R. RATHBUN. NVERTEBRATE \ Z00L0GY Crustacea — THE PREPARATION AND MOUNTING OF MICROSCOPIC OBJECTS. as aan a 4 St - 25 » #: ake wert. Lo o\ ' PREPARATION & MOUNTING R. RATHEUN. THE OF MICROSCOPIC OBJECTS. _ { ‘uy 20 THOMAS DAVIBS. Beanies Hiksy Second Gdition.— Greatly Enlarged. EpiteD By JOUN MATTHEWS, M.D., F.R.M.S. . Vice-Pres, Quekett Microscopical Club. LOD DO: ROBERT HARDWICKE, 192, PICCADILLY. "AND ALL BOOKSELLERS. i ire, te. xs) eae ja Tb a) ae . at 1 Se : iy a L$ - Fo. oh pe , - ha 7) ry: F a, Mie pas, PREFACE TO THE SECOND EDITION. HE reception accorded to this work has been so favourable as to induce the Publisher to issue a second edition, in which such new matter should be embodied as the progress of Microscopic science — might require. He therefore applied to the Author, but he found to his regret that the state of Mr. Davies’s health was such as to forbid ‘his under- taking the labour. He had, however, collected many valuable notes and memoranda, which he was willing to place at the disposal of any gentle- man who might be selected to edit the work. The Publisher then consulted the present Editor, who, after some hesitation, consented not only to use his best efforts with the ample materials placed at < vi PREFACE TO THE SECOND EDITION. his disposal, but also to make such additions as his experience might suggest in extension of the use- fulness of the book to a new class of readers,—the Medical Student, and the Junior Medical Practi- tioner. To this end, besides other matter, a brief prefatory chapter has been added, embracing the elements of preliminary histological manipulation. While claiming the indulgence of the elders of his profession,—the Editor feels that the best and truest apology for this treatise, its raison d’étre—in fact, may be found in the words of its concluding para- graph, to which the reader is now courteously referred. 4, Mytne Srreet, Myppetton Square, E.C. October, 1873. PREFACE TO THE FIRST EDITION. N bringing this Handbook before the public, the Author believes that he is supplying a want which has been long felt. Much information concerning the “ Preparation and Mounting of Microscopic Objects ”’ has been already published; but mostly as supplementary chapters only, in books written professedly upon the Microscope. From this it is evident that it was necessary to consult a number of works in order to obtain anything like a com- plete knowledge of the subject. These pages, however, will be found to comprise most of the approved methods of mounting, together with the results of the Author’s experience, and that of many of his friends, in every department of vill PREFACE. microscopic manipulation; and as it is intended to assist the beginner as well as the advanced student, the very rudiments of the art have not been omitted. As there is a diversity of opinion as to the best mode of proceeding in certain cases, numerous quotations have been made. Wherever this has been done, the Author believes that he has acknow- ledged the source from which he has taken the information ; and he here tenders his sincere thanks to those friends who have so freely allowed him to make use of their works. Should, however, any one find his own process in these pages unacknowledged, the Author can only plead over- sight, and his regret that such should have been the case. W ARRINGTON. THE PREPARATION AND MOUNTING OF MICROSCOPIC OBJECTS. CHA P LER 1: INTRODUCTION. Tais work having been written chiefly to help students, the writer does not venture to affirm of it that it is by any means complete or exhaustive. The art of microscopic manipulation is progressive, and it is scarcely possible, therefore, to say of a work on the subject, that it holds all that is known at any given time. It is an art, too, which is so inextricably mixed up with the highest branches cf scientific inquiry, that new modes of investigation are daily devised by the acutest intellects, and with these it is very difficult for a Writer to keep pace. It is a well-nigh hopeless task to attempt to teach such modes of inquiry by precept, yet it is felt that some short account of them may reasonably be expected here. Refer- ence is now made more particularly to the practical part of human and comparative histology. As this is not a treatise on histology, but is devoted mainly to the methods of pre- serving the results of researches in that science, it is scarcely possible to indicate to the student how he shall proceed in any given case; yet there are certain tests, reagents, and staining matters employed, with the uses and effects of B 2° PREPARATION AND MOUNTING which he should be familiar, so as to be able to speak with some degree of certainty of the nature of the tissues demon- strated by them. It is now, therefore, intended to give the reader a list of these aids, arranging them according to the effects which it is desired to produce. Stricker observes, “that it is to be borne in mind that it is impossible to say of any fluid that it constitutes an indifferent, 7.e., neutral, medium for fresh tissues of all.kinds. In all instances we must be prepared for changes taking place.” He gives, however, a list of fluids to which structures are generally most indifferent, 7.e., in which least alteration may be detected under examina- tion while fresh, viz. :— 1st. Fluid of the aqueous humour. 2nd. The serum of the blood. 3rd. Amniotic fluid, very fresh, in which a little iodine has been dissolved, making it of a faint yellow tint. 4th. Very dilute solutions of neutral salts, such as: phos- phate and acetate of soda and potash, &c. It is scarcely within the power of any one observer to have largely used or tested the whole of the processes here- inafter to be mentioned. The writer therefore freely admits his obligations to the treatises of Drs. Beale and Carpenter, Mr. Quekett and Mr. Fownes, as well as to those of Stricker, Frey, Klein, Schultze, Kiihne, Deiters, Leber, and others, many of whose processes he has personally verified, and of whose manuals, especially those of Beale, Stricker, and Frey, the student is advised to possess himself. He believes also, from his own early experiences, that some short rationale of the intentions of the processes and means of investigation used by well-known workers may be acceptable to the stu- dent, in repeating their experiments before embarking in any of his own. These materials and methods may be divided, then, and described according to their effects somewhat as follows; and it ts in the judicious selection of each one or more of them that the tact and discretion of the student will best OF MICROSCOPIC OBJECTS. 3 be shown. He should bear in mind, too, that the same structure may well be submitted to various modes of in- quiry, and that possibly new modes may occur to him which, though they may not serve to prove anything directly, may yet become negative proofs. Ist. Such tests and agents as render transparent or translucent some tissues but not others adjacent, or make some more conspicuous than others without colouring them, or at least but faintly. 2nd. Staining materials or fluids, which colour either all the tissues to be displayed, or some particular part or parts of them, thus making such tissues or parts more conspicuous when subsequently examined or preserved in a colourless medium. srd. Hardening agents or solutions, by the effect of ‘which tissues naturally so soft as to break down or be other- wise unmanageable under manipulation, are made firm enough for section, or for such examination as may suffice to to discriminate (or “ differentiate”) their parts, without either disturbing or confusing their structural relations. Ath. Softening agents of animal and vegetable tissues. 5th. Solvents of the same. 6th. Solvents of calcareous matter. 7th. Solvents of siliceous matter. 8th. Solvents of oily and fatty matters. 9th. Polarized light, by the agency of which structures and organs may often be optically differentiated as a pre- liminary to other modes of investigation. 10th. Electricity and heat. 11th. The moist chamber. In dealing with structures by means of the agents com- prised under the first division of our list, a very frequent _ and necessary preliminary is the teasing out or separation of fibres by means of two sharp needles set in convenient handles. But it must be remembered that an appearance of structure, where it does not really exist, may easily be thus produced. It is often necessary, also, that the object BQ 4, PREPARATION AND MOUNTING shall have been macerated in water, or some other ageut, for so long a time as may be required to loosen or dissolve the connective tissue. It is of these agents that we shall presently have to speak in detail, greater or less according to their relative importance. Boiling or steaming may often be employed with advantage. It must not be too hastily concluded that, because there is nothing at first visible, there 7s therefore nothing to be seen. There are many important tissues which are apparently structureless, or homogeneous, which yet are possessed of such diverse elements as absolutely to require some process by which . they may be optically or visually differentiated, if one may use such a phrase, 7.e., discriminated from the neighbouring tissues or organs. It is thus that their proper uses and purposes in relation to the whole organism may be correctly indicated or inferred, their histological nature decided, and their physiological relations and connections established be- yond doubt. The student is also very emphatically oabbioned against the use of objectives of very wide angle, as well as of deep eye-pieces. In the former case, the relations of structures to each other can never be well made out, since it is impossible to get a focus of any depth (z.e., of all the structures involved), in one view, because the objects in one plane only can be clearly seen, the rest, either above or below, being more or less out of focus, and therefore hazy and indistinct. 