CHAPTER IV.

Practical Microscopy: Manipulation, and Mode of Using the Microscope.

In this chapter it will be my aim to discuss the best practical methods of employing the microscope and its appliances to the greatest advantage. First, the student should select a quiet room for working in, with, if possible, a northern aspect, free from all tremor occasioned by passing vehicles. The table selected for use should be firm, and provided with drawers, in which his several appliances can be kept ready to hand. The microscope must be placed at such an inclination as will enable him to work in comfort, and without putting strain on the muscles of the neck or fatiguing the eyes. The next important point is that of light. Daylight, in some respects, is an advantage; this should come from a white cloud on a bright day, but as a rule more satisfactory results will be obtained by using a well-made lamp, as this can be controlled with ease, and used at a proper height and distance from the microscope. To have a good form of lamp is as much to be desired for the student as for those engaged in the more advanced work of microscopy.

Whatever the source of light we must on no account over-illuminate. The object having been placed on the stage of the microscope, the body should be racked down to within a quarter or half an inch of the specimen, and then, while looking through the eye-piece, should be slowly withdrawn until a sharp image comes into view. The fine adjustment may now be used for the more delicate focussing of the several parts of the field.

Accurate adjustment of focus is required when using a 1/4-inch objective; details of the object, as striæ, being brought into view when a stronger light is thrown obliquely upon them from the mirror. If a 1-inch objective is used the light often proves to be in excess of what is required, and this must be regulated by the aid of the diaphragm.

The iris diaphragm, made to drop into the under-stage, is more generally employed, as when racked up to the object it affords every necessary graduation of illumination.

To illuminate opaque objects the light should be thrown upon them from above by the bull’s-eye lens (Fig. 201). The focus of such a lens and the lamp placed at four inches from it, is about three inches for daylight, or two inches for artificial light. A large object may be placed upon the stage of the microscope at once, but smaller objects are either laid on a glass slide or held in the stage forceps.

When illuminating objects from above all light from the mirror, or that which might enter the objective from below the stage, should be carefully excluded. _Dark-field illumination_ is a means of seeing a transparent object as an opaque one. The principle, however, is that all the light shall be thrown from below the object, but so obliquely that it cannot enter the object-glass unless interrupted by the object; this is best accomplished by _Wenham’s Parabola_.

_Glass_ of any kind requires occasional cleaning; a piece of soft washed chamois leather should be used for this purpose. The fronts of the objectives may be carefully wiped, but not _unscrewed_ or tampered with; a short thick-set camel’s hair brush may be passed down to the back lens, and all dust removed without doing any harm. If the objective is an _immersion_, carefully remove the fluid from the front lens, as even distilled water will leave a stain behind. For removing oil see special directions given at page 171.

When cleaning the _eye-pieces_, which should be done occasionally, the cells containing the glasses must be unscrewed and replaced one at a time, so that they may not be made to change places.

Any dirt upon the _eye-pieces_ may be detected by turning them round whilst looking through the instrument; but if the _object-glasses_ are not clean, or are injured, it will, for the most part, only be seen by the object appearing misty.

The _object-glasses_, when in use but not on the microscope, should be stood upon the table with the screw downwards, to prevent dust getting into the lenses, and they should always be put into their brass cases when done with. A large bell-glass shade will be found the most useful cover for keeping dust from the instrument when not in use.

When looking through the eye-piece be sure to place the eye in close proximation to the cap, otherwise the whole field will not be perfectly visible; it should appear as an equally well-illuminated circular disc. If the eyelashes are reflected from the eye-glass, the observer is looking upon the eye-piece, and not through it.

_The Mirror._--The working focal distance of the mirror is that which brings the images of the window-bars sharply out upon the object resting upon the stage. In other words, the focus of the mirror is that which brings parallel rays to a correct focus on the object-glass. If employing artificial light, then the flame of the lamp should be distinguishable; a slight change in the inclination of the mirror will throw the image of the lamp-flame out of the field.

The strongest light is reflected from the concave side of the mirror, that from the flat side is more diffuse and less intense. Oblique light can be obtained by turning the mirror on one side and then adjusting it so as to illuminate the field from that position. All the necessary mechanism of the microscope is easily and quickly learned. The object-glasses or objectives are, as previously explained, designated according to the focal distance of a single lens of the same magnifying power. Thus a 2-inch objective is understood to be a combination which has the magnifying power of a single lens whose focal point is two inches from the object, and so on with reference to other powers. By the aid of different eye-pieces an extensive range of magnifying power can be obtained; for example, the 2-inch objective with a deep eye-piece will give the same amplification as the quarter objective with the ordinary eye-piece. Indeed, for certain observations, the combination of a wide-angled low-power objective, with a deep eye-piece, or _compensating eye-piece_, is considered to have an advantage.

It has been already explained that two objectives, one of much greater power than the other, but both having only the same numerical aperture, will show only the same amount of _detail_; the higher power on a larger scale. That is, supposing with a 1/4-inch objective of 1·0 numerical aperture certain structure is resolved, then a 1/8-inch substituted with exactly the same numerical aperture, but with double the magnification, no more _resolving power_ will be found in the latter objective than in the former. For this reason a doubt has been expressed as to whether high-power objectives--especially the more expensive oil-immersions, made to transmit large pencils of light through their larger apertures--are so well adapted for ordinary research as the best series of dry achromatic objectives, or even, in some instances, the medium aperture lenses; undoubtedly, for histological (physiological and pathological) work, the latter will be found to meet the students’ requirements quite as well as the former.

The student or amateur will do well to commence with moderate or medium powers, a 2-inch, a 1-inch, a 1/2-inch, a 4/10-inch, or 1/4-inch. These, together with the A and B eye-pieces, will give a range of magnification from 30 to 250 diameters.

_Penetration_ in the objective is a quality for consideration, as the adjustment of high powers is a work of delicacy, and in some cases their penetration is impaired by the arrangement made to obtain finer definition. The value, however, of penetration in an objective is always considered to be of more or less importance. It is a quality whereby, under certain conditions, a more perfect insight into structure is obtained. As a rule, the objective having the largest working distance possesses the better penetration. Theoretically, the penetration of an objective decreases as the square of the angular aperture increases. For this reason the medical student will be justified in choosing the objectives I have named, since these will be better adapted to his work and pursuits. The penetration of the objective is a relative quality assessed at a different value by workers whose aims are widely different. But for the observation of living organisms, the cyclosis within the cell of the closterium or valisneria, for instance, preference will undoubtedly be in favour of the objective with good penetration.

_Resolving Power._--This is a quality highly prized by the bacteriologist. In the case of the high-angled apochromatic oil-immersion, with its compensating eye-piece, its resolution is found to be of very considerable advantage, because of its capacity to receive and recombine all the diffraction spectra that lie beyond the range of the older achromatic objective, with its smaller angular aperture. The actual loss of resolving power consequent upon the contraction of aperture from 180° to 128-1/2° is ten per cent., if not more. Resolution depends, then, upon the quality and quantity of the light admitted, the power of collecting the greatest number of rays, and the perfection of centring. In other words, upon the co-ordination of the illuminating system of the microscope--mirror, achromatic condenser, objective and eye-piece. If diatoms are employed as test-objects, it should not be forgotten that there are great differences, even in the same species, in the distances their lines are apart. For this reason ruled lines of known value, as Nobert’s lines, are to be preferred. The following example will suffice to show the value of a dry 1/8-inch objective of 120° in defining the rulings of a 19-band plate, which is equivalent to the 1/67000th of an inch. This objective, with careful illumination, showed them all; but when cut down by a diaphragm to 110°, the eighteenth line was not separable; further cut down to 100° the seventeenth was the limit, to 80° the fourteenth, and to 60° the tenth was barely reached.

_Flatness of Field._--This quality in the objective has, by the introduction of the immersion system, lost much of the importance formerly attached to it. Some writers assume it to be an “optical impossibility.” The compensating eye-piece has had the effect of contracting the visual field, consequently the peripheral imperfections of the objective are of a less disturbing character. It has, however, not been made perfectly clear whether the highest perfection of the two primary qualities of a good objective, _defining power and resolving power_, can be always obtained in one and the same combination of lenses.

Doubtless, _defining_ power can be more satisfactorily determined by the examination of a suitable object, and the perfection of the image obtained; to assist in securing which, a solid axial cone of light equal to about three-fourths of the aperture of the objective must be employed.

To sum up, then, “the focal power of all objectives depends in their perfect _definition_, a property on which their converging power depends, and in turn their magnifying action is dependent; again, focal power is the curvature imprinted by the lens on a plane wave, and is reciprocal of the true focal length. It is appropriately expressed in terms of the proper unit of focal curvature, the _dioptric_; a unit of curvature.”[39]

It may be taken as an axiom with microscopists that “neither the penetrating power nor the high-power defining objective is alone sufficient for every kind of work. The larger the details of ultimate structure, the narrower the aperture--and the converse; the minuter the dimensions of elementary structure, the wider must be the aperture of the objective.” Every worker with the microscope must have satisfied himself of the truth of this statement, when engaged in the study of the movements of living organisms, or defining the intimate structure of the minuter diatoms, or of the podura scale.

_Test for Illumination._--Dr. C. Seiler recommends the human blood corpuscle as the best test of good illumination. He prepares the object in the following manner: Take for the purpose a clean glass slide of the ordinary kind, and place near its extreme edge a drop of fresh blood drawn by pricking the finger with a needle. Then take another slide of the same size, with ground edges, and bring one end in contact with the drop of blood, as shown in Fig. 202, at an angle of 45°; then draw it evenly and quickly across the underslide, and the result will be to spread out the corpuscles evenly throughout. Blood discs being lenticular bodies, with depressed centres, act like so many little glass-lenses, and show diffraction rings if the light is not properly adjusted.[40]

_Errors of Interpretation._--To be in a position to draw an accurate conclusion of the nature and properties of the object under examination is a matter of great importance to the microscopist. The viewing of objects by transmitted light is of quite an exceptional character, rather calculated to mislead the judgment as well as the eye. It requires, therefore, an unusual amount of care to avoid falling into errors of interpretation. Among test objects the precise nature of the structural elements of the Diatomaceæ have given rise to great divergence of opinion. Then, again, the minute scales of the podura Springtails, one of the Collembola, and their congeners _Lepisma saccharina_, the structure of which is equally debatable. Mr. R. Beck, in an instructive paper published in the “Transactions of the Royal Microscopical Society,” says that the scales of the Lepisma can be made to put on an appearance which bears little resemblance to their actual structure.

