Walter S. Colman

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 substances are:—

(1) gum, (2) celloidin, (3) paraffin or wax.

Gum.—Picked colourless 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.

Celloidin is 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, and very readily soluble in ether, or in a mixture of alcohol and ether. The last solvent is the one commonly employed.

The embedding solution is made thus:—

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. After the 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 at 110°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 just above 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 the paraffin block with a hot needle or wire and pressing it down on the wood.

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 just melts. 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.

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’s ether spray microtome (fig.4).—This, or its more recent modifications (see later), 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 about 3/8inch 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 same time 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 once transferred 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 raised by 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.

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 which the 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.

To use it, the box is filled with alternate layers of 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).

Schanze microtome (fig.7) is the pattern used in the Leipsic laboratories. It consists of a heavy 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 microtome is 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 from 1/500 to 1/3000 inch. 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.

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 sections may 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, or 3/4 per 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 are less 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.