'This objection applies far less to those of lesser angle, which are therefore the best for histological purposes. In the latter case, we have nearly the same defect to con- tend with, viz., that surface markings only, or mostly, can be seen clearly (not to speak of the loss of light). The use of the draw-tube is the true remedy for this. OF MICROSCOPIC OBJECTS. 5) Ist DtivIsIon. Under our first head may be ranged the following :— Acetic acid. Liquor potasse and sode. Concentrated sulphuric and hydrochloric acids (the latter saturated with chlorine). Tannin. Lime and baryta water. Oxalic acid. Nitric acid with chlorate of potash. Ammonia, Alcohol. Jodine. Glycerine (?) Phosphoric acid (tribasic). Acetic acid more or less dilute, e.g. one part to five of water, after a sufficiently prolonged immersion, renders transparent the following tissues, without in general des- troying their connective tissue:—some muscles (of the frog (Kolliker),) cell-walls generally (not the nucleus), epithelial structures, white fibrous tissue. Dr. Beale says that yellow fibrous tissue is unaltered by it. Many kinds of formed material, sections of preparations which have been hardened by alcohol. Dr. Beale also says that it dissolves granular matter composed of albuminous materia], and that: many tissues are quite insoluble in it, though they are not ren- dered opaque by it. Acetic acid renders some tissues trans- parent by dissolving out the phosphate or carbonate of lime, which they may contain, but it has no similar solvent power over oxalate of lime. Parts which are unaffected by this acid are then made more conspicuous. Liquor potassz and liquor sod act in ek the same way, according to the degree of their dilution, but on different structures. Albuminous tissues, epithelium, &c., are either dissolved by them or rendered so transparent as not to obstruct the view of the subjacent structures. 6 PREPARATION AND MOUNTING Concentrated sulphuric and hydrochloric acids, used cold, cause epidermic structures to swell up, so that their cells may be easily separated. Tannin, dissolved in water or rectified spirit of wine, hardens gelatinous and albuminoid tissues: it also makes them shrink. Its solution in water has been used, as men- tioned in another part of this treatise, as an injection pre- liminary to one of coloured gelatine, to prevent extravasation through the walls of the blood-vessels. It also colours the tissues a fawn-colour, or a very faint brown. | Lime water and baryta water, especially the latter, will, according to Rollet, dissolve the animal cement by which the fibres of connective tissue are held together. After a few days’ soaking such tissue, as well as tendon, may easily be teased out by needles. Oxalic acid, in a cold saturated solution (1 acid, 15 water), according to Schultze, “ causes connective tissues to swell up and become transparent, while those formed of al- buminous substances become hardened and isolated. Hx- tremely delicate elements of the body, such as the rods of the retina, &c., are thus well preserved.” Strong nitric acid mixed with chlorate of potash destroys connective tissue in a short time, and is therefore a good medium for isolating muscular fibres (Kihne). Sulphurie acid, highly diluted (1 to 1,000 parts of water), used warm, gelatinizes connective tissue, and is also useful for the isolation of muscular fibres. Strong hydrochloric acid dissolves the intercellular sub- stances of organs abounding in connective tissue. ; Ammonia acts on animal matters much in the same way as potash and soda. | Alcohol coagulates albuminous tissues, and makes them opaque. It corrugates most transparent sansa. and, thus renders them more visible. Finally, it may be affirmed that there often exists a an of making objects which are too dark more transparent by means of a fluid which permeates them wnequally, so that. OF MICROSCOPIC OBJECTS. 7 the tissues are thereby as it were “ differentiated,” yet not altered in any material degree. This may be effected by solutions of gum, sugar, glycerine, and creosote, if the tissues are moist. If dry, then turpentine, Canada balsam, benzine or benzole, and the essential oils of cloves, anise, and cassia, may be employed. Qnp DIvIsION. Under the second division of our subject come staining fluids. - Many of these will be found mentioned in the body of this work, They comprise carmine solutions, both acid and alkaline; aniline colours, indigo, carmine, hematoxyline, &c., formule for the use of which are given. ‘To these Frey adds, blue tingeing by molybdate of ammonia, and double staining by carmine and picric acid. A neutral solution of the molybdate of ammonia of the strenoth of 5 per cent. gives a blue tint to nerve-tissue, lymphatic glands, and ciliated epithelial cells, after macera- tion for 24 hours in the light. For double staining by carmine and picric acid he recom- mends a mixture containing— 1 part creosote, 10 parts acetic acid, 20 parts water. Soak the tissues in this solution while boiling for about a minute, then dry for two days. Make thin sections of them, immerse for an hour in water faintly acidulated with acetic acid, and then wash in distilled water. Next place them in a very dilute watery solution of ammoniacal car- mine, wash again in water, and place in a solution of picric acid in water, the strength of which will vary according to circumstances. The sections are then to be placed on a slide, superfluous acid allowed to drain off, and a mixture of 4 parts creosote to 1 of old resinous turpentine dropped on 8 PREPARATION AND MOUNTING them. In about half an hour they will become transparent, and may be mounted in Canada balsam. “A peculiar effect is thus obtained. Epithelial and glandular cells, muscles, and the walls of vessels show a yellowish colour, with reddened nuclei, while the connective tissue is not coloured by the picric acid, and only presents the carmine colour.” Another mode of effecting the above is by adding to a saturated and filtered solution of picric .acid in water, a strong ammoniacal solution of carmine, drop by drop, until neutralization takes place. Sections may be soaked in this - solution, more or less dilute, for a sufficient time, and treated as in the previous method. The other staining agents are:—nitrate of silver, osmic acid, chloride of gold, chloride of gold and potassium, proto- chloride of palladium. These are to be made into weak solutions in distilled water, in which the tissues, in section or otherwise, are to be placed, and then exposed to light for a sufficient time. Leber recommends a mode of staining by Prussian blue, as follows:—Immerse the specimen in a weak solution of a protosalt of iron for five minutes, more or less, according to size or the thinness of the section. Then wash and move it to and fro for a few minutes in a 1 per cent. solution of ferro-cyanide of potassium until it assumes an intense and uniform blue colour. Then wash in water, soak in alcohol, and mount as usual. The effect is that of partial tingeing; the colour penetrates very deeply, and the tissue may be subsequently stained with iodine, carmine, or fuchsine. This method has been used for the cornea of the frog. Iodine 1 part, with 3 of iodide of potassium, dissolved in 500 of water, may be used for tingeing of a brown colour animal cells, as well as all amyloid substances, animal or vegetable, sulphuric acid being added. OF MICROSCOPIC OBJECTS. 9 3RD DIVvISION. Under the third division of our list may be ranged the following agents :—alcohol, solutions of chromic acid, bi- chromate of potash, hyperosmic acid, chloride of palladium, bichloride of mercury (in Goadby’s solution), and tannin, or the substance may be dried in thin layers or small pieces, either spontaneously or in vacuo, or by carefully regulated heat; in some cases it may be boiled, or it may be frozen. Alcohol is, on the whole, the best and most convenient of the hardening agents. It acts by abstracting water and coagulating albumen, and its uses as a preservative fluid per se are well known. It enters also into many of the preservative fluids, and is especially convenient and useful when it is desired to mount specimens quickly out of watery fluids in Canada balsam, without drying them previously. After a longer or shorter soaking in it, according to their size or thinness, preparations may be at once placed in tur- pentine, and then easily and speedily put up for examina- tion in balsam. Dr. Beale recommends a mixture of alcohol and a solu- tion of caustic soda for the preservation of delicate tissues. He observes, “‘ that alcohol alone tends to coagulate albu- minous textures and render them opaque, at the same time that it hardens them. The alkali, on the other hand, will render them soft and transparent, and would dissolve them if time were allowed. These two fluids, in conjunction, harden the texture, and at the same time make it clear and transparent.” Chromic acid in solution, 0'°25—0'5 to 1 and 2 per cent. of distilled water is much used. On account of its deli- quescence, it is most conveniently kept in a saturated solu- tion, which may be diluted as desired; and very often the weaker this solution the better. When it has had the de- sired effect on the tissue, the preparation should be removed into diluted alcohol, on account of the readiness with which fungi and confervoid growths are formed in chromic acid 10 : PREPARATION AND MOUNTING solutions. There are some precautions needed for perfect success with this agent, for which the reader is referred to Frey’s “ Microscopic Technology.” Bichromate of potash, in solutions of similar strength to those of chromic acid, may be used in the same way, but is far slower in producing its effect, and therefore inferior in the opinion of many. Stricker, however, says, “that it has the great advantage that tissues saturated with it do not become friable, and that the time occupied by this agent, as well as by the preceding, may be much shortened by