A well-known skilled observer of test objects41 says: “Practically the resolving power of our achromatic objectives on lined objects reached their maximum in the late Dr. Woodward’s hands. Amphipleura pellucida was then, as now, the finest known regular structure of the diatoms. There appeared then nothing more to be gained in resolution when one of the apochromatic ¹⁄₁₂-inch objectives of Zeiss, with its entire absence of colour, passed into my hands, and I soon became convinced that it possessed the power of separating the different layers of structure in the valve, beyond the grasp of the dry-objective. The result of this increase of power enabled me to split up, as it were, the one plate of silex forming the valve of Pleurosigma formosum into three layers, and which had never before appeared to be possible; proving, in fact, that magnification without corresponding aperture is of little or no account.”

“The intimate structure of these test objects,” says Mr. Smith, “is built up on one plan, each being composed of two or more layers, (1) a valve with two layers, as in Pleurosigma balticum; (2) two layers with a grating and secondary markings placed diagonally, as in Pleurosigma formosum; (3) with two layers of a net-like structure, as in Pleurosigma angulatum, the fineness of the striæ or gratings of which measure the ¹⁄₅₀₀₀₀th of an inch. Five other diatoms afford evidence of this compound structure. The presence of beads or hemispheres in one of the focal planes, and depressions or pits in another, are emphasised in the micro-photograph itself; reduced portions of the valve are represented in Figs. 204 and 204a.”

A portion of a diatom valve, Pleurosigma angulatum, micro-photographed on a higher scale of magnification, 4,500 diameters, is given further on.

Errors of interpretation arise either from the small cones of illumination afforded by the dry-objective, or the oblique illumination formerly resorted to for the resolution of these difficult test objects, and several of the lights and shadows resulting from the refractive power of the object itself. But the most common error is that produced by the reversal of the lights and shadows resulting from the refractive powers of the object itself. To make this clear, I reproduce two reduced photographs of a small section of an old-fashioned glass tumbler, covered externally with numerous hemispheres, illuminated by transmitted light (Fig. 205).

This illustration well emphasises the difficulty there is in determining structure under precisely similar conditions to those we are accustomed to of examining valves of diatoms under the microscope. If these photographs be held in front of a strong light, they at once convey different impressions to the mind, the hemispheres appearing depressions in the one, and raised beads in the other. Both are prints from the same negative, but in mounting are reversed; and therefore the apparent dissimilarity is due to a slight inequality of illumination, which the mind accepts as light and shade.

Very similar appearances to those described will result if a thin plate of glass were studded with minute, equal, and equi-distant plano-convex lenses, the foci of which would very nearly lie in the same plane. If the focal surface, or plane of vision, of the objective be made to coincide with this plane, a series of bright points will result, from the excess of light falling on each lens. If the plane of vision be next made to coincide with the surfaces of the lenses, these points would appear dark, in consequence of the rays being refracted towards points now out of focus. Lastly, if the plane of vision be made to coincide with the plane beneath the lenses that contain their several foci, so that each lens may be, as it were, combined with the object-glass, then a second series of bright points will result from the accumulation of the rays transmitted at those points. Moreover, as all rays capable of entering the objective are concerned in the formation of the second series of bright focal points, the first series being formed by the rays of a cone of light only, it is evident that the circle of least confusion must be much less, and therefore the bright points better defined in the first than in the last series.

There are no set of objects which have given rise to more discussion as to their precise character than the scales of the podura (Lepidocyrtus cervicollis), to the intimate structure of which Mr. Smith turned his attention, and succeeded, I am inclined to think, in his attempt to settle the structure of these very minute scales, and which heretofore have been described as “notes of exclamation.” By the aid of the same power as that employed in the examination of the pleurosigma formosum, the old conventional markings have disappeared, and well-defined “featherlets” have taken their place. By careful focussing up and down, a series of whitish pin-like bodies is to be seen, with an intervening secondary structure. A micro-photograph of a portion of a scale taken by Mr. Smith shows that these pin-like bodies are inserted in a fold of the basement membrane, which, in his opinion, furnish unmistakable evidence of the fact that these projecting bodies are real, and must no longer be looked upon as mere ghosts. Quite recently, a micro-photograph of a portion of a podura scale was placed in my hands, taken by Mr. J. W. Gifford with a Swift’s ¹⁄₁₂-inch apochromatic objective, of numerical aperture 1·40, and a deep eye-piece, having a combined magnifying power of 3,827 diameters. Fig. 206 shows a portion of the photograph which, it will be admitted, supports Mr. Smith’s view of the structure of the podura scale.