In the more abundant kind of scales the prominent markings appear as a series of double lines. These run parallel and at considerable intervals from end to end of the scale, whilst other lines, generally much fainter, radiate from the quill, and take the same direction as the outline of the scale when near the fixed or quill end; but there is, in addition, an interrupted appearance at the sides of the scale, which is very different from the mere union, or “cross-hatchings,” of the two sets of lines (Fig. 203, Nos. 1 and 2, the upper portions).

The scales themselves are formed of some truly transparent substance, for water instantly and almost entirely obliterates their markings, but they reappear unaltered as the moisture leaves them; therefore the fact of their being visible at all, under any circumstances, is due to the refraction of light by superficial irregularities, and the following experiment establishes this fact, whilst it determines at the same time the structure of each side of the scale, which it is otherwise impossible to do from the appearance of the markings in their unaltered state:--

“Remove some of the scales by pressing a clean and dry slide against the body of the insect, and cover them with a piece of thin glass, which may be prevented from moving by a little gum at each corner. No. 3 may then be taken as an exaggerated section of the various parts. A B is the glass slide, with a scale, C, closely adherent to it, and D the thin glass-cover. If a very small drop of water be placed at the edge of the thin glass, it will run under by capillary attraction; but when it reaches the scale, C, it will run first between it and the glass slide, A B, because the attraction there will be greater, and consequently the markings on that side of the scale which is in contact with the slide will be obliterated, while those on the other side will, for some time at least, remain unaltered: when such is the case, the strongly marked vertical lines disappear, and the radiating ones become continuous. (_See_ No. 1, the lower left-hand portion.) To try the same experiment with the other, or inner surface of the scales, it is only requisite to transfer them, by pressing the first piece of glass, by which they were taken from the insect, upon another piece, and then the same process as before may be repeated with the scales that have adhered to the second slide, the radiating lines will now disappear, and the vertical ones become continuous. (_See_ No. 2, left portion.) These results, therefore, show that the interrupted appearance is produced by two sets of uninterrupted lines on different surfaces, the lines in each instance being caused by corrugations or folds on the external surfaces of the scales. Nos. 1 and 2 are parts of a camera lucida drawing of a scale which happened to have opposite surfaces obliterated in different parts. No. 4 shows parts of a small scale in a dry and natural state; at the upper part the interrupted appearance is not much unlike that seen at the sides of the larger scales; but lower down, where lines of equal strength cross nearly at right angles, the lines are entirely lost in a series of dots, and exactly the same appearance is shown in No. 5 to be produced by the two scales at a part where they overlie each other, although each one separately shows only parallel vertical lines.”

A well-known skilled observer of test objects[41] 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 204_a_.”

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 1/2-inch, a 4/10-inch, and a 1/4-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 1/2-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, 216_a_) 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. Cole[44]:--(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 3-1/2 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

Staining Animal Structures.

Specific stains are chiefly employed to assist the eye in distinguishing one elementary tissue from another. It is therefore necessary to stain all structures, as certain parts are seen to have a special affinity for one colouring agent rather than another, whereby they become more deeply stained, and consequently more clearly differentiated. For staining animal structures, borax, carmine, and hæmatoxylin are more frequently employed than others. The formulæ for each will be found in the Appendix “Formulæ and Methods.”

_Staining Process._--Place the section in distilled water to wash away the alcohol; place a little of the carmine in a watch glass, and immerse the section in it for four or five minutes; then remove it to a solution composed of methylated spirit five parts, hydrochloric acid one part; shake well together. This solution should be kept ready for use. Immerse the section in this solution and leave it to soak for about five or ten minutes if over-stained, until the desired tint has been obtained. Sections of skin and fibrous tissue may be left until nearly all colour is removed, the glands and hair follicles will then be brought out more clearly. The section must be transferred to methylated spirit to remove all traces of acid, then to oil of cloves contained in a watch glass, lift the section from the methylated spirit by one of the _lifters_ (Fig. 250), and carefully float it on the oil, in which it should be allowed to remain for about five minutes. This is the clearing process, the object of which is to remove the spirit and prepare the section for mounting in Canada balsam. First, however, place the section in filtered turpentine to wash away the oil of cloves; this is found to answer better than another plan adopted, that of removing the section from the oil of cloves and mounting it in balsam direct. The oil, however, has a tendency to darken the balsam.

_Logwood or Hæmatoxylin Stains_ (see Appendix for the several formulæ). Staining by this agent is effected as follows:--

After the specimen has been hardened in any of the chromic acid solutions in use, transfer it to a seven per cent. watery solution of bicarbonate of soda for about five minutes, then wash well in distilled water. Spirit prepared preparations do not require to be transferred to the soda solution, but all sections must be washed before they are transferred to the logwood stain. To a watch glass nearly full of distilled water add ten or twenty drops of the logwood stain, in which it should remain for twenty or thirty minutes. Wash well with the ordinary tap water, which will fix the dye and cause it to become blue. Dehydrate in methylated spirit, clear in clove oil, and mount in dammar or Canada balsam.

_Double-staining with Hæmatoxylin and Rosin._--Stain the section as directed above, then place it in an alcoholic solution of rosin, about one gramme of rosin to an ounce of methylated spirit, and let it soak for a few minutes; wash well in methylated spirit, clear in oil of cloves, and mount in balsam.

_Canada balsam_ should be prepared for use as follows:--One ounce of dried balsam to one fluid ounce of pure benzole; dissolve, and keep in an _outside_ stoppered bottle. Clear the section in clove oil, and place it in turpentine, clean a cover-glass and slide, place a few drops of balsam on the centre of the latter, take the section from the turpentine on a _lifter_, allow the excess of turpentine to drain away, and with a needle-point lift the section on to the balsam slide. Now take up the cover-glass with a pair of forceps (Fig. 236), and bring its edge in contact with the balsam, ease it down carefully as shown in Fig. 237, so that no air bubbles are enclosed, and with the needle point press the surface of the cover until the section lies quite smoothly and flat, and the excess of balsam is pressed out. The slide should now be transferred to the _warm-chamber_, and there allowed to remain for a day or two, or until set and hardened.

Any exuded balsam may be washed away with benzole and a soft camel’s hair brush; then dry the slide with an old piece of linen cloth, and apply a ring of cement or Japanner’s gold size. Other methods for staining and mounting will be found to answer quite as well--that of Beneke’s is a useful one for staining connective tissue.

For staining connective tissue a modification of Weigert’s method of staining fibrine is resorted to. Portions of tissue that have been fixed in alcohol having been embedded in paraffin and cut, the sections are detached and placed on glass slides, and stained for ten or twenty minutes with gentian violet, ten parts, well shaken with water 100 parts; filter, and add five to ten parts of a concentrated alcoholic gentian violet solution. Afterwards treat for one minute with lugol solution, of a port wine tint, dry with filter paper and decolourise with aniline xylol (aniline oil two parts and xylol three parts). Decolourisation having been stopped at the right point (judged from experience) mount the sections in xylol balsam. The fibres of the connective tissue should appear stained of various shades of violet.

_Double Staining_ nucleated blood corpuscles. Two kinds of staining agents are required. Stain A: dissolve five grammes of rosin in half an ounce of distilled water, and add half an ounce of rectified alcohol. Stain B: dissolve five grammes of methyl green in an ounce of distilled water. Place a drop of frog’s blood on a glass slide, and with the edge of another slide spread it evenly over the centre of the slip, and put it away to dry; when quite dry flood the slide with stain A for three minutes, and wash with water, now flood the slide with Stain B for five minutes, wash again with water, and allow the slide to dry. Apply a drop of the prepared Canada balsam and a cover-glass.

The blood of such mammals as are non-nucleated should be treated in a slightly different way. Spread a drop or two of blood on a slide and dry it quickly; then put the slide on Shadbolt’s turn-table (Fig. 238) and run a ring of cement around it; allow this time to dry, and then apply a second coating, and before this becomes quite dry place on it a clean glass cover, and press it down gently with one of the fine needles (Fig. 229), until firmly adherent.

_Epithelium_.--Remove from the mouth of a frog by scraping some _squamous_ epithelium; the columnar must be taken from the stomach; place it in glycerine, or Farrant’s solution on the slide; apply a cover-glass, and with the point of the needle press it down until the epithelium cells are separated and spread evenly over the slide. Set this aside for a day or two, then wash away any of the medium which may have escaped; dry the slide, and run a ring of cement around the edges, on the turn-table. Portions of the intestine of a rabbit or other animal may be treated in the same way. If it is wished to make permanent specimens of such structures, the intestine must be hardened in a two per cent. solution of bichromate of potash for a couple of days, then washed until all colour is discharged, and removed to a solution of picro-carmine for twenty-four hours, after which allow the stain to drain away, when it will be ready for mounting.

By the aid of the handy little spring clip (Fig. 239), objects of delicacy when mounted may be left to dry and harden for any length of time.

_Striped muscular fibre_, taken from the pig, must be teased out in a two per cent. solution of bichromate of potash, in which it should remain for two or three weeks, when it may be transferred to methylated spirit, and allowed to remain until required for mounting. Soak a piece in water to remove the spirit, place a small fragment on a slide in a few drops of water, and with a couple of needles tease the tissue up, so as to separate the fibres. Drain away the water, and apply a drop or two of Farrant’s medium and a cover-glass, which cement down as before directed.

_Fibrous tissue_ may be served in the same way. _Yellow elastic tissue_ must be first placed in a solution of chromic acid and spirit for ten days, and then treated as directed for muscular fibre.

_Non-striated Muscle._--A piece of the intestine of a rabbit should be steeped in chromic acid and spirit for ten days, then washed in water; strip off a thin layer of the muscular coat, and stain in hæmatoxylin solution. Well wash in ordinary water until the colour changes to blue, when it will be fit for mounting. Place a fragment on a slide and a drop of water, and carefully separate the fibres with a pair of needles. Drain off the water, as it is now ready for mounting, place on slide, and add a drop or two of Farrant’s medium, and place on the cover-glass.