re- moving the preparation into alcohol for twenty-four hours.” It is always advisable to divide the substances to be har- dened into portions as small as convenient, since the larger often putrefy in the centre, though they harden at the sur- face. It is quite certain that many of the more delicate structures, such as the rods of the cochlea of the ear (Pritchard), those of the eyes of insects, &., are better prepared with this than by the preceding agent. One great element of success in these two processes is, that the volume of the solution should be very large in proportion to the size of the object; another, that the action should be commenced with a weak solution, and continued with a stronger. It sometimes happens that objects may be hardened too much by these solutions, though there is less risk by the bichromate of potash. In such cases Frey recommends that they be soaked in glycerine for a few days, and even that it be added to the solutions at first. He, with Deiters, Arnold, Schultze, and Kihne, claims for these solutions an effect of the most important kind, viz., that of “ preserving the finest textural relations, while exerting a somewhat macerating action on them, so that very delicate organizations, especially in nerve tissues, may be made visible which were previously hidden, or not visible in examination of the fresh tissue.” Hyperosmic acid and chloride of palladium are sometimes used for this purpose also. Their solutions may contain from one-fifth to one-tenth per cent. of distilled water. OF MICROSCOPIC OBJECTS. 1t Bichloride of mercury acts, in hardening tissues (like most of the preceding, probably), by combining and forming an insoluble compound with their albuminoid elements. It is not much employed for this purpose, but is principally of use in certain preservative solutions mentioned elsewhere in these pages. Tannic acid forms insoluble compounds with a great variety of organic and especially animal substances, as solu- tions of starch and gelatine, solid muscular fibre and skin, &c., which then acquire the power of resisting putrefac- tion. It scarcely colours animal membrane. Dr. Beale says that its action upon red blood corpuscles is “ very peculiar.” The solution used is three grains to an ounce of water. Other uses of tannin (tannic acid) will be found elsewhere in this work, and the intelligent student will easily thence infer its action and properties. Drying may be effected either in a current of warm dry air, or under a bell-glass over sulphuric acid, or over a layer of parched oatmeal; or a cheap form of water bath may be employed, such as will be found described in this work. Another very speedy method is to soak the specimen in strong alcohol for a sufficient time, remove it, and expose to a current of warm dry air. Boiling.—Tissues may be hardened by boiling in a fluid consisting of 8 parts water, 1 part creosote, and 1 part vinegar, for two or three minutes. They may then be laid out to dry. After two or three days they acquire a firm- ness admirably adapted for section; but if they remain too long uncut they become of a consistence unfit for that pur- pose. On the whole, boiling is not to be recommended, though Stricker says that it has its occasional uses. Freezing may be employed for otherwise unmanageable structures, such as brain, spinal cord, &c. (though there seems to be an objection of a theoretical kind to this use of it, viz., that it may injure or alter the cells), or other tissues which will not admit the use of chromic acid, or which it may be.desired to view under other aspects. 12 PREPARATION AND MOUNTING The writer has little or no experience of this plan, he therefore quotes from Frey as follows :— “The preparation is allowed to freeze (by contact, it is presumed, with a freezing mixture or solution) until it as- sumes a consistency which will permit fine sections to be made with a cooled razor. The object is more convenient to handle if it is allowed to freeze on a piece of cork. Nerves and muscles have been treated in this manner with good results. Glands (salivary), livers, spleens, the lungs, skin, and the bodies of embryos (see Beale’s process for the same. in this work), also ganglia, afford excellent appearances. Indifferent (or neutral) media, such as iodine serum, are to be used in examining such sections. Or the preparation may be held in paraffine wax (diluted or not with oil), or tallow, which have been melted, and the object suspended or plunged in them until they cool, and the cooling may be carried further, if needed, by freezing.” In reference to this subject, Mr. Kesteven informs the author that he has found the parafiine composition more useful for brain than spinal cord. The former can be cut into any angular shape, and be so held steady for slicing; but the cord, being round, becomes loosened in its setting of wax (or paraffine), and revolves with the pressure of the knife. ¥For either brain or cord he prefers hand-cutting with a very sharp razor, after the manner of Lockhart Clarke (see Mr. Kesteven’s paper in St. Bartholomew’s Hospital Reports). If many sections are to be made from a brain, machine- cutting saves much time. The razor should have some spirit of wine dropped on it, so as to prevent the sections adhering. The cutting machines are generally graduated (by a screw and index) on the upward movement, so as to enable one to judge of the thickness of the section; but as the brain substance and paraffine are both yielding to a cer- tain extent, the reading must be taken with allowance. OF MICROSCOPIC OBJECTS. 13 47H DIvIsIon. This includes glycerine, liquor potassee and sods, heat (as regards some substances), maceration (carried to incipient putrescence), nitric and chlor-hydric acids, either pure or dilute (in the case of bones, nails, &c.). The writer is in doubt whether glycerine ought or not to be included under this section or the first, its uses and effects being so various and interesting. Indeed, there is scarcely any agent to which histology is more indebted for its present status and progress, since there is now no doubt that elementary tissue can be more readily discriminated in this medium—perhaps, too, by it—than any other. It has also the valuable property of preserving the tissues, if it be ' not too much diluted, and even then it is generally effectual if camphor water be employed as the diluent. The strongest and best glycerine should always be employed. ‘The first effect on tissues immersed in it is that they shrink, owing to the abstraction of their water; but Dr. Beale speaks in the highest terms of its uses and advantages, and declares that the tissues gradually regain their original volume if left in it for a sufficient time. They then soften, and even swellup. His practice is first to immerse the specimen in weak glycerine solution, and then gradually to increase the density of the fluid. He recommends, also, “in order that tissues may be uniformly permeated with a fluid within a very short time after the death of an animal, that the fluid should come quickly in contact with every part of the texture.” This, he says, may be effected in two ways, by A. Soaking very thin pieces iu the fluid; B. By injecting the fluid into the vessels of the animal. He thinks that these properties more particularly apper- tain to glycerine than to any other medium, and affirms that “ cerebral tissues, delicate nervous tissues like the retina or the nerve-textures of the internal ear, may be saturated with it, and dissection then carried to a degree of minuteness 14 PREPARATION AND MOUNTING impossible in any other mediwm. All that is required is, that the strength of the fluid should be increased very: gradually until the whole tissue is thoroughly penetrated by the strongest that can be obtained;” and “ that thus very hard textwres may be softened, so that by gradually increas- ing pressure and careful manipulation exceedingly thin layers can be obtained, without the relation of the anatomi- cal elements to each other being much altered, or any of the tissues destroyed.” He also takes occasion to observe, “ that tissues immersed in water are destroyed by even moderate pressure; but that in a viscid medium (such as glycerine or syrup) the requisite pressure can be borne not only without injury or impairment of the discrimination of their parts, but with advantage to their detail.” One very great advan- tage which results from the use of glycerine for the prepara- tion of textures is, that however they may swell in it after prolonged immersion, a sufficient soaking in water will always restore them to their normal condition. Another is, that on account of its very high refractive power, it is peculiarly fitted for the preparation of structures to be in- vestigated by polarized light, with the same advantage as in the preceding case, that they are still amenable to all other modes of inquiry. The caustic alkalies—potash, soda, and ammonia, are solvents of all animal textures except chitine, and perhaps bone. Asin nearly all cases a softening action, with little or no alteration of tissue, precedes the solvent action, these agents, and especially the first two, have their uses. Under their influence “a condition is induced very favourable to the imbibition of water, which afterwards penetrates very rapidly, so that cells swell up and burst.” They may be used either with or without heat, and more or less dilute. There is one disadvantage attending their use, that objects can with difficulty be preserved after soaking in them. Heat, applied either by the aid of hot water or steam, or the sand-bath, or a bath of fusible metals, or of melted lead, is a very efficient means of softening horny substances, whale- OF MICROSCOPIC OBJECTS. 