Many other errors of interpretation are not unknown to the experienced operator with the microscope, arising, for the most part, from an influence exerted by peculiarities in the internal structure of certain objects; for example, that offered by the human hair, and which, when viewed by transmitted light, presents the appearance of a flattened-out band, with a darkish centre, due to the refractive influence of the rays of light transmitted through the hair. That it is a solid or tubular structure is proved by making a transverse section of the hair-shaft, when it is seen filled up by medullary matter, the centre being somewhat darker than the outer part. It is, in fact, a spiral outgrowth of the epithelial scales, overlapping each other, imparting a striated appearance to the surface. A cylindrical thread of glass in balsam appears as a flattened, band-like streak, of little brilliancy. Another instance of fallacy arising from diversity in the refractive power of the internal parts of an object is furnished by the mistakes formerly made with regard to the true character of the lacunæ and canaliculi of bone structure. These were long supposed to be solid corpuscles, with radiating opaque filaments proceeding from a dense centre; on the contrary, they are minute chambers, with diverging passages—excavations in the solid osseous structure. That such is the case is shown by the effects of Canada balsam, which infiltrates the osseous substance.

Air bubbles are a perplexing source of trouble. The better way of becoming accustomed to deceptive appearances of the kind is to compare the aspect of globules of oil in water with bubbles of air in water, or Canada balsam.

The molecular movements of finely divided particles, seen in nearly all cases when certain objects are first suspended in water, or other fluids, are a frequent cause of embarrassment to beginners. If a minute portion of indigo or carmine be rubbed up with a little water, and a drop placed on a glass slide under the microscope, it will at once exhibit a peculiar perpetual motion appearance. This movement was first observed in the granular particles seen among pollen grains of plants, known as fovilla, and which are set free when the pollen is crushed. Important vital endowments were formerly attributed to these particles, but Dr. Robert Brown showed that such granules were common enough both in organic and inorganic substances, and were in no way “indicative of life.”42

Professor Jevons succeeded in throwing light on these curious movements. He showed that they were not due to evaporation, as some observers contended, as they continue when all possibility of evaporation is cut off, when the fluid is surrounded by a layer of oil, and enclosed in an air-tight case: but as Professor Jevons pointed out, these movements are greatly affected by the admixture of various substances with water, being increased by a small quantity of gum, and checked by a drop of sulphuric acid, or a few grains of some saline substance, which increases the conducting power of water for electricity. The Brownian movement, now termed pedesis, much depends upon the size of the particles, their specific gravity, and the nature of the liquid in which they are immersed.

The correct conclusions to be drawn by the microscopist regarding the nature of an object will necessarily depend upon previous experience in microscopic observations, a knowledge of the class of bodies brought under observation, and the skill of the observer in the use of the instrument—that is, in securing the best focus possible with any objective brought into use. I am indebted to Messrs. Beck for the following series of illustrations, showing the effect of under and over correction of the objective.

Directions for finding the best Focus.

The method of finding and determining when the screw-collar adjustment of the high-power objective has arrived at a point of perfect definition and magnification is as follows:—

Select any dark speck of dust, or an opaque portion of the object, and carefully focus this small particle by working the screw of the fine adjustment, move the screw up and down until you are satisfied the image is the sharpest and blackest that can be obtained, then once more test the focus a little above and a little below while closely scrutinising the effect on the image. It will now be seen that whereas in focussing on one side of the best focus the object disappears in a fog, by focussing on the other side it remains in view for a longer period, but alters its appearance; it is now no longer a black dot, but a bright dot of light surrounded by a black margin. The effects being thus dissimilar on different sides of the best focus, show that the objective is not perfectly adjusted for the cover-glass in use.

The next step is to find out whether the bright image is above or below the best focus, as on this depends the direction in which the adjustment-collar should be turned. To determine this it is only necessary to ascertain which way the slow-motion milled head of the microscope turns when moving the objective upwards.