_Nerve Tissue._--Dissect out the large sciatic nerve from a frog’s thigh, and stretch it on a small piece of wood, to which pin both ends of the nerve, and transfer it to a one per cent. solution of osmic acid for an hour or two. Wash in distilled water; tease up a small fragment on a slide (as shown in Fig. 240), and apply a drop or two of Farrant’s solution and cover-glass.

Tissues containing air should be soaked in water that has been boiled for ten minutes; this will displace the air. (For Farrant’s medium, see Appendix.)

_Glycerine Jelly._--Dissolve one ounce of French gelatine in six ounces of distilled water, and melt together in a hot-water bath. When quite dissolved, add four ounces of glycerine, and a few drops of creosote or carbolic acid. Filter through white filtering paper while warm, and keep in a capped bottle. This may be used instead of Farrant’s solution.

_Nitrate of silver_ darkens by exposure; it is used in a half per cent. watery solution. Specimens to be acted upon should be washed in distilled water, to remove every trace of sodium chloride, and then steeped in the silver solution for some two or three minutes, after which they should be again washed until they cease to turn milky; then place them in glycerine and expose them to the action of light until they assume a dark brown colour, when they should be mounted in glycerine or glycerine jelly.

By means of this stain the endothelial cells of the lymphatics, blood vessels, &c., and the nodes of Ranvier, constrictions in medullary nerves, are rendered visible. Sections of any of these may subsequently be stained by logwood or carmine.

Several methods have been adopted for staining with gold chloride. Dr. Klein’s and Professor Schäfer’s are among the best.

Dr. Klein’s method of showing the nerves of the cornea is as follows:--Remove the cornea within fifteen minutes of death; place it in a half per cent. chloride of gold solution for half an hour, or an hour; wash in distilled water, and expose to the light for a few days; in the meantime occasionally change the water. Then immerse it in glycerine and distilled water, in the proportion of one to two; lastly, place it in water, and brush gently with a sable pencil to remove any precipitate, when it will be fit for mounting in glycerine. The colour of the cornea should be grey-violet.

Schäfer adopts another method--a double chloride of gold and potassium solution.

Osmic acid, first used by Schultze, is useful for the demonstration of fatty matters, all of which it colours black; it is also valuable for certain nerve preparations. Specimens should be allowed to remain in a one or two per cent. aqueous solution of the acid from a quarter to twenty-four hours, when the staining will be completed; but if it is desired to harden specimens at the same time, they should remain in it for some few days. Osmic acid does not penetrate very deeply, therefore small portions should be selected for immersion. This is a useful stain for infusorial animals.

Chloride of palladium, another of Schultze’s staining fluids, is used to stain and harden the retina, crystalline lens, and other tissues of the eye, the cornified fat and connective tissues remaining uncoloured. The solution should be used very weak:--Chloride of palladium, one part; distilled water, 1,000 parts. Specimens should be mounted in glycerine at once, or further stained with carmine.

Dr. Schäfer employs a silver nitrate and gelatine solution for demonstrating lung epithelium; this is made as follows:--Take of gelatine ten grammes, soak in cold water, dissolve, and add warm water to 100 cc. Dissolve a decigramme of nitrate of silver in a little distilled water, and add to the gelatine solution. Inject this with a glass syringe into the lung until distension is pretty complete. Leave it to rest in a cool place until the gelatine has set; then cut sections as thin as possible, place them on a slide with glycerine, and expose to light till ready for mounting.

Of the double stains Mr. Groves prefers only those where the double colour is produced by a single process--or stains in which one colour is first employed, and then another. Single stains are picro-carmine, carmine and indigo carmine, aniline blue and aniline red.

Picro-carmine is specially useful for staining sections hardened in picric acid. It is prepared in several ways:--

1. Add to a saturated solution of picric acid in water a strong solution of carmine in ammonia to saturation.

2. Evaporate the mixture to one-fifth its bulk over a water bath, allow it to cool, filter from deposit, and evaporate to dryness, when picro-carmine is left as a crystalline powder of red-ochre colour.

Sections can be stained in a one per cent. aqueous solution, requiring only ten minutes for the process; wash well in distilled water, and transfer them to methylated alcohol, then to absolute alcohol, after which they are rendered transparent by immersing in oil of cloves or benzole, before mounting in balsam or dammar.

To summarise Mr. Groves’ recommendations:--

1. Let the material be quite fresh.

2. (_a_) Take care that the hardening or softening fluid is not too strong. (_b_) Use a large bulk of fluid in proportion to the material. (_c_) Change the fluid frequently. (_d_) If freezing be employed, take care that the specimen is thoroughly frozen.

3. (_a_) Always use a sharp razor. (_b_) Take it with one diagonal sweep through the material. (_c_) Make the sections as thin as possible; and (_d_) Remove each one as soon as cut, for if sections accumulate on the knife or razor they are sure to get torn.

4. (_a_) Do not be in a hurry to stain, but (_b_) Remember that a weak colouring solution permeates the section better, and produces the best results; and (_c_) That the thinner the section the better it will take the stains.

5. (_a_) Always use glass slips and covers free from scratches and bubbles, and chemically clean. (_b_) Never use any but extra thin circular covers, so that the specimens may be used with high powers. (_c_) Always use cold preservatives, except in the case of glycerine jelly, and never use warmth to hasten the drying of balsam or dammar, but run a ring of cement round the cover.

6. Label specimens correctly; keep them in a flat tray, and in the dark.

Double and Treble Staining.

Dr. W. Stirling[46] furnishes a brief but useful account of the methods he has employed with much success.

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

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

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

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

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

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

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

Injecting Small Animal Bodies.

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

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

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

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

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

_Prussian or Berlin Blue and Gelatine._--Take 1-1/2 ounces of gelatine, place it in a vessel and cover it with water; allow it to stand until all the water is absorbed and the gelatine is quite soft, then dissolve in hot water. Dissolve one drachm (60 grains) of Prussian or Berlin blue in six ounces of water, and gradually mix it with the gelatine solution, stirring well with a glass rod during the mixing; then filter as before.

_Watery Solution of Berlin Blue._--Dissolve 2-1/2 drachms of the blue in 18 ounces of distilled water, and filter. This staining fluid is used for injecting the lymphatic system.

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

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

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

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

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

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

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

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

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

Cutting, Grinding, and Mounting Hard Structures.

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

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

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

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

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

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

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

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

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

For _Kleinenberg’s_ solution many different formulæ exist. Squire’s improved formulæ for both stains is given in the Appendix, “_Formulæ and Methods_.”

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

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

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

_Picro-carmines_ are also largely used. The following formulæ will be found the most useful:--

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

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

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

The usual process is to stain in 1/4 or 1/2 per cent. aqueous solutions and wash in methylated spirit. Methylene blue and methyl green have the reputation of being so readily washed out in the methylated spirit as to be worthless. This is obviated by washing the sections (when removed from the stain) in distilled water, previous to the differentiation in methylated spirit. Treated in this manner, the nuclear staining is very beautiful. This also applies to Hoffmann’s blue and partly to vesuvine; with the latter, however, it is not a necessity. Safranine and gentian violet worked better by transferring the sections directly from the stain into 90 per cent. alcohol.

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

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

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

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

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

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

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

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

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

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

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

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

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

Although alkaline glycerine has been recommended for several purposes in micro-technique, it is not so well known as it should be how serviceable it is as an extempore mounting solution in vegetable histology. The best mixture for general use is composed of glycerine 2 ozs., distilled water 1-1/2 oz., solution of potash, B.P., 1/2 oz. This combines the refringent property of the glycerine with the clearing action of the caustic potash, while the swelling action of the potash is considerably diminished.

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

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

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

Bacteria Cultivation, Sterilising, and Preparing for Microscopical Examination.

That branch of mycology which is now looked upon as a separate department of science, termed bacteriology, took shape in the years 1875-9, when its founder, the veteran botanist Cohn, who recognised that the protoplasm of plants corresponded to the animal sarcode, published his exact mode of studying bacteria. But it was a pupil of his, Dr. Koch, who a year later discovered that a specific cattle disease, anthrax, was due to a bacillus, and it was he also who gave us the useful modification of gelatine as a medium in which to grow bacteria; he hit upon the method of pouring melted gelatine containing distributed germs on to plates, and thus isolating the colonies and ensuring the further isolation of the spores, and so facilitate the preparation of pure cultures on a large scale, and with great saving of time.

The difficulty of isolating a bacterium and tracing its life history under the microscope must at first sight appear great. A further objection that such work is slow and difficult has no more weight here than in any other department of science, as will be seen on proceeding to follow out the directions I am about to furnish for the use of the student.

Apparatus, Material, and Reagents employed in Bacteriological Investigations.

A good microscope with a wide-angled sub-stage condenser, and objectives of an inch, 1/4-inch, or 1/6-inch, and a 1/12-inch homogeneous oil-immersion.

A large bell-glass for covering the same when fuming acids are in use in the laboratory.

About a square foot of blackened plate-glass.

A white porcelain slab, or a shallow photographic dish of some size.

Glass bottles with ground stoppers for alcoholic solutions and aniline dyes.

Glass bottles with funnels for filtering solutions of stains, with pipettes.

A specialised form of pipette for the micro-chemical filtration of solutions (Fig. 246).

A small stoppered bottle of cedar oil (Fig. 247).

Set of small glass dishes or watch-glasses for section staining.

Stock of glass slides sterilised, together with round thin glass-covers, in boxes (Fig. 248).

Needle holders and platinum needles, with a packet of ordinary sewing needles (Fig. 249).

Platinum, or plated copper section-lifters (Fig. 250).

Glass rods, drawn out to a fine point, for manipulating sections when acids are employed.

A pair of small spring steel platinum-pointed forceps for holding glass-covers (Fig. 251).

One or two pairs of fine-pointed forceps (Fig. 251_a_).

Collapsible tubes for containing Canada balsam and dammar.

Soft rags or old pocket handkerchiefs for removing cedar oil from lenses and cover-glasses. Chamois leather for wiping lenses and removing dust.

_Reagents_, _alcohol_, bergamot oil, _celloidin_, dissolved in equal parts of ether and alcohol.

_Ebner’s_ solution. (See Appendix.)

_Formalin_, glycerine, gelatine, _Klebs’_ and _Kleinenberg’s_ solutions. (See Appendix.) The latter consisting of a watery solution of picric acid 100 parts; strong sulphuric acid two parts; filter, and add distilled water 300 parts.

_Muller_ fluid. (See Appendix.)