15 bone, &c., and rendering them plastic. Very thin laminz of these substances may also be procured by the employment of a well-sharpened scraper, such as that used by cabinet- makers. This plan applies more to longitudinal than to transverse sections; yet even the latter may be obtained by fixing the object while soft in a piece of hard wood, and scraping both together. Long continued slow boiling softens and eventually disintegrates nearly all animal and vegetable tissues. Muscular fibre and many other textures may thus be isolated, such as spiral vessels, &c., in vegetables. Prolonged maceration in water, for the preparation of anatomical structures, generally bony, is a process too well known to need description here. The addition of very dilute nitric, hydrochloric, and acetic acids is much em- ployed for the separation of muscular fibres, both striated and smooth. ‘Two or three days are required, or even more. Nails may be softened very quickly by hot concentrated sulphuric acid—or, still better, by liquor potassz, strength about 25 to 27 per cent.—so as to show isolated and dis- tended cells by solution of the intercellular substance. Bones are softened, %.e. decalcified, by boiling or, still better, by slow maceration in weak solutions of nitric and hydrochloric acids, by the action of which the phosphate and carbonate of lime may be entirely removed. This pro- cess isolates the animal matter, 7.e. the osseine—sometimes miscalled gelatine—with all its peculiar fibres and processes. But bones may be treated in another way, so as to show or isolate the bone corpuscles with their processes, by removal or destruction of the intercellular substance. Though this can scarcely be called softening them, yet it may be most fitly mentioned here. For this purpose, a Papin’s digester is necessary. When the boiling of bones has been for a long time carried on by means of one of these machines, they seem to be dissolved; but on examination a coarse powder, consisting of the isolated corpuscles and their processes, is found at the bottom of the vessel, which will amply repay the trouble of examination. 16 PREPARATION AND MOUNTING Teeth may be treated in the same manner as bones, except for the examination of the enamel, which is best effected by sections and grinding. For that purpose developing teeth should be chosen, as in them the enamel prisms are most easily isolated. 5rH DIvIsION. As the solution of animal and vegetable tissues generally means the confusion or destruction of their histological elements, not much can or need be said of it here, except that it may be as well to indicate the special solvents and tests of the special components of all tissues, since it is upon a correct knowledge and appreciation of the degrees and differences of the action of these, that effective histological research must chiefly depend. Albumen, when pure, is nearly insoluble in water, wholly so when coagulated by heat. In dilute caustic alkali it dissolves with facility. Solution of nitrate of potash, acetic aud tri-basic phosphoric acids, and pepsine, dissolve the pur- est form of albumen procured from white of egg. Fownes observes, “that it must be remembered that a considerable quantity of alkali and very minute quantities of the mineral acids, prevent coagulation by heat, and that the addition of acetic acid, indispensable to the test by mer- cury, produces the same effect.” Fibrine of blood is insoluble in both hot and cold water, but is partly dissolved by long-continued boiling. ‘Fresh fibrine, wetted with concentrated acetic acid, forms after some hours a transparent jelly, which slowly dissolves in water. Very dilute caustic alkali dissolves fibrine completely. Phosphoric acid produces a similar effect. Fibrine of flesh, which is not identical with that of blood (Liebig), is soluble in cold water containing one-tenth of hydrochloric acid. Casein is only soluble in water in the presence of free alkali in very small quantities. It is partly soluble also in very dilute acids. OF MICROSCOPIC OBJECTS. 17 Gelatin, chondrin, and osseine are the result of the boiling of animal membranes, skin, tendons, and bones, respectively at a high temperature for a sufficient time. They are insoluble in cold water, but easily dissolved by the use of heat. Alcohol, corrosive sublimate in excess, nitrate of mercury, and, most characteristically, tannin, precipitate gelatine—the latter when it is very largely diluted. * Skin and tendons contain a substance which resists the action of boiling water for many hours. It is insoluble in cold concentrated acetic acid, but by long-continued boiling in it, is gradually dissolved, and more easily in hydrochloric acid.” (Fownes.) Horny substance—keratin, found in hair, nails, feathers, and epithelium, is obtained by finely dividing them, treat- ing them with hot water, and afterwards by boiling alcohol and ether. The horny substance is then very soluble in caustic potash. Of bones we have already spoken. It has been mentioned elsewhere in this work, that all the internal organs of insects may easily be dissolved out by boiling in liquor potassz, leaving their external chitinous structures, limbs, &c., unaffected. But this is a proceeding much to be deprecated, for various reasons which it is scarcely necessary to give here... It is far better to treat them in another way, by which these organs may be ex- amined im situ, at least to a very great extent, as will presently be shown. The parenchyma of leaves and many other vegetable structures may be decomposed by prolonged maceration in water, and then easily be washed away. Nitric acid, vary- ingly diluted, will produce the same effect more speedily, the objects not requiring the same amount of bleaching subse- quently. But by far the best and most speedy method is, to place them in the liquid manure tank of the gardener for a sufficiently long maceration. The results of this plan are exquisitely beautiful. 18 PREPARATION AND MOUNTING 6rH Drvision. \ The proper solvents of calcareous animal matters are nitric, hydrochloric, and sulphuric acids. The earth of bones consists of a combination of two tribasic phosphates of lime, both of which are entirely soluble in nitric and hydrochloric acids. Sulphuric acid abstracts a part of the lime of bones, leaving a superphosphate—a substance much used in agriculture as a manure. JF lnoride of calcium, existing in small quantity in bones, but in larger in the enamel of teeth (and of the ganoid scales of fish ?), is de- composed by sulphuric acid, which combines with the cal- cium, allowing the hydrofluoric acid to fly off in a gaseous state. Carbonate of lime dissolves in nitric and hydro- chloric acids. The shells of mollusca, and testz of echino- dermata, consisting principally of carbonate of lime, are also soluble in the same acids, as well as those of nummulites foraminifera, &c., which have been infiltrated with siliceous matter. These present the most beautiful “ casts,” which are exactly of the shape of the Sarcode body and canal sys- tem, thus enabling their internal organs to be studied with much accuracy. Dr. Carpenter says that they are of “ won- derful completeness.” - 7TH DIvIsION. Silica is nearly altogether insoluble in water, but dissolves freely in strong alkaline solutions. Its only acid solvent is hydrofluoric acid, Its combinations with a larger proportion of alkali are soluble in water, and from such solutions silica may be precipitated in a gelatinous or colloid form by acids, or separated by dialysis, in the form of colloid silica. This substance may be used for procuring certain modifica- tions of crystals of salts for the polariscope, such as.sulphate of magnesia, sulphate of copper, boracic acid, sulphate of zinc, &c. In its combination with a smaller proportion of alkali, forming glass, it is attacked by hydrofluoric acid and its vapour, and advantage may be taken of this property te OF MICROSCOPIC OBJECTS. 19 engrave names, numbers, &c., neatly upon slides, for classifi- cation in the cabinet. The glass to be engraved must be coated with an etching ground of oily varnish or wax, and the necessary writing effected upon it by a point, which must pierce through the protective material. A shallow basin, made by bending up the edges of a piece of sheet-lead, is then prepared, a little powdered fluor spar placed in it, and enough sulphuric acid added to form a thin paste. The glass is then placed in any convenient way over the basin, the waxed side downwards. A gentle heat is next applied, whereby the vapour of hydrofluoric acid is disengaged. This acts upon the glass exposed by the point in a very few minutes, removing a portion of its surface. The wax must then be removed by turpentine. If the lines which result are then rubbed over with any coloured varnish, and the varnish gently wiped off by a soft piece of rag, a sufficient portion will most probably remain in the etched marks to render them easily visible and legible. Of course it will be as well to prepare many slides in this way atonce. It is not necessary to coat the whole surface of the slides with the protective varnish, if the leaden basin be covered with a thin piece of wood or sheet-lead perforated with holes slightly larger than the surface to be etched, over which holes the slides must be inverted for a sufficient time. This latter hint applies more particularly to finished slides requir- ing to be labelled. 