In the case under consideration, the bright image will be above the best focus, which shows that the cover-glass in use is thicker than that for which the objective is adjusted, consequently the adjustment-collar must be moved in the opposite direction.

If the collar be turned too far in the opposite direction, it will be found that the bright image is below the best focus, and the cover-glass is then thinner than that for which the objective is adjusted. The collar must then be turned back again until the effect on each side of the best focus is exactly similar. This effect in the case of a circular speck of dust will be that the object disappears equally rapidly on either side, and does not instantly vanish into fog, on either side presenting the bright spot appearance, though not in so marked a degree on either side. When the object is in perfect adjustment the expansion of the outline is exactly the same, both within and without the focus.

A different indication, however, is afforded by such test-objects as the finer diatoms, and the podura scale, in which we have to do with a set of distinct dots and other markings. If the dots have a tendency to run into lines when the object is without the focus, the glasses should be brought closer together; on the contrary, if the lines appear when the object is within the focal point, the glasses should be farther separated.

The adjustment of the objective by the screw-collar in the case of the podura scale should be carried out in the way described, when the following effects will be observed to take place, usually in the order of their arrangement.

Fig. 1 shows the appearance of a podura scale when the adjustment of the object-glass is correct, and Fig. 2 shows the effect produced on each side of the exact focus. Fig. 3 shows the way in which the markings individually divide when all the adjustments are correct, and when the focus is altered the least possible amount only each way.

Figs. 4 and 5 show the two appearances on one and the other side of the best focus when the adjustment is incorrect, Fig. 6 showing the appearance of the same at its best focus.

The scales are magnified 1,300 diameters, and each square measures ·001 of an inch.

This method, however, of finding the best focus of an objective can scarcely be accomplished without a sub-stage condenser. It may therefore be of service to the student, and to those who are not disposed to purchase expensive forms of condensers, to know that either an inch or an inch and a half objective, or convex-lens mounted on a simple wooden ring with a flange, can be arranged to slip in the place of the diaphragm under the stage. This kind of condenser will prove to be of considerable value with a ½-inch, a ⁴⁄₁₀-inch, and a ¼-inch; while a still more excellent achromatic condenser can be made out of a Steinheil’s aplanatic-loup arranged to drop into the central fitting of the sub-stage. As without a condenser of some kind it is hardly possible to enter upon any course of histological or scientific research.43

Working Accessories.

Troughs—Live-cages—Compressors.

A glass plate with a ledge, and some pieces of thin glass, although applicable for many purposes, are specially designed for objects in fluid. Thus a drop of fluid containing the object sought for is placed upon the slide and covered by a piece of thin glass; or, the object being put upon the glass slide and the thin glass over it, the fluid is applied near one side, and runs under by capillary attraction.

Troughs and Live-box.—These are made of various materials, glass, vulcanite, brass, &c., expressly for examining infusoria and live animals. They should be so constructed as to admit of the use of a medium power, a ½-inch at least, under the microscope. They should also admit of being easily cleaned and repaired when broken; matters rarely thought of by those who construct them. An early devised live-box (Varley’s, Fig. 208) consists of two circular pieces of brass tubing, one sliding over the other carrying a disc of glass and fitting over another glass with bevelled edges to prevent the fluid flowing away.

The Compressorium is used for similar purposes. By a graduated pressure the fluid is thinned out and a higher power can be employed for the examination of the object. Ross’s early compressorium consists of a plate of brass about three inches long, having in its centre a circle of glass like the bottom of the live-box. This piece of glass is set in a frame, B, which slides in and out so that it can be removed for the convenience of preparing any object upon it—under water if desirable. The upper movable part, D, is attached to a screw-motion at C; and at one end of the brass plate, A, which forms the bed of the instrument, is an upright piece of brass grooved so as to receive a vertical plate, to which a downward motion is given by a single fine screw, surrounded by a spiral spring, which elevates the plate as soon as the screw-pressure is removed.

Beck’s Parallel-plate Compressor (Fig. 210) affords a more exact means of regulating the pressure, and can be used for a variety of purposes. It is also easily cleaned.