_Osmic acid_, a five per cent. solution.

_Paraffin_, _spermaceti_ and _xylol_, _acetic acid_, _hydrochloric acid_, a one per cent. solution with _alcohol_.

_Ammonia_ liquid, _ether_, _picro-lithium carmine_, _potash solution_.

_Safranine_, concentrated alcoholic solution of, and a watery solution.

_Turpentine_, _vesuvin_, water distilled and sterilised.

Aqueous solutions of the several dyes may be kept in bottles ready for use.

To both aqueous and alcoholic solutions a few drops of phenol, or a crystal of thymol, should be added as a preservative. For the rapid staining of cover-glass preparations, it is convenient to have the most frequently used stains--fuchsine, methyl-violet, &c.--in bottles provided with pipette stoppers.

_Clearing Agents._--Oils of cedar wood, cloves, origanum, aniline, terebene, toluol and xylol, benzol and spirits of turpentine.

_Mounting Media._--Acetate of potash solution concentrated, benzole, balsam, glycerine jelly, Fanant’s medium, dammar and mastic, Canada balsam in xylol, Hollis’s glue, zinc white.

Cement for fixing small specimens temporarily to a glass slide. Remove all traces of moisture, place upon it a drop or two of a medium prepared as follows:--Dissolve over a water bath 15 grammes of white lac in 100 grammes of absolute alcohol, decant off the clear liquid, and stand it by for a while.

As the alcohol evaporates from the warmed surface of the glass slide a hard transparent coating is left. This may be slightly softened at any time by means of a drop of oil of lavender. After arranging the objects the heat of a spirit-lamp will cause the oil to evaporate, leaving them firmly attached. Objects may be mounted on cover-glasses in a similar way. A resinous mounting medium may then be employed in the usual manner. If glycerine or glycerine jelly is the mounting medium employed, collodion diluted with two or three times its volume of oil of lavender may be found preferable as the fixing agent. The section should be placed in position before the preparation dries and the oil is evaporated.

Methylated spirit is often so largely adulterated with rock-oil as to render it unsuitable for technical purposes. Even to varnishes it imparts a fluorescent appearance as it dries off.

The needles and instruments used must not be passed through a Bunsen burner flame, which is most destructive, but enclosed in a sheet-iron box made for the purpose (Fig. 252), and placed in the hot-air steriliser for an hour at 150°C. The box can be opened at the side, and each instrument withdrawn with a pair of sterilised forceps when required for use.

_Glass plates_ are sterilised in the same iron box, and the _platinum needles_ for inoculating nutrient media, examining cultivations, &c., are served in the same manner before being used. The needles consist of two or three inches of platinum wire fixed to the end of a glass rod. Several of these needles should be made by fixing pieces of wire into a glass rod about six inches long. The glass rod must be heated at the extreme end in the flame of a Bunsen burner, or blow-pipe, and the platinum wire held near one extremity with forceps, and fused into the end of the glass rod. Some of these rods should be straight, and some bent, and others provided with a loop, and kept especially ready for inoculating test-tubes of nutrient jelly.

_Glass Dishes._--Several shallow glass dishes are required for preparing damp chamber cultivations, the upper covers fitting over the under (as in Fig. 253), in the centre of which culture-plates are stacked one above the other, and when necessary placed in the incubator.

Apparatus for Incubation and Cultivations in Liquid Media.

_Lister’s Flasks._--Lister devised a globe-shaped flask with two necks, a vertical and a lateral one, the lateral being a bent spout, tapering towards the extremity. When the vessel is restored to the erect position after pouring out some of its contents, a drop of liquid remains behind in the end of the nozzle, and thus prevents the regurgitation of air through the spout. A cap of cotton-wool is tied over the orifice, and the residue left in the flask for future use. The vertical neck of the flask is plugged with sterilised cotton-wool in the ordinary way.

Sternberg advocates the use of a glass bulb, provided with a slender neck drawn out to a fine point and hermetically sealed. Special forms of tubes, bulbs, and pipettes were devised by Pasteur, and are still in use at the Bacteriological Institute, Paris, and known as the Pasteur’s bulb pipette (Fig. 254).

Others are provided with lateral or with curved arms, one of which is drawn out to a fine point, and the slender neck plugged with cotton-wool, as in Fig. 255.

THE WARM CHAMBER, STERILISER, AND INCUBATOR.

_The Warm Chamber._--This is an accessory of importance in bacteriological work. For the continuous heating of specimens during cultivation it is an absolute necessity. Pfeiffer’s warm chamber (Fig. 256) is suitable for microscopical work generally. It consists of a hard-wood box, made air-tight, with doors and glass windows to allow of the specimen being moved from time to time, and kept under constant observation. The box is mounted on a metal plate tripod stand, and is heated from below by a small gas burner, with a thermo-regulator. A paraffin lamp will do as well, so long as it maintains a temperature of from 25° to 45°C., and without danger of injury to the stand and lenses of the microscope. A thermometer is placed in the air space to mark the temperature.

_Hot-air Incubators and Sterilisers_ are usually made of sheet-iron, in the form of a cubical chest, with double walls, supported on four legs, as that of Dr. Crookshank’s (Fig. 257). They are heated by gas or a lamp from below, while the temperature is indicated by a thermometer inserted through a hole in the top, as in that of the Hearson’s incubator. Test-tubes, flasks, funnels, cotton-wool, &c., must be sterilised by exposure to a temperature of 150°C. for an hour or more.

_Wire Cages_ or crates are used for containing test-tubes, especially when they are to be sterilised in the hot-air steriliser, or for lowering tubes of nutrient jelly into the steam steriliser. All instruments, needles, scalpels, &c., before using must be carefully sterilised.

_Steam Sterilisers_ are made either of iron or tin, jacketed with thick felt, and provided with a conical cap or lid perforated at the apex to receive a thermometer (Fig. 258). Inside the vessel is an iron grating or diaphragm about two-thirds of the way down, which divides the interior into two chambers, the upper or steam chamber, and the lower or water chamber. A gauge outside marks the level of the water in the lower chamber; this should be kept about two-thirds full. The apparatus stands upon three legs, and is heated from below with a Bunsen burner or a lamp. It is employed for sterilising nutrient media in tubes or flasks, for cooking potatoes or hastening the filtration of agar-agar. When the thermometer indicates 100° C. the lid is removed, and test-tubes are lowered in a wire-basket by means of a hook and string, and the lid quickly replaced. Potatoes or small flasks are lowered into the cylinder in a tin receiver with a perforated bottom, which rests upon the grating, and admits of the contents being exposed to the steam generated.

One of the most efficient forms of incubators introduced into the bacteriological laboratory is that known as Hearson’s (Fig. 259). This consists of a chamber surrounded by a water-jacket, with water space below, to afford room for the pipe, L, which conveys the heated products from the flame of the lamp, T, through the water and back again to the lantern. A is the water-jacket surrounding the chamber containing the cultures; O, the pipe through which the water supply is admitted; N, the tap for employing the same; M, the overflow pipe; S, the capsule in a case attached by a tube to the lower plate outside; D, a lever pivoted on the left, carrying at its free end a damper, F, which, when resting on the chimney, V, effectually closes it; P, a screw for adjusting the damper when starting the apparatus; H, a lead weight for bringing more pressure on the capsule; K, a thermometer, the bulb of which is inside and the scale outside the chamber.

The treated products of combustion move in the direction indicated until the water and chamber are sufficiently heated to distend the capsule. When this point is reached the wire between S and P is pushed up by the capsule, and the lever causes the damper to rise more or less off the chimney, V, and on examining the thermometer the inside of the chamber is at length found to remain steadily at the required temperature.

When the thermometer registers the desired temperature, the lead weight must be damped to the lever by means of the milled-head screw which goes through it. After having been once adjusted the heat in the interior will remain constant, notwithstanding the utmost changes of temperature occurring in these latitudes, nor will very great alterations in the size of the lamp-flame seriously interfere with the results. The milled-head screw, P, must be turned, after the first adjustment, during the whole time that the incubator is in use. Observe the temperature before opening the door; observations taken afterwards are worthless.

Preparation of Nutrient Media--Separation, and Cultivation of Bacteria.

To cultivate micro-organisms artificially they must be supplied with the proper nutrient material, perfectly free from pre-existing organisms. The secret of Koch’s methods greatly depends upon the possibility, in the case of starting with a mixture of micro-organisms, of being able to isolate them completely one from another, and to obtain an absolutely pure growth of each cultivable species. When sterile nutrient gelatine has been liquefied in a test-tube and inoculated with a mixture of bacteria in such a way that the individual micro-organisms are distributed throughout it, and the liquid is poured out on a glass plate and allowed to solidify, the individual bacteria, instead of moving about freely as in a liquid medium, are fixed to one spot, where they develop their own species. In this way colonies are formed, each possessing its own biological characteristics and morphological appearances (Fig. 260).

To maintain individuals isolated from each other during growth, and free from contamination, it is only necessary to thin out the cultivation to protect the plates from the air, and to have facilities for examining them from time to time, and observing the characteristic microscopical appearances. The colonies on nutrient gelatine examined with a low power (Fig. 260), if micro-organisms such as _Bacillus anthraces_ and _Proteus mirabilis_, the naked eye appearances in test-tubes of the growth of the bacilli of anthrax and tubercle, and the brilliant growth of micro-coccus prodigiosus, may be given as examples in which the appearances are often very striking and sometimes quite characteristic. I must, however, first direct attention to a well-recognised fact, that bacteriology only touches animal pathology at a few points, and that so far from bacteria being synonymous with _disease germs_, the majority of these remarkable organisms appear to be beneficent rather than inimical to man. This is of immense importance to science, as I shall attempt to show further on; although even a brief description of all the useful ferments due to bacteria and brought into use would occupy a volume to themselves, and call for a school of bacteriology quite apart from that involved in the medical aspect of the question, for the purpose of fully investigating problems raised by the agriculturist, the forester, the gardener, the dairyman, brewer, dyer, tanner, and other industries, which open up vistas of practical application, and to some extent are already being taken advantage of in commerce.