8tH DIVISION. The proper solvents of the fixed oily and fatty matters are ether, benzole (or benzine), turpentine, and the essen- tial oils generally. Castor oil is nearly the only one which is soluble in alcohol, the rest being only slightly so. They are all capable of saponification with caustic alkalis, and so become indirectly soluble in soft or distilled water, other- ' wise they are wholly insoluble in it. The volatile or essential oils mix in all proportions with fatty oils, and are wholly soluble in ether and alcohol. c2 20 PREPARATION AND MOUNTING Camphor dissolves in a only very small proportion in water, but freely in alcohol, ether, and strong acetic acid. 9TH DIVISION. It is by no means intended to speak here of the general properties and uses of polarized light. But in relation to its special powers in the “ differentiation ” of tissues, there is very much to be learned. To be fitted for examination by this method, objects must be made more or less trans- parent or translucent; and in effecting this it is advisable, | perhaps necessary, to employ media of high refractive power. Even when so prepared, it may be further necessary in some cases to employ selenite or mica films, still more to enhance their colour. Not the least indication can be afforded to the observer as to what colours he should employ generally, yet it is a matter of frequent observation that what are called “neutral tints” are to be preferred, such as result from the judicious use of compound selenite stages adjusted properly for that special effect. The media most suitable for the preparation of objects to be examined in this way are glycerine, syrup, turpentine, dammar and benzole—or the latter alone, Canada balsam, and the essential oils. Of course sections must be made of tissues otherwise too thick. Of the advantages of employing the first of these we have already spoken; but to these must be added this important one, that it does not spoil the object for examination by other methods, if the glycerine be soaked out by maceration in water; and this is true also of syrup, though it is far less useful. For preparation by the other methods, tissues must have been soaked in alcohol, and then removed into the turpentine, &c. For the examination of insects by polarized light, two preliminaries are necessary. Firstly, that they be made transparent or translucent by prolonged soaking in one of the above-named media, preferably in turpentine or the essential oils, or benzole. Secondly, that as in (most?) many of them their chitinous case is too deeply coloured for OF MICROSCOPIC OBJECTS, 21 any amount of soaking to render them sufficiently trans- parent, some bleaching process should be premised. A for- mula for such a process may be found in another part of this work, where the preparation of the antennz of insects is described. If that should not prove successful, some modification will easily occur to the student. Of course it is not all insects that can be treated in this way, the size and deep colour of very many quite preventing a good result; but when they have been successfully prepared by any of the methods of which we have spoken, it is then possible to discriminate their internal organs by the differ- ences of colour which they present. The use of the binocular microscope, and of objectives of low angular aperture, will also much facilitate this mode of examination, by increasing the depth of focus, and enabling the organs to be seen more or less in connection with each other, even if they be super- posed. It is also possible to examine the muscles of the limbs and bodies of insects, so as to decide upon their forma- tion, origin, and insertion, and probable mode of action; and this is only one of many such uses. What a mistake must it be, then, to prepare insects for mounting’ by boiling in liquor potasse, and so dissolving out their viscera, and squeezing them flat! In the case of living insects, especially those of the more transparent salt and fresh water species, the results of their - examination by polarized light are exquisitely beautiful and interesting, because their organs and circulation may be more clearly discriminated while in motion. 10TH DIvisIon. Electricity has been employed in histology partly for its electrolytic effects, but chiefly as a means of producing certain variations of temperature in objects under examina- tion. Stricker says “that the tissues become altered by it as they would be were they subjected to the action of weak acids or alkalias,” and he describes a rather complicated apparatus for this purpose, of which it is impossible to give - ¢ 22 PREPARATION AND MOUNTING an account here; but the author believes that most, if not all, of the same effects may be produced by the employment either of a thick plate of metal placed upon the stage, or of a thin water-bath, which may be heated by a spirit or gas flame, after the glass slide shall have been placed on it. They should both be properly fitted with thermometers. Of the decomposition of salts by electricity, and their re- duction to the metallic state, it is not necessary to speak here, but such effects are very beautiful, and the resulting crystalization may easily be watched ; Dr. Beale speaks very favourably of the inverted micro- scope devised by Dr. Lawrence Smith, U.S.A., by which © objects may be viewed from their under instead of from their upper surface, and at the same time heated (or re-agents applied to them) without any risk of dimming or injuring the object-glass by vapours thus raised. The optical part is so fitted to the base that it may be drawn away from be- neath the stage (to make room for the application of the lamp, or) for the sake of changing the powers. 11TH Dryiston. It is evident that in all these plans an amount of evapo- ration is constantly going on, which will eventually dry and so spoil the object, unless obviated. Frey, therefore, describes a “moist chamber” invented by Recklinghausen for this purpose. It consists of a glass ring, more or less high, which has been cemented by its edge to a broad glass slide. A tube of thin rubber is then firmly fastened about the ring. The upper end of this tube is also fastened around the tube of the microscope. In order to keep the place thus enclosed saturated with moisture, some small pads of wetted bibulous paper, or pieces of elder pith also saturated with fluid, are to be enclosed with the object, which in this case need not be covered with thin glass in the usual manner. It is con- ceivable also that this apparatus may easily be converted into a gas chamber, by fixing two small, light vulcanized OF MICROSCOPIC OBJECTS, ’ 29 tubes into that which embraces. the glass ring and the end of the microscope tube—one for the entrance, the other for the exit of the gas. This is a simpler and less costly plan than that devised by Stricker. Frey observes that it is most advantageous to use wnmersion lenses and the moist chamber with the hot stage. 24, PREPARATION AND MOUNTING CHAPTER wt. APPARATUS. BEFORE entering into the subject of the setting of Objects for the Microscope, the student must be convinced of the necessity of cleanliness in everything relating to the use of that instrument. In no branch is this more apparent than in the preparation of objects; because a slide which would be considered perfectly clean when viewed in the ordinary way is seen to be far otherwise when magnified some hun- dreds of diameters; those constant enemies, the floating particles of dust, are everywhere present, and it is only by unpleasant experience that we fully learn what cleanli Ness 78. An object which is to be viewed under the microscope must, of course, be supported in some way—this is now usually done by placing it upon a glass slide, which on account of its transparency has a great advantage over other substances. These “slides” are almost always made of one size, viz., three inches long by one broad, generally having the edges ground so as to remove all danger of scratching or cutting any object with which they may come in contact. The glass must be very good, else the surface will always present the appearance of uncleanliness and dust. This dusty look is very common amongst the cheaper kinds of slides, because they are usually made of “sheet” glass ; but is seldom found in those of the quality known amongst dealers by the name of “ patent plate.” This latter is.more expensive at first, but in the end there is little difference in the cost, as so many of the cheaper slides cannot be used for delicate work if the mounted object is to be seen in per- fection. These slides vary considerably in thickness ; care OF MICROSCOPIC OBJECTS. 25 _ should, therefore, be taken to sort them, so that the more delicate objects with which the higher powers are to be used may be mounted upon the thinnest, as the light em- ployed in the illumination is then less interfered with. To aid the microscopist in this work, a metal circle may be pro- cured, having a number of different sized openings on the outer edge, by which glass slides can be measured. ‘These openings are numbered, and the slides may be separated according to these numbers; so that when mounting any object there will be no need of a long search for that glass which is best suited to it. When fresh from the dealer’s hands, these slides are generally covered with dust, &c., which may be removed by well washing in clean rain-water; but if the impurity is obstinate, a little washing soda may be added, care being taken, however, that every trace of this is removed by sub- sequent waters, otherwise, crystals will afterwards form upon the surface. Sometimes, however, a certain greasiness is very obstinate upon the glass. It is then necessary to use a little liquor potassze with a small piece of linen, rubbing the slide with some pressure, and then washing as before to remove all remains. A clean linen cloth should be used to dry the slides, after which they may be laid by for use. Immediately, however, before being used for the reception of objects by any of the following processes, all dust must be removed by rubbing the surface with clean wash-leather or a piece of cambric, and, if needful, breathing upon it, and then using the leather or cambric until perfectly dry. Any small particles left upon the surface may generally be removed by blowing gently upon it, taking care to allow no damp toremain. A very efficient remedy, also, is a mixture of equal parts of sulphuric ether and alcohol, with which the glass must be rubbed by the aid of a tuft of clean cotton-wool until no stain appears after breathing upon it. A strong infusion of nutgalls may be used in the same way, and is preferred by many to all other applications; or, a mixture of equal parts