Rousselet’s Compressorium (Fig. 211) is a very effective form for general use. It is so arranged that the student has perfect control over the pressure to which the specimen should be subjected. The cover-glass is large in comparison with that beneath; being bevelled causes evaporation to go on very slowly while the pressure between the two glass surfaces is kept perfectly parallel.

Botterill’s Live-trough (Fig. 212) consists of two brass plates screwed together by binding screws, and holding between them two plates of thin glass, which are maintained at a proper distance by inserting a semicircular flat disc of india-rubber.

Glass troughs for chara and polypes (a sectional view of one shown at Fig. 213) are made of three pieces of glass, the bottom being a thick strip, and the front (a) of thinner glass than the back (b); the whole is cemented together with Jeffery’s marine-glue. The method adopted for confining objects near the front glass varies according to circumstances. The most convenient is to place in the trough a piece of glass wide enough to stand across diagonally, as at c; then, if the object be heavier than water, it will sink until stopped by the glass plate. At other times, when used to view chara, the diagonal plate may be made to press it close to the front by means of a wedge of glass or cork. When using the trough the microscope should be placed in a nearly horizontal position.

Cells for viewing living objects, and watching their movements, take many forms, usually determined by the makers for the purposes they are required to serve. The smaller glass troughs (Figs. 216, 216a) are made for examining the small infusoria, rotifers, &c., some of which take special forms, as the double or divided trough (Fig. 217) intended for viewing the circulation of the blood in the tail of a small fish, and at the same time keep up a supply of water and air.

The Frog-plate consists of a strip of plate-glass, or wood, pierced with holes on either side, through which tapes are passed to secure the frog in its place. At the extreme end is a shallow glass trough, made to hold a sufficient quantity of water to keep the web of the foot moist while under examination. In this way a continuous view of the circulation of the blood of the animal is obtained.

Growing Cells have received more attention from those who devote attention to the lower forms of life, the construction of which, for the purpose of maintaining a continuous supply of fresh water to objects under observation, and for sustaining their vital energy for a long period, is of some importance. The employment of live-cells is resorted to by microscopists, as doubtless there is much to be discovered concerning the metamorphoses which some of the lower micro-organisms, both of plant and animal life, pass through.

Holman’s life slide consists of a 3 × 1 inch glass slide, with a deep oval cavity in the middle to receive the specimen for observation. A shallow oval is ground and polished around the deep cavity, forming a bevel. From this bevel a fine cut extends, to furnish fresh air to the living low forms of life which invariably seek the bevelled edge of the cavity, thus bringing them within reach of the highest powers. He also contrived a convenient form of “moist chamber,” or animalcule-cage (Fig. 220), for the purpose of studying the growth of minute organisms, without in any way disturbing them for a lengthened period. This is also found useful as a dry chamber for holding minute insects.

Zentmayer’s Holman Syphon Slide is used either as a hot or cold water cell. It should be deep enough to hold a small fish or newt, and retain it without any undue pressure. When in use it is only necessary to place the animal into it (as shown in Fig. 221), with some water, and secure it with a glass cover; then immerse the upper tube in a jar of water, while another, at a lower level, maintains a current. When the slide is on the stage of the microscope, one jar should stand on a lower level than the other, the slide being made the highest part of the syphon. The pressure of the atmosphere is sufficient to keep the cover-glass in its place.

The examination of the various kinds of infusorial life—rotifers, for instance—is facilitated by the addition of the smallest particle of colouring matter, either carmine or indigo. A small quantity of either of these colours should be rubbed up in a little water in a watch-glass, and a portion taken up on the point of a brush, and the brush run along the edge of the cover-glass; sufficient will be left behind to barely tinge the water with the colour, and this gradually distributes itself over the rotifers. Under the microscope this minute quantity will be seen like a rising cloud of dust, and as it approaches a rotifer it is whirled round in different curves, showing at once the action of its wonderfully rapid cilia. This colouring matter appears to be devoured, as it may be traced from the mouth to the digestive canal. Monads may be detected by this means, and the smaller forms of algæ, Euglena viridis and Protococcus pluvialis.