_The Preparation of Nutrient Gelatine and Agar-agar._--Take half a kilogramme (one pound) of beef as free as possible from fat, chop finely, transfer to a flask or cylindrical vessel, and shake up well with a litre of distilled water. Place the vessel in an ice-pail, or ice-cupboard, or in winter in a cold cellar, and leave for the night. Next morning commence with the preparation of all requisite apparatus. Thoroughly wash and rinse with alcohol about 100 test-tubes, and allow them to dry. Plug the mouths of the test-tubes with cotton-wool, place them in their wire cages in the hot-air steriliser, to be heated for an hour at a temperature of 150°C. In the same manner cleanse and sterilise several flasks, and a small glass funnel. In the meantime, the meat infusion must be well shaken, and the liquid portion separated by filtering and squeezing through a linen cloth or a meat press. The red juice thus obtained must be brought up to a litre by transferring it to a large measuring glass and adding distilled water. It is then poured into a sufficiently large and strong beaker, and set aside after the addition of ten grammes of peptone, five grammes of common salt, and 100 grammes of best gelatine.

In about half an hour the gelatine is sufficiently softened, and subsequent heating in a water bath causes it to be completely dissolved.

The next process requires the greatest care and attention. Some micro-organisms grow best in a slightly acid, others in a slightly alkaline, medium. For example, for the growth and characteristic appearances of the _comma bacillus_ of Asiatic cholera a faintly alkaline soil is absolutely essential. This slightly alkaline medium will be found to answer best for most micro-organisms, and may be obtained as follows:--With a clean glass rod dipped in the mixture, the reaction upon litmus-paper may be obtained, and a concentrated solution of carbonate of soda must be added drop by drop until red litmus-paper becomes faintly blue. If it is too alkaline, it can be neutralised by the addition of lactic acid.

Finally, the mixture is heated for an hour in the water-bath. Ten minutes before the boiling is completed the white of an egg beaten up with the shell is added, and the liquid is then filtered while hot.

During filtration the funnel should be covered over with a plate of glass, and the process of filtering must be repeated, if necessary, until a pale straw-coloured, perfectly transparent filtrate results. The sterilised test-tubes are filled to about a third of their depth by pouring in the gelatine carefully and steadily. The object of this care is to prevent the mixture touching the part of the tube with which the plug comes into contact; otherwise, when the gelatine sets, the cotton-wool adheres to the tubes and becomes a source of embarrassment to subsequent procedures. As the tubes are filled they are placed in a basket, and then sterilised. They are either lowered into the steam steriliser, when the thermometer indicates 100 cc., for twelve minutes, for four or five successive days, or they may be transferred to the test-tube water-bath, and heated for an hour or two for three successive days.

If the gelatine shows any turbidity after, it must be poured back into a flask, boiled for ten minutes, and filtered again, and the process of sterilisation repeated.

_Nutrient Agar-agar_ is a substance prepared from seaweed which grows on the coasts of Japan and India, and is supplied in long crinkled strips. It boils at 90° C., and remains solid up to a temperature of about 45° C. It is therefore substituted for gelatine in the preparation of a jelly for the cultivation of those bacteria which will grow best in the incubator at the temperature of the blood, and also at ordinary temperature for bacteria which lignify gelatine. The preparation is conducted on much the same principles as those already described. Instead, however, of 100 grammes of gelatine, only about twenty grammes of agar-agar (1·5 to 2 per cent.), and to facilitate the solution it must be allowed to soak in salt water overnight. Flannel is substituted for filter paper. The hot-water apparatus is invariably employed. The final results, when solid, should be colourless and clear; but if slightly milky, it may still be employed.

_Wort-gelatine_ is used in studying the bacteria of fermentation. It is made by adding from five to ten per cent. of gelatine to beer-wort.

_Glycerine Agar-agar._--This is made by adding five per cent. of glycerine to nutrient agar-agar, after the boiling and before the filtration.

_Test-tube Cultivations._--To inoculate test-tubes containing nutrient jelly, the cotton-wool plug is removed. A sterilised needle, charged, for example, with blood or pus containing bacteria, is thrust once in the middle line into the nutrient jelly, and steadily withdrawn. The tube should be held horizontally or with its mouth downwards, and the plug replaced as quickly as possible, and an india-rubber cap fitted over the mouth of the tube.

The appearance produced by the growths in the test-tubes can be in most cases sufficiently examined with the naked eye (Fig. 261). In some cases the jelly is partially liquefied, while in others it remains solid. The growths may be abundant or scanty, coloured or colourless. When liquefaction slowly takes place in the needle tracts, the appearances which result are often very delicate and in some very characteristic. The appearance of a simple white thread with branching lateral filaments, of a cloudiness, or of a string of beads in the track of the needle, may be given as examples. In some cases much may be learnt by means of a magnifying-glass.

Beneke recommends that gelatine culture tubes should be inoculated by making a puncture quite at the side of the medium, close to the glass. The advantage of this method over the plan of inoculating the mass in the middle is that the growing culture can be microscopically examined from the outside, and various details made out, such as the nature of the growth, the comparative appearance of colonies near the surface and those situated more deeply, and the presence of one or more distinct organisms. If the tubes used have the opposite sides flat and parallel, such examinations will be still further facilitated.

_Plate Cultivations._--By this method a mixture of bacteria, whether in fluids, excreta, or in cultivations on solid media, can be so treated that the different species are isolated one from the other, and perfectly pure cultivations of each of the cultivable bacteria in the original mixture established in various nutrient media. We are enabled also to examine under a low power of the microscope the individual colonies of bacteria. The same process, with slight modification, is also employed in the examination of air, soil, and water.

In order to spread out the liquid jelly evenly on the surface of a glass plate, and to hasten its solidification, it is necessary to place the plate upon a level and cool surface. The glass plates are sterilised in an iron box placed in the hot-air steriliser, at 150° C., from one to two hours.

The damp chambers for the reception of the inoculated plates are prepared by cleansing and washing out with one in twenty carbolic acid the shallow glass dish and bell-cover (Fig. 253). A piece of filter-paper should cover the bottom of dish, moistened with the same solution.

“In a glass-beaker with pad of cotton-wool at bottom place tube containing cultivation, the three tubes to be inoculated, three glass rods which have to be sterilised, and a thermometer. Liquefy the gelatine in the three tubes by placing them in a beaker containing water 30° C. Keep the tubes, both before and after the inoculation, in the warm water to maintain the gelatine in a state of liquefaction. Remove the plug from the culture and also the plug of test-tube with liquefied jelly. With the needle take up a droplet of the cultivation and stir it round in the liquefied jelly. Replace both plugs, and set aside the cultivation. Hold the freshly-inoculated tube almost horizontally, then raise it to the vertical, so that the liquid gelatine gently flows back. By repeating this motion, and rolling the tube, the micro-organisms which have been introduced are distributed throughout the gelatine. Any violent shaking, and consequent formation of bubbles, must be carefully avoided. Inoculate the second tube, and also third, in the same way, but with three droplets from a sterilised needle. The next process consists in pouring out the gelatine on glass plates and allowing it to solidify.

“Remove cover of box containing sterilised plates, withdraw a plate with sterilised forceps, and rapidly transfer it to the filter-paper under the bell-glass and quickly replace cover of box. Remove plug from the test-tube which was first inoculated, and the contents are poured out on the plate. With a glass rod the gelatine must be then rapidly spread out in an even layer within about half an inch of the margin of the plate, the bell-glass is replaced, and the gelatine is allowed to set. Meanwhile a glass bench is placed in damp chamber, upon which the plate is placed when the gelatine is quite solid; precisely the same process is repeated with the other tubes.

“The colonies will be found to develop in the course of a day or two, the time varying with the temperature of the room. The lower plate will contain a countless number of colonies, which, if the micro-organisms liquefy gelatine, speedily commingle, and produce in a very short time a complete liquefaction of the whole gelatine. On the middle plate the colonies will also be very numerous, but retain their isolated positions for a longer time; while on the uppermost plate the colonies are completely isolated from one another, with an appreciable surface of gelatine intervening.

“The microscopical appearances of the colonies are best studied by placing the plate on a slab of blackened glass, or on a porcelain slab if the colonies are coloured. A small diaphragm is used, and the appearances studied principally with a low power. A much simpler method of plate-cultivation is to pour the liquefied jelly into shallow flat dishes; they take up much less room, and in many ways are more convenient.

“Nutrient agar-agar can also be employed for the preparation of plate-cultivations, but it is much more difficult to obtain satisfactory results.”

Microscopical Examination of Bacteria.

_Bacteria in Liquids, Cultures, and Fresh Tissues._--In conducting bacteriological researches, the importance of absolute cleanliness cannot be too strongly insisted upon. All instruments, glass vessels, slides, and cover-glasses should be thoroughly cleansed before use. The same applies to the preparation and employment of culture media; any laxity in the processes of sterilisation, or insufficient attention to minute technical details, will be followed with disappointing results by contamination of the cultures, resulting in the loss of much time.

For the preparation of microscopical specimens it will be found convenient to use a platinum inoculating needle, sterilised, as before directed, in the sheet-iron box; in a few moments it will be cool enough not to destroy the bacteria with which it is brought into contact.

_Unstained Bacteria._--The bacteria in liquids, such as blood and culture-fluids, can be investigated in the unstained condition by transferring a drop with a looped platinum needle, or a capillary pipette, to a slide, covering it with a clean cover-glass, and examining without further treatment. If it is desirable to keep the specimen under prolonged observation, a drop of sterilised water or salt solution must be run in at the margin of the cover-glass to counteract the tendency to dry.

Cultures on the solid media can be examined by transferring a small portion with a sterilised needle to a drop of sterilised water on a slide, thinning it out, and covering with cover-glass as already described. Tissues in the fresh state may be teased out with needles (Fig. 249) in sterilised salt solution, and pressed out into a sufficiently thin layer between the slide and cover-glass. Glycerine may in many cases be substituted for salt solution, especially for such as actinomyces and mould fungi.

Very small bacilli and micro-cocci are distinguished from granular matter or fat-crystals, or _vice versâ_, by the fact that the latter are altered or dispersed by the addition of acetic acid, and changed by solution of potash; ether dissolves out fatty particles, while micro-organisms remain unaffected. Baumgarten demonstrated tubercle bacilli in sections by treating them with potash, which clarified the tissues and brought the bacilli clearly into view. In examining unstained bacteria the iris-diaphragm should be used, and the sub-stage condenser carefully centred and focussed.