of alcohol, benzole, and liquor sodz- 26 PREPARATION AND MOUNTING may be employed, which thoroughly and speedily cleanses glass from all traces of grease or balsam. We have before said that any object to be viewed in the microscope must have its support; but if this object is to be preserved, care must be taken that it is defended from dust and other impurities. For this purpose it is necessary to use some transparent cover, the most usual at one time being a plate of mica, on account of its thinness; this sub- stance is now, however, never used, thin glass being substi- tuted, which answers admirably. Sometimes it is required to “ take wp” as little space as possible, owing to the short- ness of focus of the object-glasses. It can be procured of any thickness, from one-fiftieth to one-two-hundred-and- fiftieth of an inch. On account of its want of strength, and probable defect of due annealing, it is difficult to cut, as it is very liable to “fly” from the point of the diamond. To overcome this tendency as much as possible, it must be laid upon a thicker piece, previously made wet with water, which causes the thin glass to adhere more firmly, and conse- quently to bear the pressure required in cutting the covers. The process of cutting being so difficult, especially with the thinner kinds, little or nothing is gained by cutting those which can be got from the dealers, as the loss and breakage is necessarily greater in the hands of an amateur. It is convenient, however, to have on hand a few larger pieces, from which unusual sizes may be cut when required. If the pieces required are rectangular, no other apparatus will be required save a diamond and a flat rule; but if circles are wanted, a machine for that purpose should be used (of which no description is necessary here). There are, however, other contrivances which answer tolerably well. One method is, to cut out froma thick piece of cardboard a circle rather larger than the size wanted. Dr. Carpenter recommends metal rings with a piece of wire soldered on either side; and this, perhaps, is the best, as cardboard is apt to become rough at the edge when much used. A friend of mine uses thin brass plates with circles of various sizes OF MICROSCOPIC OBJECTS. 27 “turned” through them, and a small raised handle placed at one end. The diamond must be passed round the inner edge, and so managed as to meet again in the same line, in order that the circle may be true, after which it may be readily disengaged. The sizes usually kept in stock by the dealers are one-half, five-eighths, and three-quarters inch diameter; but other sizes may be had to order. For the information of the beginner it may be mentioned here that the price of the circles is a little more than that of the squares; but this is modified in some degree by the circles being rather lighter. If appearance, however, is cared for at all, the circles look much neater upon the slides when not covered with the ornamental papers; but if these last are used (as will shortly be described) the squares are equally serviceable. As before mentioned, the thin glass is made of various thicknesses, and the beginner will wish to know which to use. For objects requiring no higher power than the one- inch object-glass, the thicker kinds serve well enough; for the half-inch the medium thickness will be required ; while, for higher powers, the thinnest covers must be used. The * test-objects ” for the highest powers require to be brought so near to the object-glass that they admit of the very thinnest covering only, and are usually mounted betwixt glasses which a beginner would not be able to use without frequent breakage; but if these objects were mounted with the common covers, they would be really worthless with the powers which they require to show them satisfactorily. It may be desirable to know how such small differences as those betwixt the various thin glass covers can be measured. Jor this purpose there are two or three sorts of apparatus, all, however, depending upon the same principle. The description of one, therefore, will be sufficient. Upon a small stand is a short metal lever (as it may be termed) which returns by a spring to one certain position, where it is in contact with a fixed piece of metal. At the other end this lever is connected with a “ finger,” which moves round 28 PREPARATION AND MOUNTING a dial like that of a watch, whereupon are figures at fixed distances. When the lever is separated from the metal which is stationary, the other end being connected with the “finger,” of the dial, that “ finger” is moved in proportion to the distance of the separation. The thin glass is, there- fore, thrust betwixt the end of the lever and fixed metal, and each piece is measured by the figures on the dial in stated and accurate degrees. This kind of apparatus, however, is expensive, and when not at our command, thin glass may be placed edgewise in the stage forceps, and measured very accurately with the micrometer, or by the calliper eye- piece described by Dr. Matthews in No. 8, for October, 1869, of the Journal of the Quekett Microscopical Club. Cleanliness with thin glass is, perhaps, more necessary than with the sides, especially when covering objects which are to be used with a high power; but it is far more difficult to attain, on account of the liability to breakage. The usual method of cleaning these covers is as follows :—T'wo discs of wood, about two inches in diameter, are procured, one side of each being perfectly flat and covered with clean wash-leather. ‘To the other side of these a small knob is firmly fixed as a handle, or where practicable, the whole may be made out of a solid piece. In cleaning thin glass, it should be placed betwixt the covered sides of the discs, and may then be safely rubbed with a sufficient pressure, and so cleaned on both sides by the leather. If, however, the glass be greasy, as is sometimes the case, it must be first washed with a strong solution of potash, infusion of nutgalls, or any of the commonly used grease-removing liquids; and with some impurities water, with the addition of a few drops of strong acid, will be found very useful, but this last is not often required. This method of cleaning thin glass should always be used by beginners; but after some experience the hand becomes so sensitive that the above apparatus is often dispensed with, and the glasses, however thin, may be safely cleaned betwixt the fingers and thumb with a cambric handkerchief, OF MICROSCOPIC OBJECTS. 29 having first slightly damped the ends of the fingers employed to obtain firm hold. When the dirt is very obstinate, breathing upon the glass greatly facilitates its removal, and the sense of touch becomes so delicate that the breakage is inconsiderable; but this method cannot be recommended to novices, as nothing but time spent in delicate manipulation can give the sensitiveness required. It has been before mentioned that ordinary glass sides are sometimes worthless, especially for fine objects, from having a rough surface, which presents a dusty appearance under the microscope. This imperfection exists in some thin glass also, and is irremediable; so that it is useless to attempt to cleanse it; nevertheless, care should be taken not to mistake dirt for this roughness, lest good glass be laid aside for a fault which does not really belong to it. When any object which it is desired to mount is of con- siderable thickness, or will not bear pressure, it is evident tkat a wall must be raised around it to support the thin glass—this is usually termed a “cell.” There are various descriptions of these, according to the class of objects they are required to protect; and here may be given a description of those which are most generally used in mounting “ dry” objects, leaving those required for the preservation of liquids until we come to the consideration of that mode of mounting. Many have used the following slides. Two pieces of hard wood of the usual size (3 in. by 1 in.), not exceeding one- sixteenth of an inch in thickness, are taken, and a hole is then drilled in the middle of one of these of the size required. The two pieces are then united by glue or other cement, and left under pressure until thoroughly dry, when the cell is fit for use. Others substitute cardboard for the lower piece of wood, which is less tedious, and is strong enough for every purpose. This class of “cell” is, of course, fitted for opaque objects only where no light is required from below ; and as almost all such are better seen when on a dark background, it is usual to fix a small piece of black paper at the bottom of the cell upon which to place them. For 30 PREPARATION AND MOUNTING very small objects the grain which all such paper has when magnified detracts a little from the merit of this background; and lately I have used a small piece of thin glass covered on the back with black varnish, and placed the object upon the smooth untouched side; but a solution of the best Egyptian asphalt in benzole of moderate thick- ness may be painted on with this further advantage, that im mounting such opaque objects as foraminifera, &c., it will be sufficient to arrange them in the positions they are to occupy, when by slightly warming the slide they will adhere to the asphalt. Another method of making these cells is as follows :— Two punches, similar to those used for cutting gun-wads, are procured, of such sizes that with the smaller may be cut out the centre of the larger, leaving a ring whose side is not less than an one-eighth of an inch wide. These rings may be readily made, the only difficulty being to keep the sides parallel; but a little care will make this easy enough. For this purpose close-grained cardboard may be conveniently used. It must have a well-glazed surface, else the varnish or cement used in affixing the thin glass cover sinks into the substance, and the adherence is very imperfect. When this takes place it is easily remedied by brushing