Dipping-tubes.—In dealing with infusorial or monad life it is convenient to keep a stock-bottle ready for their reception, and in a light favourable to health. When a live specimen is required for examination, the dipping-tube is brought into requisition. These tubes are open at both ends, and vary in length and diameter. Their ends should be nicely rounded off in the flame of a blow-pipe; in form either straight, or bent and drawn out to a fine point, as represented in Fig. 222. When any special specimen is required for examination, then one of the tubes must be passed down into the water, the upper orifice having been previously closed by the forefinger, and kept tightly pressed, until its lower orifice comes in contact with the object. On the finger being removed, the water rushes up and carries the creature sought for with it. The finger is once more replaced at the top of the tube; it is then lifted out, and the contents deposited in one or other of the glass cells described. Tubes with india-rubber covers can be had.

Moist and Warm Stages.—In addition to the moist cells and chambers described it is often found necessary in working out the histories of minute organisms to keep them for some time under observation, and as far as possible in an undisturbed condition, and it is equally necessary to prevent evaporation of the water in which they are immersed. One of the best warm stages is that known as Maddox’s growing stage; this can be had of any optician. More elaborate adaptions are required for the study of special organisms, and for experimental research.

In that case Bartley’s Warm Stage (Fig. 224) is recommended. There are other forms of warm stages in use, many of an inexpensive kind and readily adaptable to any stage. Bartley’s has proved useful; it consists of a vessel, E, three parts filled with water and supported on a ring stand. This may be kept at any temperature by the small spirit-lamp, C; a syphon tube d conveys the warm water along f, and through the bent tubing which surrounds the object under observation on the stage, D, and then passes off through the open end, C, into the receptacle, B, placed to receive the overflow. Steam can be used for heating, or iced water for observing the effects of cold upon the organism.

A simple form of warm stage may be made of an oblong copper plate, two inches long by one wide, from one side of which a rod of the same material projects. The plate has a round aperture, the centre half an inch in diameter, and is fastened to an ordinary slide with sealing-wax. The drop or object to be examined is placed on a large-sized cover-glass and covered over with a smaller one. Olive oil or vaseline is painted round the edge of the smaller one to prevent evaporation, and the preparation is placed over the aperture in the plate. The slide bearing the copper plate is clamped to the stage of the microscope. The flame of the spirit-lamp is applied to the extremity of the rod, and the heat is conducted to the plate and thence transmitted to the specimen. In order that the temperature of the copper plate may be approximately that of the body, the lamp is so adjusted that a fragment of cacao butter and wax placed close to the preparation is melted.

Professors Stricker and Schäfer have constructed warm stages for accurate observations, and which fully answer every purpose.

Stricker’s Stage (Fig. 225) consists of a rectangular box with a central opening, C, permitting the passage of light through the specimen under examination. The water makes its exit and entrance at the side tubes B B′, and the temperature is indicated by a thermometer in front. In this apparatus either warm or cold water can be continuously used.

Schäfer’s apparatus (Fig. 226) consists of a vessel filled with water (seen near the stage) which has been first boiled to expel the air, and then heated by means of a gas flame. The warm water ascends the india-rubber tubing to the brass box on the stage. The box is pierced by a tubular aperture to admit light to the object, and has an exit tube by which the cooled water from the stage returns by another piece of tubing to be reheated by the gas flame. There is a gas-regulator, by means of which any temperature can be maintained.

Methods of Preparing, Hardening, Staining and Section Cutting.

Numerous methods are employed for the preparation, hardening, staining, and section cutting of animal and vegetable tissues for the microscope, the details of which are modified, or varied as may be found needful, from time to time, by those whose intimate acquaintance with the subject entitles them to make innovations and changes in this very important department of microscopy. In the hands of the original worker, formulæ and methods will only be regarded as finger-posts pointing out a means of saving time in turning over pages to find this or that special method of staining. For this particular reason I have collected all the most accredited formulæ together in an Appendix at the end of the book, and arranged them alphabetically for ready reference.

As to section cutting, the student will do well to practise himself in making dissections, thick and thin sections, of vegetable and animal substances. The medical student will require no advice on this point, as the use of the scalpel, and those instruments needed for microscopical work, form an important part of his education. Of all the instruments contrived for delicate dissections, none are more serviceable than those which the student may make for himself out of ordinary needles. These may be fixed in handles as represented in Fig. 229, in addition to which, a pair of scissors and forceps, and a few small knives, such as those used in eye-operations, will prove most suitable. The double-bladed scissors represented in Fig. 227, with curved blades, are brought into use for cutting vegetable and other soft structures, the disadvantage attendant upon the use of which is owing to the curvature of the blades; when dealing with flat surfaces, the middle of the section is left too thick to exhibit structure.