His’s Method of Staining.--A slide is prepared as for bacteria in the fresh state; the reagents are then applied by placing them with a pipette drop by drop at a margin of the cover-glass, and causing them to flow through the preparation by means of a strip of filter-paper placed at the opposite margin.

Babès’ Method is as follows: A little of the growth spread out on a cover-glass into as thin a film as possible; when almost dry, apply a drop or two of a weak aqueous solution of methyl-violet from a pipette to the film; any excess of the stain must be removed by gentle pressure with a strip of filter-paper.

_Cover-glass Preparations._--A cover-glass is smeared with the substance to be examined spread out into a sufficiently thin layer; in the case of cultures on solid media, diffuse the bacteria in a little sterilised water. By means of another cover-glass the juice or fluid is squeezed out from between them into a thin layer, and on sliding them apart each cover-glass bears on it a thin film of the material. The cover-glass is then placed with its film side upwards and allowed to dry. After a few minutes it is passed from above downwards through the flame of a Bunsen burner three times. Apply two or three drops of an aqueous solution of fuchsine or methyl-violet to cover the film, wash away any surplus stain after a few minutes with distilled water. The cover-glass is then allowed to dry, when the preparation may be mounted in Canada balsam, or while still wet, turned over on a slide, and the excess of water removed with filter-paper.

If necessary to apply stain for a much larger period, pour staining solution into a watch glass and allow cover-glass to swim on surface with prepared side downwards.

Crookshank, instead of watery solutions of aniline dyes, prefers to use stronger solutions, and to reduce the staining by a momentary immersion in alcohol. The method is as follows: cover-glass preparations are stained with carbolised fuchsine (_Neelsen’s solution_) for about two minutes, rinsed in alcohol for a few seconds, and quickly washed in water. This method is specially valuable for sarcinæ and streptococci.

_Gram’s Method._--The whole film is first stained violet with gentian-violet, fixed by a solution of iodine, in iodide of potassium in the bacilli, but not in any débris, pus cells, or tissue elements present. Transfer cover-glass to alcohol, the bacilli alone remain stained, the violet colour being changed to blue. By employing a contrast colour, such as eosin, a double staining is obtained.

For staining preparations with gentian-violet Crookshank employs the following useful method:--Place four or five drops of pure aniline in a test-tube, add distilled water to three-quarters full, close mouth with thumb, shake thoroughly. Filter the emulsion twice, pour filtrate into watch-glass. To the perfectly clear aniline water thus obtained, add, drop by drop, a concentrated alcoholic solution of gentian-violet till precipitation commences. Cover-glasses must be left in this solution ten minutes, transferred to iodine-potassic-iodide until the film becomes uniformly brown, then rinsed in alcohol. The decolourisation may be hastened by dipping the cover-glass in clove oil and returning to alcohol. Again immerse cover-glass in clove oil, dry by gently pressing between two layers of filter-paper, and mount in Canada balsam.

_Double-staining_ of cover-glass preparations.--They can be treated by Ehrlich’s method for staining tubercular sputum, or by Neelsen’s modification, or by staining with eosin after treatment by the method of Gram.

Ehrlich’s Method is as follows: Five parts of aniline oil are shaken up with one hundred parts of distilled water, and the emulsion filtered through moistened filter-paper. A saturated alcoholic solution of fuchsine, methyl-violet, or gentian-violet, is added to filtrate in watch-glass, drop by drop, until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen minutes to half an hour, then washed for a few seconds in dilute nitric acid (one part of nitric acid to two of water), then rinsed in distilled water.

Neelsen’s Solution and Methylene Blue.--Ziehl suggested the use of carbolic acid as a substitute for aniline blue. Neelsen recommended a solution of carbolic acid, absolute alcohol and fuchsine. (See Appendix.)

_Gram’s Solution and Eosin._--After using Gram’s method as above and decolourising in alcohol, the cover-glass is placed in a weak solution of eosin for two or three minutes, washed in alcohol, immersed in clove oil, dried, and mounted in balsam.

_Staining of Spores._--The cover-glass preparation must be heated to 210° C. for half an hour, or passed about twelve times through the flame of a Bunsen burner, or exposed to the action of strong sulphuric acid for several seconds, then a few drops of a watery solution of aniline dye applied in the usual way. To double-stain spore-bearing bacilli the cover-glass preparation must be floated from twenty minutes to an hour on Ehrlich’s fuchsine-aniline-water, or on the Ziehl-Neelsen solution. The stain must be heated until steam arises.

Staining of Flagella.

Koch first stained flagella by floating the cover-glass on a watery solution of hæmatoxylin, transferring them to a five per cent. solution of chromic acid, or to Müller’s fluid, by which they obtained a brownish-black coloration.

_Löffler’s Method._--Add together aqueous solutions of ferrous-sulphate and tannin (twenty per cent.) until the mixture turns a violet-black colour, then add three or four cc. of a one-in-eight aqueous solution of logwood; a few drops of carbolic acid may be added before transferring to a stoppered bottle; that is the mordant. The dye consists of 1 cc. of a one per cent. solution of caustic soda, added to 100 cc. of aniline water, in which four or five grammes of either methyl-violet, methylene blue, or fuchsine, are dissolved. A cover-glass preparation is made in the usual way, then the film is covered with mordant, and cover-glass held over flame until steam rises, the mordant is then washed off with distilled water. The stain is filtered and a few drops allowed to fall on film, after a few minutes the cover-glass is again warmed until steam rises. The stain is then washed off with distilled water, and the preparation is ready to be mounted for examination.

As Löffler’s process is somewhat complicated, a modification has been said to afford more satisfactory results. A specimen is taken from a recent gelatine culture and diluted with water. A little of the fluid is then transferred to a warm cover-glass by means of a pipette and allowed to dry, after which a drop of the following mordant is applied:--Aqueous solution of tannin (twenty per cent.), ten cc.; cold saturated solution of ferrous sulphate, five cc.; saturated solution of fuchsine in absolute alcohol, one cc. The cover is next heated gently for a short time until vapours are given off, then washed carefully. This process is repeated two or three times, and the specimen washed after each application. Subsequently, staining is effected by means of Ziehl’s fuchsine solution, the cover is afterwards warmed once or twice for about fifteen seconds, then washed, and the specimen examined in water to ascertain if the colour is sufficiently intense. If satisfactory, the preparation may then be dried and finally mounted in Canada balsam or dammar.

_Preservation of Preparations._--After examining a cover-glass preparation with an oil-immersion objective the cedar oil must be carefully wiped off, and the slide set aside for the Canada balsam to set. At a convenient time these preparations should be sealed with a ring of Hollis’s glue.

Bacteria in Sections of Tissues.

_Method of Hardening and Decalcifying Tissues._--To harden small organs, such as the viscera of a mouse, they should be placed on a piece of filter-paper at the bottom of a small wide-mouthed glass jar, and covered with about twenty times their volume of absolute alcohol. Larger organs are treated in the same way, but must be cut up into small pieces. Müller’s fluid, methylated spirit, or formalin may be used.

Teeth, or osseous structures, must first be placed in a decalcifying solution, as Kleinenberg’s. When sufficiently softened, soak in water, to wash out picric acid, and transfer to weak spirit. Ebner’s solution gives good results.

Methods of embedding, fixing, and cutting.--Crookshank finds that after hardening, the pieces of tissue are embedded in a mixture of ether and alcohol for an hour or more, then transferred to a solution of celloidin in equal parts of ether and alcohol, and left there for several hours.

The piece of tissue is then placed in a glass capsule, and some of the celloidin solution poured over it. The capsule can be placed bodily in 60 to 80 per cent. alcohol, and left there until the following morning. The celloidin should be of the consistency of wax. The piece of tissue is next cut out, and after trimming is put into water until it sinks, then transferred to gum, and cut with the freezing microtome.

Sections of fresh tissues are to be floated in ·8 per cent. salt solution, and then carefully transferred by a platinum lifter to a watch-glass containing absolute alcohol.

_Staining Bacteria in Tissue Sections._--Weigert’s method is as follows:--Place sections for from six to eighteen hours in a one per cent. watery solution of any of the basic aniline dyes. To hasten, place the capsule containing solution in the incubator, or heat it to 45° C., or a stronger solution may be used. In the latter case the sections must be treated with a half-saturated solution of carbonate of potash, as they are easily over-stained. In either case the sections are next washed with distilled water, passed through sixty per cent. alcohol into absolute alcohol. When almost decolourised, spread out on a platinum lifter and transfer to clove oil, or stain with picro-carmine solution (Weigert’s) for half an hour, wash in water, alcohol, and treat with clove oil, and transfer to clean glass slide.

_Gram’s Method._--Sections are stained for ten minutes in a capsule containing aniline-gentian-violet solution, then placed in the iodine and iodide solution until uniformly brown, then placed in absolute alcohol, and washed by carefully moving sections in the liquid with a glass rod. When completely decolourised, they are transferred to clove oil and then to a slide.

Double-staining is obtained by transferring the sections after decolourisation to eosin, Bismarck brown, or vesuvin (Crookshank).

_Formalin_ is an excellent preservative fluid; one part to 20,000 is sufficient to prevent fermentation. For the preservation of vegetable sections, a one per cent. solution is required; even the fresh appearance of vegetable structures is preserved for some time when immersed in it. In the nutrient gelatine for biological specimens, if used early, will arrest the liquefaction of the gelatine by bacteria. For hardening it saves time, and is even better than alcohol, chromic acid, pot. bich., and many others. It does not cause shrinkage of the cells. Tissue 1/2 to 3/4 inch thick hardens in twenty-four hours in pure formalin; five to ten per cent. is best for loose tissue. In another method, by which time can be saved, instead of placing the specimen in the _formalin_ and afterwards in mucilage, prior to cutting sections, make the mucilage with two per cent. (or stronger) formalin water, and it will then answer both purposes at the same time.

Preparing, Mounting, Cementing and Collecting Objects.

Various materials are required for preparing and mounting microscopic objects, as slips of glass, patent flatted plate measuring 3 × 1 inch, thin glass covers, glass cells, preservative media, varnishes, cements, a glazier’s diamond, and a Shadbolt’s turn-table.

The glass slides and covers, although sent out packed ready for use, should be immersed in an alkaline solution to ensure perfect freedom from any greasiness derived from touching by the fingers. Dr. Seller recommends a particular solution for this purpose. (See _Formulæ_, Appendix.)