over the sur- face of the cardboard a strong solution of gum or isin- glass; and this application, perhaps, closes also the pores of the card, and so serves a double purpose. But, of course, the gum must be perfectly dried before the ring is used. For cardboard, gutta-percha has been substituted, but cannot be recommended, as it always become brittle after, a certain time, never adheres to the glass with the required firmness, and its shape is altered when worked with even a little heat. Leather is often used, and is very convenient ; it should be chosen, however, of a close texture, and free from oil, grease, and all those substances which are laid upon it by the dressers. Rings of cardboard, &c., have been rejected by persons of OF MICROSCOPIC OBJECTS. Sr great experience, because they are of such a nature that dampness can penetrate them. This fault can be almost, if not totally, removed by immersing them in some strong varnish, such as the asphalt varnish hereinafter mentioned; but they must be left long enough when affixed to the glass slide to become perfectly dry, and this will require a much longer time than at first would be supposed. There has, however, been lately brought out what is termed the ivory cell, This isa ring of ivory-like substance, which may be easily and firmly fixed to the glass slide by any of the commonly used cements, and so forms a beautiful cell for any dry objects. They are made of different sizes, and are notexpensive. Flat rings of brass turned down to the sizes of the circular discs of covering glass and of varying thickness are very neat and useful for mounting opaque objects: they can also be obtained in tin and zinc. Some of our best microscopic men have stated that they have been frequently disappointed by an accumulation of encrusted matter upon the inner surface of thin glass used to cover the cell enclosing any dry object, and therefore use a shallow pillbox, made expressly for this purpose, which must be strongly cemented to the slide. For examination the lid must be removed, whilst it must be closed to protect the object from dust when laid aside. Another worker of experience recommends a cell in a mahogany slide, over which, by aid of a stud as on a pivot, a bone disk can be turned: this is termed, “ Piper’s Revolving Cover Slide,” and can be procured at the opticians’. Sometimes slides are used which are made by taking a thin slip of wood of the usual size (3 in. by 1 in.), in the centre of which is cut a circular hole large enough to receive the object. A piece of thin glass is fixed underneath the slide forming a cell for the object, which may then be covered and finished like an ordinary slide. This has the advantage of serving for transparent objects for which the before-mentioned wooden slides are unsuitable. A slight modification of this plan is often used where the thickness o2 PREPARATION AND MOUNTING of the objects is inconsiderable, especially with some of the Diatomaceze, often termed “ test-objects.” The wooden: slide is cut with the central opening as above, and two pieces of thin glass are laid upon it, betwixt which the diatoms or other objects are placed, and kept in their proper position by a paper cover. This arrangement is a good one, insomuch as the very small portion of glass through which the light passes on its way to the microscope from the reflector causes the refraction or interference to be reduced to the lowest point. A novice would naturally think the appearance of some cof the slides above mentioned very slovenly and unfinished ; but they are often covered with ornamental papers, which may be procured at almost every optician’s, at a cost little more than nominal, and of innumerable patterns and colours. How to use these will be described in another place. It is very probable that a beginner would ask his friend what kind of slides he would advise him to use. Almost all those made of wood are liable to warp more or less, even when the two pieces are separate or of different kinds; those of cardboard and wood are generally free from this fault, yet the slides, being opaque, prevent the employment of the Lieberkuhn. ‘To some extent glass sides, when covered with ornamental papers, are liable to the same objection, as the light is partly hindered. And sometimes dampness from the paste, or other substance used to affix the papers, penetrates to the object, and so spoils it, though this may be rendered less frequent by first attaching the thin glass to the slide by some harder cement. Much time, however, is taken up by the labour of covering the slides, which is a matter of consideration with some. Certainly the cost of the glass slides was formerly great; but now they are reasonable enough in this respect, so that this objection is removed. It is, therefore, well to use glass slides, except where the thin glasses are employed for tests, &c., as above. When the thin glass circles are placed upon the slides, and OF MICROSCOPIC OBJECTS. 30 the edge is varnished with black or coloured rings, the appearance of finish is perfect. The trouble is much less than with most of the other methods, and the illumination of the object very slightly impaired. To varnish the edges of these covers, make circles of any liquid upon the glass slide, and perform any other circular work mentioned hereafter, the little instrument known as * Shadbolt’s turntable” is almost indispensable. It is made as follows:—At one end of a small piece of hard wood is fixed an iron pivot about one-eighth inch thick, projecting half an inch from the wood, which serves as a centre upon which a round brass table three inches in diameter revolves. On the surface of this are two springs, about one and a half- inch apart, under which the slide is forced and so kept in - position, whilst the central part is left open to be worked upon. The centre is marked, and two circles half an inch and one inch in diameter are usually deeply engraved upon the table to serve as guides in placing the slide, that the ring may be drawn in the right position. When the slide is placed upon the table underneath the springs, a camel- hair pencil is filled with the varnish, or other medium used, and applied to the surface of the glass; the table is then made to revolve, and a circle is consequently produced, the diameter of which it is easy to regulate. Mr. Hislop places two equidistant pins at opposite sides of the centre of the revolving plate, against which the opposing edges of the slip are made to bear, so that the instrument is self- centering, The springs are turned in contrawy directions and are screwed on the pins, or the screws are made into the pins against which the sides of the slide bear. The form of this “turntable” has been modified by many manipulators to suit their several wants. Almost all slides used are of nearly the same size—3 in. by 1 in.; and therefore the centres of all are equidistant from the edge. On this account one of my friends has a thin brass bar screwed upon the side of his turntable in such a position that the centres of the slides and table always coincide. The rings of varnish D 3o4 PREPARATION AND MOUNTING upon the slides and thin glass upon the cell are thus kept uniform. Dr. Matthews, a gentleman of no little experience, has given us an improvement as follows :—Take two “jaws” of the average thickness of a glass slide, ¢ inch wide, 25 long. Each of these is pivoted on the face of the turntable . by a screw through its centre, each screw being placed . exactly equidistant from the centre of the turntable, so that - the jaws are separated by a space as wide as an average slide; i.e. a full inch. Outside of that space, on one side of the centre of one of the jaws, is a wedge fixed by a screw in’ such a way as to be capable of motion in the direction of its length by a slotted hole. This is all the machinery. AB and CD are the two jaws, E is the wedge. On placing a slip between the jaws they probably at first do not touch it. If the wedge be then pushed so as to approximate B to C, the jaws move on their centres, so that, however far B may be pushed towards (and moving) C, the other end of C—i.e. D—is moved eaactly as much in the opposite direc- tion until they approach near enough to grasp the slide by its edges. The length of the wedge must, of course, be such as to provide for about 4 inch variation in the width of slides. It will readily be seen that the slip may be pushed | in either direction excentrically lengthwise, so as to allow of , the formation of any number of cells, all of which must needs be central as regards their width, if the instrument has been accurately made, which is a very easy matter. I have added also a rest for the hand, F, which may be turned aside on a centre at will, and which I have found to be a great convenience. OF MICROSCOPIC OBJECTS. V CL DR. MATTHEWS’ TURNTABLE. 39 36 PREPARATION AND MOUNTING Mr. Spencer slightly modifies the above, using wood jaws and wedge, which the following engraving will best explain. DR. MATTHEWS’ TURNTABLE, TWO-THIRD SIZE. Many objects for the microscope may be seriously injured by allowing the fingers to touch them—many more are so minute that they cannot be removed in this way at all, and often it is necessary to take from a mass of small grains, as in sand, some particular particle. To accomplish this, there are two or three contrivances recommended: one by means of split bristles, many of which will readily be found in any shaving-brush when it has been well used. The bristles when pressed upon any hard surface, open, and when the pressure is removed close again with a spring; but the use of these is limited. Camel-hair pencils are of great service for this, and many other purposes, to the microscopist. In very fine work they are sometimes required so small that all the hairs, with the exception of one or two finer pointed OF MICROSCOPIC OBJECTS. 