The double-bladed knife of Professor Valentin was formerly held in high estimation by the microscopist, but this has been almost superseded by the microtome, which has taken the place of all other instruments, since by its aid uniform series of nearly all substances can be cut. The standard unit of a perfect section cutter, of any kind, has been fixed by the Royal Microscopical Society at the one-thousandth of a millimetre.

The use of the razor for cutting sections has not been wholly abandoned, the method of using which is as follows:—Take the tissue between the thumb and finger of the left hand, hold the finger horizontally, so that its upper surface may form a rest for the razor to glide upon, take the razor firmly, and keep the handle in a line with the blade, then draw it through the tissue from heel to point and towards yourself. While cutting keep the razor well wetted with diluted methylated spirit.

Some preparation is required for cutting sections with the single microtome. The substance to be cut must be embedded in some other material, as carrot, turnip, potato, alder pith, paraffin, or thick gum, with either of which the cylinder or well of the microtome must be so nearly filled as to leave only an excavation in the centre for the specimen to be operated upon to occupy. The various forms of microtomes in use, and the selection of the most suitable, is therefore a matter of some difficulty. I must content myself by particularising two or three typical forms in general use. As all the substances intended for cutting require preparation, it will be first necessary to attend to the following directions given by one experienced in section cutting, Mr. M. J. Cole44:—(1) Always use fresh tissues. (2) Cut the organs into small pieces with a sharp knife. (3) Never wash a specimen in water; when it is necessary to remove any matter, allow some weak salt solution to flow over the surface of the tissue, or wash it in some hardening re-agent. (4) All specimens should be hardened in a large quantity of the re-agent; too many pieces should not be put into the same bottle, and keep them in a cool place. (5) In all cases the hardening process must be completed in spirits. (6) Label the bottles, stating the contents, the hardening fluid used, and when changed. Attention to details is necessary, as if hardening is neglected, good sections cannot be made.

Embedding in Paraffin Wax or Lard.—Melt together, by the aid of gentle heat, four parts of solid paraffin and one part of lard. A quantity of this may be made and kept ready for use. Melt the paraffin mass over a water bath, take the specimen, and dry it between the folds of a cloth to remove the spirit, so that the paraffin may adhere to its surface, place it in a small chip-box, in the desired position, and pour in enough melted paraffin to cover it, then set aside to solidify; when quite cold break away the box, and cut sections from the embedded mass with a sharp razor.

To infiltrate a tissue with paraffin, place the specimen in absolute alcohol or chloroform for an hour or two, then transfer to a bath of melted paraffin, at its melting point (about 110° F.), and keep it at this temperature for several hours, so that the paraffin may penetrate to the middle of the tissue. Then remove the specimen from the paraffin and put it into a small chip-box, pour in enough paraffin to cover it, and set aside to cool. When quite cold, make sections as before, with a razor, or fix it into a microtome, with a little melted paraffin. The sections when cut must be placed in turpentine to remove the paraffin, and then into absolute alcohol to remove the turpentine, and finally in distilled water to remove the alcohol, when they may be forthwith stained. It is often found better to stain the tissue in bulk before embedding. In this case the sections will only require the turpentine to dissolve away the paraffin, and may then be mounted in Canada balsam.

Hardening and Preparing Animal Tissues for section cutting and microscopical examination.—Fresh tissues are not well suited for microscopical examination, but it is sometimes advisable to observe the appearances of a fresh specimen, especially if it is suspected to contain amaloid bodies or parasites. It will then be necessary to tease out a small portion of the tissue immersed in a weak solution of salt and water by the aid of a pair of fine needles (Fig. 229) and the dissecting microscope (Fig. 230).

The most important point in connection with an instrument of this kind is, that it affords firm and convenient rests for the hands, and should not be raised too high from the table.

The stage should either be made of glass, or provided with a glass dish for dissecting under water, or preservative fluid. A pair of aplanatic lenses, mounted on a focussing bar as shown in Fig. 230, will be found the most convenient to work with.