Varnishes and cements must be selected with care, as these are not only expected to adhere firmly to the glass slide, but also to resist the action of the preservative fluid in which the specimen may be mounted. Among the numerous preparations employed, I may enumerate Canada balsam, gum dammar, Venice turpentine, Japanners’ gold size, used for closing up cells, asphalte varnish, Brunswick black, shellac, glue and honey, Hollis’ liquid glue, and marine glue. To give a finish to the mounted specimen, coloured varnishes are sometimes resorted to. A red varnish of sealing-wax is made by digesting powdered sealing-wax in strong alcohol. Filter, and place the solution in a dish, and evaporate by means of a sand bath to reduce it to a proper consistency. This is said to resist the action of cedar oil. For white, zinc, cement is the best. This is made of benzole, gum dammar, oxide of zinc, and turpentine. Cole gives another formula, but either of these may be obtained of Squire, who supplies every kind of staining and mounting material.

_Cells for Mounting._--The minuter forms of life should be mounted in thin cells, which may be readily made with Japanners’ gold size, dammar or asphalte, and a Shadbolt or Walmsley’s turntable. The glass slide being placed under the metal springs in such a manner that its two ends shall be equi-distant from the centre (a guide to the position is afforded by the circles traced out on the brass), take a camel’s hair pencil and dip it into the Japanner’s gold-size, holding it firmly between the finger and thumb, and set the wheel in motion, when a perfect circle will be formed; put it aside to dry, or place it in the warm chamber to harden. To cut cover-glasses place a sheet of thin glass under the brass springs, and substitute for the pencil a cutting diamond. A cutting diamond is not only useful to the microscopist for the above purpose, but also for writing the names of mounted objects on one end of the slide.

It will be found convenient to make a number of such cells, and keep a stock ready for use. There are many objects whose structure is very transparent. These should be mounted dry. Scales from the wings of butterflies and moths, of the podura and lepisma, and some of the diatomaceæ are of this class. All that is necessary in preparing objects for dry mounting is to take care that they are free from extraneous matter, and fix them permanently in the position in which their structure will show to the best advantage.

For mounting specimens of greater thickness it is desirable to use deeper cells. It will then be found convenient to make a second or a third application of the gold-size, allowing sufficient time between applications for the varnish to dry. Cells of a still deeper kind are made up by cementing rings of glass or metal to the glass-slides with marine glue or Brunswick black. The latter will be rendered more durable by mixing in a small quantity of indiarubber varnish (made by dissolving small strips of caoutchouc in gas-tar). The process of mounting in glass-cells is similar to that employed in making varnish-cells, except that a somewhat larger quantity of cementing medium is required. Objects mounted in this way should be kept for a time in the horizontal position, and a little fresh varnish must be applied if the cement shows a tendency to crack. In mounting objects in balsam, care must be taken to have the specimen _quite dry_ before transferring it to turpentine. Objects mounted in cells should become _perfectly saturated_ with the mounting fluid before being finally cemented down.

It is preferable to mount and preserve specimens of animal tissues in shallow cells, to avoid undue pressure on the preparation. Cells intended to contain preparations immersed in fluid must be made of a substance impervious to the fluid used, such as here represented (Fig. 263). The surface of the fixed glass-circle should be slightly roughened before applying the cement.

Different modes of mounting may be employed with advantage; for instance, entomological specimens, as legs, wings, spiracles, tracheæ, ovipositors, stings, tongues, palates, corneæ, should be mounted in balsam; the trachea of the house-cricket, however, should be mounted dry. Sections of bone may either be mounted dry or in a fluid. Other objects, as sections of wood and stones of fruit, exhibit their structure best in Canada balsam.

In mounting entomological specimens, the first thing, of course, is the dissection of the insect. This is best accomplished by the aid of a dissecting microscope, a pair of small brass forceps, and finely-pointed scissors; the parts to be prepared and mounted should first be carefully detached from the insect with the scissors, then immersed in a solution of caustic alkali (_liquor potassæ_) for a few days, to soften and dissolve out the fat and soft parts. The length of time necessary for their immersion can only be determined by experience, but, as a general rule, the objects assume a certain amount of transparency when they have been long enough in the alkali; when this is ascertained, the object must be placed in a proper receptacle and put by to soak for two or three hours in soft or distilled water. It should then be placed between two slips of glass, and gently pressed till the softer parts are removed. Should any adhere to the edge of the object, it will be necessary to wash the specimen carefully in water, a process that will be much assisted by the delicate touches of a camel’s-hair brush. Place the object now and then under the microscope to see that all extraneous matter is removed, and when this is accomplished take the specimen up carefully with the camel’s-hair brush, or a lifter, and place it on a piece of very smooth paper (thick ivory note is the best for the purpose), arrange it carefully with the brush and a finely pointed needle, place a second piece of paper over it, and press it flat between two slips of glass, and compress it by a small spring clip (Fig. 264). A dozen clips may be had for a few pence. When _thoroughly_ dry (which it will probably be in about twenty-four hours, if in a warm room), separate the glasses, and gently unfold the paper; then, with a little careful manipulation, the object may be readily detached, and placed in a little spirit of turpentine, where it should be allowed to remain until rendered transparent and fit for mounting. The time during which it should remain in this liquid will depend on the structure; some objects, such as wings of flies, will be quickly permeated, while horny and dense objects require an immersion of a fortnight or even longer. A pomatum pot with a _concave_ bottom and well-fitting lid will answer admirably for conducting the soaking process in; and it is well, in preparing several specimens at a time, to have two pots, one for large and medium, the other for very small objects, otherwise the smaller will adhere to the larger.

In mounting objects in fluid, the glass cover should come nearly, but _not quite_, to the edge of the cell, a slight margin being left for the cement, which should project slightly over the edge of the cover, in order to secure it to the cell.

_Media for Preserving Algæ._--The most useful preservative media for algæ are chrome-alum, formalin, and camphor water. The solution should consist of one per cent. of chrome-alum and one per cent. of formalin; this will render the gelatinous sheath and matrix form clear, while it will retain the colour of the algæ in most cases. The Chlorophyceæ do well in any of these media; but other species, as _Ulva Lactuca_, are rendered somewhat brittle. For such use formalin alone. The Phæophyceæ should be placed while fresh in the formalin; the larger forms are better fixed by placing them for an hour or two in chrome-alum solution. The Florideæ do well in any of the three solutions, but the more delicate species, _Griffithsia_, require a two per cent. formalin solution in sea-water; the plant preserves its natural appearance in this medium.

To preserve and mount diatomaceæ in as nearly as possible a natural condition, they should be first well washed in distilled water and mounted in a medium composed of one part of spirits of wine to seven parts of distilled water. The siliceous coverings of the diatoms, however, which show various beautiful forms under the higher powers of the microscope, require more care in preparation. The guano, or infusorial earth containing them, should first be washed several times in water till the water is colourless, allowing sufficient time for precipitation between each washing. The deposit must then be put into a test tube and nitro-hydrochloric acid (equal parts of nitric and hydrochloric acids) added to it, when a violent effervescence will take place. When this has subsided, the whole should be subjected to heat, brought nearly to the boiling point for six or eight hours. The acid must now be carefully poured off, and the precipitate washed in a _large_ quantity of water, allowing some three or four hours between each washing, for the subsidence of some of the lighter forms. The sediment must be examined under the microscope with an inch object-glass, and the siliceous valves of the diatoms picked out with a coarse hair or bristle.

Dr. Rezner’s Mechanical Finger (Fig. 265) for selecting and arranging diatoms, adaptable to any microscope, is made to slip on to the objective far enough to have a firm bearing, and so that the bristle point can be brought into focus when depressed to its limit. It is clamped in its place by a small thumb-screw. The bristle holder slides into its place, and is carefully adjusted to the centre of the field. When using the finger, the bristle is first raised by means of the micrometer screw till so far within focus as to be nearly or quite invisible, then the objective is focussed on to the slide, and the desired object sought for and brought into the centre of the field; the bristle point is then lowered by the screw until it reaches the object, which usually adheres to it at once, and can then be examined by rotating the bristle wire by means of the milled head.

The medium used for mounting diatomaceæ is of considerable importance, inasmuch as their visibility is either diminished or much increased thereby. Professor Abbe, experimenting with the more minute test objects, diatoms, &c., found monobromide of naphthaline gave increased definition to most of them. This liquid is colourless, somewhat of an oleaginous nature, and is soluble in alcohol. Its density is 1·555, and refractive index 1·6. Its index of visibility is about twice that of Canada balsam.

Taking the refractive index of air as 1·0, and diatomaceous silex as 1·43, the visibility may be expressed by the _difference_ ·43.

The following table may be constructed :--

Refractive indices Visibility of silex (taken approximately). (Refr. index = 1·43). Water = 1·33 10 Canada balsam = 1·54 11 Bisulphide of carbon = 1·68 25 Sol. of sulphur in bisulph. = 1·75 32 " phosphorus " = 2·11 67

These data relating to visibility must be taken in connection with the numerical aperture of the objectives and of the illuminating pencil. The effect produced on diatoms is very remarkable, the markings on their siliceous frustules being visible under much lower powers.

So that the visibility of the diatom mounted in phosphorus as compared with balsam is as sixty-seven to eleven; in other words, the image is six times more visible. Mr. Stephenson’s phosphorus medium is composed of a solution of solid or stick phosphorous dissolved in bisulphide of carbon. Great care is required in preparing the solution owing to the very inflammable nature of the materials. So small a quantity of the bisulphide of carbon is required to dissolve the phosphorus that the diatom may be said to be mounted in nearly pure phosphorus. Remarkable enough, this medium has the reverse effect upon such test-objects as podura and lepisma scales. These lose their characteristic markings.

For mounting minute objects, carbolic acid solution will be found a useful medium--the purest crystals of carbolic acid dissolved in just sufficient water to render them fluid. No more should be dissolved than may be wanted for the time being, as if left standing exposed to the light it changes colour. Small crustacean foraminifera, the palates of moluscs, after boiling a short time in liquid potash and well washing to remove all traces of alkali, may be preserved in carbolic acid solution. Should the specimens appear cloudy gently warm the slide over a spirit lamp.

