and other volatile oils. It is then allowed to cool to a yellow vitreous mass. Take of
Dried Canada balsamequal parts.Xylol
Leave till dissolved, stirring occasionally.
Unless the solution be perfectly clear, it must be filtered through a very thin paper, previouslywetted with xylol. If the medium be too thick more xylol should be added, if too thin, the xylol should be allowed to evaporate until the medium is of the consistence of a thin syrup.
If the medium is made too thin much annoyance will be caused by its evaporating at the edge of the cover-glass, leaving an air-space, which will increase daily until the section is left quite dry. This should be remedied by putting another drop of balsam at the edge of the coverslip and allowing it to run in and displace the air. A ring of cement should be put on as early as possible afterwards.
The bottle in which the balsam is preserved must be very carefully dried before being filled and then rinsed out with absolute alcohol, and afterwards with xylol. Turpentine or benzol are often used instead of xylol in the preparation of the medium, and in the same proportion, but the latter is less apt to dissolve out the aniline colours from the sections.
To mount sections in Canada balsam they must be transferred first to a watch glass containing absolute alcohol or an alcoholic solution of somestaining reagent,e.g., eosine (p.72) and left in it, no attempt being made to spread it out, until it is perfectly dehydrated,i.e., in about two minutes. It should then be transferred to the clarifying oil on a mounted needle, or on a section lifter, which must be perfectly dry as any spot of moisture that gets on to the section will resist the clarifying action of the oil, and will cause unsightly opaque areas when the section is mounted. Even breathing on the section on its way to the clarifying agent will prevent uniformity of clearing. Should white spots appear in the section while in the oil it must be taken out with as little oil as possible, and again dehydrated in absolute alcohol.
The process ofclarifyingmust be performed in some medium in which Canada balsam is readily soluble, and which is also readily miscible with alcohol. Those most frequently employed are oil of cloves, xylol, oil of bergamot, oil of cedar, and origanum oil. The first named has always been much used because of its agreeable odour, its cheapness, and the ease with which it can be obtained. But it has the disadvantage of dissolving out many important staining reagents,especially eosine and the various aniline colours. In addition as it dissolves celloidin, sections cut in this medium tend to fall to pieces when transferred to oil of cloves, and one of the other oils (which have no solvent action on celloidin) should always be employed with celloidin sections. Oil of bergamot is the most generally useful, but rather expensive. Where there are special reasons for employing other dehydrating agents, they will be indicated in the special directions for particular staining methods in ChaptersVI. andVII.
As soon as the section is plunged into the oil, the alcohol rapidly diffuses out, so that the edges of the section fly out with it, and the section floats quite flat on the surface of the oil. When it is completely clarified (in about a minute), as shown by its sinking in the oil, it should be transferred to the slide by the section lifter, and the oil drained off. Excess of oil may be removed by pressing gently on the section with a flat piece of filter paper folded several times. If carefully performed this manœuvre will not injure the section, but it requires practice.
If the tissue is very delicate, and likely to beinjured 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. The section will float on the oil at first, but the latter will gradually come through and appear on the top of the section. When this occurs the clarification is complete, and the oil may be run off by tilting the slide and the section mounted in Canada balsam.
Cementing of cover-glasses.—The cover-glasses may be cemented down to prevent their shifting and spoiling the specimen. If the cover-glass be circular, a Shadbolt’s turntable should be used. It consists simply of a horizontal heavy brass disc, rotating easily on a pivot. There are a number of circles traced on the disc concentrically. The slide is then fixed on the disc bymeans of the clips, so that the circumference of the cover-glass corresponds to one of the circles. The disc is then rotated and the cement applied to the edge of the cover-glass with a brush.
Many materials are employed. The most suitable are:—(1) Canada balsam, which is almost colourless and transparent and looks very neat. (2) Gold size. (3) Marine glue.
When these are dry a finished appearance may be given to the slide by laying on a ring of zinc white. This is made asfollows:—
Oxide of zinc1/2drachm.Benzolehalf an ounceof each.Gum dammar
Preservation of sections.—They should be keptflat, and preserved from both light and dust. Very useful cardboard trays are now sold by almost all dealers in boxes made to contain twenty-four dozen slides for about eight shillings, or suitable cabinets may be constructed by a carpenter.
Much information may be obtained from unstained sections, and in most cases one section should be examined unstained, but the specimens mounted in this way are so transparent that it is difficult to study the details of the tissue. 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. Sections should bestained with several reagents, as their effect on individual specimens varies a good deal.
The following are the most useful stains for generalpurposes:—
Logwood.—This or its purified principle hæmatoxyline is the most useful general stain. The hæmatoxyline itself is preferable, giving more constant results, and less diffuse staining.
For general staining purposes the following formula will be found to give excellent results:—Hæmatoxyline. Schuchardt’s formula.—