Investigations of this nature should be always carried out in the manner described, but preparations of the kind cannot be preserved any length of time, unless properly hardened in spirit or Formalin solution. The method of teasing out under the light of a condensing lens is shown in Fig. 231.

It may be as well to state at the outset that physiological and pathological tissues can be hardened by immersion in methylated spirit alone, or a saturated solution of picric acid in methylated spirit in about a week, and it is said to yield satisfactory results, even some of the tissues being ready in twenty-four hours. The only drawback is that sections thus quickly hardened must be stained with picro-carmine. But, whatever method of hardening adopted, the tissue should be washed by means of a stream of water for half an hour, to remove all traces of the hardening agent, and on its removal pressed between folds of cotton cloth or fine Swedish filtering paper.

The principal hardening re-agents usually kept in bulk ready for use are the following:—

Absolute Alcohol.—This is suitable for the internal organs of animals, glands, &c. These organs must be perfectly fresh, and should be cut into small pieces, so that the alcohol may penetrate them as quickly as possible. The hardening is usually complete in a short time.45

Chromic Acid and Spirit.—Chromic acid one-sixth per cent., water solution two parts, and methylated spirit one part. This reagent hardens in about ten days. Then transfer to methylated spirit, which should be changed every day until all colour is discharged from the tissue. This is a suitable reagent for the preparation of cartilage, nerve trunks, heart, lips, blood vessels, trachea, lungs, tongue, intestines, and gullet.

Potassium Bichromate.—-Make a two per cent. water solution of this salt. This will harden specimens in about three weeks. Then transfer the preparation to methylated spirit, and change it every day until all colour is discharged. This is suitable for spinal cord, medulla, cerebellum, and cerebrum.

Müller’s Fluid.—Bichromate of potash 30 grains, sulphate of soda 15 grains, distilled water 3½ ounces. This hardens in from three to six weeks. Then transfer, as before, to methylated spirits, and change it every day until colour ceases to appear. Most suitable for lymphatic glands, eye-ball and its internal structures, as well as for tendons, and thymus gland.

Methylated Spirit may be generally employed, but it has a tendency to shrink some tissues too much; it hardens in about ten days. It is usual to change the spirit daily, for the first three days at least. Skin, mammary gland, supra-renal glands, tonsils, and all injected organs may be hardened in it. (See note on the adulteration of methylated spirit with rack-oil, which utterly spoils it for use.)

Decalcifying solution for bones and teeth. Take one-sixth per cent. watery solution of chromic acid, and to every measured ounce add five drops of nitric acid. This reagent will soften the femur of any small animal in about three weeks; larger require a longer time. Change the fluid several times, and test its action by running a needle through the thickest part of the bone. Should it not pass through easily, then continue the process until it does. When soft enough transfer to water, let it soak for an hour or two, then pour off the water and add ten per cent. solution of carbonate of soda, and soak for twelve hours to remove all trace of acid. Wash again in water, and transfer to methylated spirit until required. Teeth require a large quantity of the decalcifying solution for softening.

Microtomes.—The simplest form of “hand-cutting machine” is that worked by a screw, which raises the preparation, and at the same time regulates the fineness of the section. When a number of sections are required, or when a complete series of sections of an organ is desired, Cole’s simple microtome (Fig. 233) is in every way adapted.

The method of using it is as follows:—Screw the microtome firmly to the table, and with the brass tube supplied with the microtome, punch out a cylinder of carrot to fit into the well. Cut this in half longitudinally, and scrape out enough space in one half of the carrot to take the specimen; then place the other half of carrot in position, and make sure that the specimen is held firmly between them, but it must not be crushed. Now put the cylinder of carrot and specimen into the well of the microtome and commence cutting the section. A good razor will do, but it is better to use the knife which Messrs. Watson supply with the microtome. While cutting keep the knife and plate of the microtome well wetted with dilute methylated spirit, and as sections are cut place them in a saucer of dilute spirit. A number of sections may be cut and preserved in methylated spirit until required for examination or mounting.

When a specimen has a very irregular outline, it cannot be very successfully embedded in carrot; paraffin will then be found to be more suitable. Place the tissue in the well of the microtome in the proper position, pour in enough melted paraffin to cover it, and put it by to get cold and hard before attempting to cut sections.