_Preserving and Killing Rotatoria with cilia in situ._--Mr. C. Rousselet’s method of preserving and mounting the Rotatoria[47] has been attended with so much success that the old difficulty attendant upon the preservation of these various beautiful forms of infusorial life has been practically overcome. The process resorted to consists of four stages, namely, narcotising, killing, fixing, and preserving. In dealing with rotifers hitherto, the difficulty has been that of successfully killing them with their rotating organs fully extended. It has been found needful to have recourse in the first instance to a narcotising agent, and one that acts slowly. The most suitable is a weak solution of the hydrochlorate of cocaine, a one per cent. solution, or even weaker. This was first proposed by Mr. Weber for keeping these active little bodies quiet while under observation. Mr. Rousselet carries this agent further; he applied it to narcotise them prior to killing, and this it does most effectually. The rotifers are seen to sink to the bottom of the live-cell, and the cilia gradually to slacken in motion, and the time for killing has arrived. This is effected by Flemming’s chromo-aceto-osmic acid. A rather weak solution must be employed--consisting of 1 per cent. solution of chromic acid, 15 parts; 2 per cent. osmic acid, 4 parts; glacial acetic acid, 1 part--which is at the same time a killing and fixing medium. The word “fixing” must not be taken to imply simply fixing, as it includes rapidly _killing_ and _hardening_ and preventing further change in the tissues of the rotifers by subsequent treatment, as mounting. The animal, therefore, must remain quietly for a few minutes, and then taken out and washed in five or six changes of distilled water, and hence transferred to the preservative fluid. All this must be effected with great care. The best preservative fluid is simply distilled water, rendered antiseptic by a trace of the fixing solution (about eight drops to an ounce of water) giving the slightest tinge of yellow to the solution. This slight tinge of colour is imparted to the rotifers, otherwise they remain transparent and unchanged, while the nervous tissue throughout the body is brought out to perfection.

Some slight difference in treatment is required by certain species, as that of _Asplanchna priodonta_; after the application of the cocaine solution, which should be added slowly, that is, by letting a few drops trickle down the side of the live-trough; this, being heavier than water, sinks to the bottom, thus narcotising the rotifers, and assisting to kill them with the cilia fully expanded. They should be left quietly for fifteen minutes, then thoroughly washed with distilled water. On further experimenting, Mr. Rousselet found that a weaker solution of osmic acid alone, 1/4 per cent., answers quite as well as, if not better than, Flemming’s fluid; even this must be allowed to act for only a very short time--a minute at most; the rotifers then remain white and transparent, excepting the ova, in which a fat-like substance, _lecithene_, is secreted. If they become too much stained, they may be decolourised by passing them through peroxide of hydrogen. For narcotising the following solution has been found most useful:--Take a 2 per cent. solution of cocaine hydrochlorate, 3 parts; methylated spirit of wood naphtha, 1 part; and distilled water, 6 parts. This must be added as before directed, drop by drop, watching the effect upon the rotifers under the microscope.

All the rotatoria may be killed and preserved in the same way. For mounting, Mr. Rousselet prefers a slightly _hollowed-out_ glass cell, the advantage of which is that the rotifers are kept to the centre, and cannot move to the edge. A little difficulty at first presents itself to exclude air-bubbles, but this, with a little care, can be overcome by placing a drop of a two or three per cent. solution of formalin, just sufficient to fill the cell. Then transfer the rotifers with a dipping pipette to the cell, and lower the cover-glass down very gently, removing any excess of fluid by blotting-paper. The best cement for the cover-glass is gold-size.

_Method of Cementing._--After many years’ experience, I have arrived at the conclusion that for cementing down the cover-glass there is nothing better than either gold size or gum dammar varnish. The latter, for some preparations, will be improved by the addition of a small proportion of indiarubber dissolved in naphtha. (See Appendix.)

Should glycerine be preferred, carefully wash away any surplus quantity by gently syringing; then apply a ring of waterproof cement round the cover-glass. An inexpensive one can be made by dissolving ten grains of gum-ammoniac in an ounce of acetic acid, and adding to this solution two drachms of Cox’s gelatine. This liquid flows easily from the brush and is waterproof, rendered more so if subsequently brushed over with a solution of ten grains of bichromate of potash in an ounce of water. An especial recommendation to this cement is its adhesiveness to glass, even should there be a little glycerine left behind on the cover. After the gelatine ring is thoroughly dry any kind of cement may be employed.

A useful cement for fixing minute objects, diatoms, &c., temporarily to thin glass covers, before permanently mounting them in Canada balsam, is made as follows:--Dissolve, without heat, two or three grains of gum arabic in one ounce of distilled water, then add glacial acetic acid, three minims, and the least trace of sugar. Filter carefully through filter paper, and repeat this in the course of three or four weeks. This cement will be unaffected by the balsam.

_Mounting Chara._--It is often found difficult to preserve and mount the fruit of chara, but this can be successfully accomplished in glycerine jelly, by taking the following precautions. After cleaning the specimen place it in 92 per cent. of alcohol for several hours, then transfer it to a mixture of equal parts of spirit and glycerine for several hours longer, pour off nearly all the mixture, and add pure glycerine at intervals till the glycerine becomes concentrated. The specimen is then mounted in glycerine jelly in a cell just deep enough to take it without pressure.

There are some objects much more difficult to prepare than others, and which tax the patience of the beginner in a manner which can hardly be imagined by any one who has never made the attempt. The structure of many creatures is so delicate as to require the very greatest care to prevent mutilation, and consequent spoliation, of the specimen. The beginner, therefore, must not be discouraged by a few failures in commencing, but should persevere in his attempts, and constant practice will soon teach him the best way of managing intricate and difficult objects. The room in which he operates should be free from dust, smoke, and intrusion, and everything used should be kept scrupulously clean, since a very small speck of dirt, which may be almost invisible to the naked eye, will assume unpleasant proportions under the microscope, and not only mar, but possibly spoil a fine and delicate preparation.

Few students on commencing to work with the microscope will fully realise the fact that under medium or high powers the natural appearance of almost all objects is changed by the refractive nature of the fluid medium in which they are immersed and which enters more or less into their composition. The remarkable changes effected by the law of diffusion, when alkaloid substances enter into their composition, show the necessity of taking every precaution in the employment of preservative fluids. Glycerine affords an example of the chemical change induced, should the preparation have been passed through an alkaline solution.

_Air Bubbles_ are a constant source of annoyance both in preparing and mounting. These may be removed from the specimen by gently warming the under part of the slide over a spirit lamp, or placing the slide in the warm chamber, when the bubbles will move towards the edge of the cover-glass and ultimately disappear. The air-pump is preferred by many microscopists.

Collection of Objects.

_Infusorial Life_, with all its fascinations, was fully unveiled to naturalists by the celebrated Ehrenberg. It was he who termed it infusorial, because he first met with the more interesting forms of minute life in infusions of hay and other vegetable substances. Since his day it is a well-known experience of those who take up the microscope that the most interesting objects to commence with are infusorial living creatures of sufficient dimensions to be easily understood and seen with moderate magnifying powers. Moreover, infusoria are more readily found in almost any pool or running stream of water, either near the surface or clinging to the under surfaces of aquatic plants. At one time all the small shallow pools in the neighbourhood of London--Hampstead Heath, Clapham, Wandsworth, and other commons--abounded in the most interesting forms of life, were famous hunting grounds for the marvellous volvox, the charming dismid and diatom, the wonderful budding and self-dividing hydra. A few hours’ ramble furnished the microscopist with a bountiful supply of these and many other forms of life. Now all is changed; our commons have been devoted to other purposes, and with the general _levelling_ up all the little pools have disappeared, and the microscopist has been warned off and driven further afield, or seeks the good offices of a country friend for an occasional peep into pond life.[48]

A teaspoonful, however, judiciously taken from a well-chosen locality will often be found to contain a variety of living forms, every one of which will deserve a careful and patient study.

Of the microscopic organisms, the collection of which requires no other methods than those ordinarily pursued by the naturalist, most of them must be sought for in pools or running waters, basking in the sunshine, clinging to leaves and rootlets of all aquatic plants; some freely moving about, others clinging to stones or pieces of wood at the bottom. Dismids congregate in shallow waters or rise to the surface in a quiet nook, while the diatomaceæ are seen covering the bottom of clear water, to which they give a yellowish-brown tinge of colour.

Infusorial animal life, as vorticellæ, stentors, rotifers, and various polyzoa, cling, as also do hydra, in colonies to vallisneria, duck-weed, frogbit, or small branches dipping down under water; and if some of the water-weed is brought home the little creatures will live and thrive for several weeks. No waters, however, are so full of minute animal life as the sphagnum bog. A number of species of diatoms, as well as protozoids and the smaller molluscs, will be found in all peat bogs. It is remarkable, too, that the same species, everywhere, are associated with this kind of moss. Lord Sidney Godolphin Osborn supplied his friends with moss growing in a damp part of the garden walk of his rectory; this always furnished the same species of rotifers. These proved to be most interesting objects to my friends, and in an early communication I described them as _indestructible_, since they will bear any amount of desiccation; nevertheless, they were revived when a drop of water was introduced into the glass-cell.

The Thames mud always furnishes a number of beautiful forms of triceratum. Lower down the river, as brackish water is reached, greater varieties of diatoms appear. But to secure them the collector must be provided with a collecting stick. A convenient form is furnished by Messrs. Baker (Fig. 266). This consists of an ordinary walking-stick, together with a lengthening rod, a cutting hook to clear away weeds, ringed bottles with screw tops, and a net with a glass tube attached. Their uses are too obvious to need further description.

The siliceous skeletons of diatoms are met with in the fossil state. Among the first discovered of the infusorial strata were the polishing slates of Bilin and Tripoli, the berg-mehl or mountain meal, the entire mass of which is composed of the siliceous skeletons of different species of diatoms. Richmond, Virginia, is rich in the same organisms, while the great mass of our chalk cliffs are composed of foraminiferous shells, xanthidiæ, &c. One remarkable fact in connection with fossil infusoria is that most of the forms are still found in the recent state. The beautiful engine-turned discs, _Coscinodisci_, so abundant in the Richmond earth, may be met with in our own seas, and in great profusion in the deposits of guano on the African and American coasts, and in the stomachs of the oyster, scallop, and other salt-water molluscous animals common to our shores.

A great number of infusorial earths may be mounted as dry objects, while others require careful washing and digesting in appropriate media. The finer portions of the sediments will be found to contain the better and more perfect siliceous shells.

Preparing and Mounting Apparatus.