37 ones, are removed. A few of various sizes should always be kept on hand, Equally necessary are fine-pointed needles. They are very readily put up for use by thrusting the eye end into a common penholder, so as to be firm. The points may be readily renewed, when injured, on a common whetstone; and when out of use they may be protected by being thrust into a piece of cork. In laying out animal tissues that have been stained by nitrate of silver or chloride of gold, it is advisable to employ a small rod of glass drawn out to a_point, as the use of a metallic point causes a deposit of gold or silver at the place of contact, which disfigures the preparation. Knives of various kinds are required in some branches of microscopic work; but these will be described where dissection, &c., is treated at some length, as also various forms of scissors. In the most simple objects, however, scissors of the usual kind are necessary. Two or three sizes should always be kept at hand, sharp and in good order. A set of glass tubes, kept in a case of some sort to prevent breakage, should form part of our fittings, and be always cleaned immediately after use. These are generally from six to ten inches long, and from one-eighth to a quarter of an inch in diameter. One of these should be straight and equal in width at both ends; one should be drawn out gradually to a fine point; another should be pointed as the last, but slightly curved at the compressed end, in order to reach points otherwise unattainable. It is well to have these tubes of various widths at the points, as in some waters the finer would be inevitably stopped. For other purposes the fine ones are very useful, especially in the transfer of preservative liquids which will come under notice in another chapter. Forceps are required in almost all microscopic manipu- lations, and consequently are scarcely ever omitted from the microscopic box, even the most meagrely furnished ; but of 38 PREPARATION AND MOUNTING these there are various modifications, which for certain purposes are more convenient than the usual form. The ordinary metal ones are employed for taking up small objects, thin glass, &c.; but when slides are to be held over a lamp, or in any position where the fingers cannot con- veniently be used, a different instrument must be found. Of these there are many kinds; but Mr. Page’s wooden forceps serve the purpose very well. ‘T'wo_ pieces of elastic wood are strongly bound together at one end, so that they — may be easily opened at the other, closing again by their own elasticity. Through the first of these pieces is loosely passed a brass stud, resembling a small screw, and fastened in the second, and through the second a similar stud is taken and fixed in the first—so that on pressure of the studs the two strips of wood are opened to admit a slide or other object required to be held in position. The wood strips are generally used three or four inches long, one inch wide, and about one-eighth inch thick. Again, some objects when placed upon the glass slide are of such an elastic nature that no cement will secure the thin glass covering until it becomes hard. ‘This difficulty may be overcome by various methods. The following are as good and simple as any. ‘Take two pieces of wood about two inches long, three-quarters wide, and one-quarter thick ; and a small rounded piece one inch long, and one-quarter in diameter; place this latter betwixt the two larger pieces. Over one end of the two combined pass an india-rubber band. This will give a continual pressure, and may be opened by bringing the two pieces together at the other end; the pressure may be readily made uniform by paring the points at the inner sides, and may be regulated by the strength of the india-rubber band. These bands may be made cheaply, and of any power, by procuring a piece of india-rubber tubing of the width required, and cutting off certain breadths. Another very simple method of getting this pressure is mentioned in the “ Micrographic Dictionary.” Two pieces of whalebone of the length required are tied OF MICROSCOPIC OBJECTS. 39 together firmly at each end. It is evident that any object placed betwixt them will be subject to continual pressure. The power of this may be regulated by the thickness and length of the whalebone. This simple contrivance is very useful. Almost every scientific man, however, has his own model, and it may be as well to examine one or two of them. Mr. Goode uses the following: A, a piece of wood 8 in. long and # in. thick. B, a spring made with thin iron wire. The end of the spring is driven into the table, as at C. A piece of 3-in. iron wire is then run through the springs, which forms an axis to work upon, and also keeps them in their places. He inserts a pin at the side of the spring, so that it will fall on a given spot, and not rub the cover from side to side. The springs are made by binding the thin wire round the j-in. rod about four or five times. PREPARATION AND MOUNTING 40 WIRE CLIP. OF MICROSCOPIC OBJECTS. 41 Mr. J. B. Spencer’s model is made thus:—It is formed of thin sheet steel (obtainable at any instrument maker’s), and cut out in one piece, of the form above, with a stout pair of scissors, and then bent the required shape with a pair of pliers. When used, the fore and middle fingers are applied on the under side, and the thumb on the spring. If great pressure is required, two clips may be used,—one at each end of the slide,—and for any delicate work the width of the steel can be reduced. My STEEL CLIP. The American wooden spring clips are occasionally very useful, and wire clips of the kind described by Dr. Carpenter are now commonly sold and are indispensable. Common watch-glasses should always be kept at hand. They are certainly the cheapest, and their transparency makes them very convenient reservoirs in which objects may 4.2, PREPARATION AND MCUNTING be steeped in any liquid; and the use of them saves much trouble in examining cursorily under the microscope, whether the air-bubbles are expelled from insects, &c. &c. They are readily cleaned, and serve very well as covers, when turned upside down, to protect objects from dust. For this latter purpose Dr. Carpenter recommends the use of a number of bell-glasses, especially when one object_must be left for a time (which often happens) in order that another may be proceeded with. Wine-glasses, when the legs are broken, may thus be rendered very useful. As heat is necessary in mounting many obejects, a lamp will be required. Where gas is used, the small lamp known as “ Bunsen’s” is the most convenient and inexpensive. It gives great heat, is free from smoke, and is readily affixed to the common gas-burner by a few feet of india-rubber tubing. The light from these lamps is small, but this is little or no drawback to their use. Where gas is not avail- able, the common spirit-lamps may be used, as they are very clean and answer every purpose. In applying the required heat to the slides, covers, &c., it is necessary in all cases to ensure uniformity, otherwise there is danger of the glass being broken. For this purpose a brass plate at least three inches wide, somewhat longer, and one-eighth of an inch thick must be procured. It should then be affixed to a stand, so that it may be readily moved higher or lower, in order that the distance from the lamp may be changed at will, and thus the degree of heat more easily regulated. This has also the advantage of enabling the operator to allow his slides, &c., to cool more gradually, which, in some cases, is absolutely necessary,— as in fusing some of the salts, &c. | In order to get rid of air-bubbles, which are frequently disagreeable enemies to the mounter of objects, an air-pump is often very useful. This is made by covering a circular plate of metal with a bell-glass, both of which are ground so finely at the edges that greasing the place of contact renders it air-tight. The pump is then joined to the metal plate \ OF MICROSCOPIC OBJECTS. 43 underneath, and worked with a small handie like a common syringe. By turning a small milled head the air may be ' allowed to re-enter when it is required to remove the bell- glass and examine or perform any operation upon the object. The mode of using this instrument will be described here- after, but it may be here stated that substitutes have been devised for this useful apparatus; but as it is now to be obtained at a low cost, it is hardly worth while to consider them. Much time is, in many instances, certainly saved by its use, as a very long immersion in the liquids would be required to expel the bubbles, where the air-pump would remove them in an hour. The next thing to be considered is what may be termed Cements, some of which are necessary in every method of mounting objects for the microscope. Of these will be given the composition where it is probable the young student can use it; but many of them are so universally kept as to be obtainable almost anywhere; and when small quantities only are required, economy suffers more from home manu- facture than from paying the maker’s profit. Amongst these, Canapa Batsam may, perhaps, be termed the most necessary, as it is generally used for the preserva- tion of many transparent objects. It is a thick liquid resin of a light amber-colour, which on exposure to the atmosphere becomes dry and hard even to brittleness. For this reason it is seldom used as a cement alone where the surface of contact is small, as it would be apt to be displaced by any sudden shock, especially when old. In the ordinary method of using, however, it serves the double purpose of preserving the object and fixing the thin glass cover; whilst the com- paratively large space upon which it lies lessens the risk of displacement.. By keeping, this substance becomes thicker ; but a very little warmth will render it liquid enough to use, even when to some extent this change has taken place. When heated, however, for some time and allowed to cool, it becomes hardened to any degree, which may be readily regulated by the length of time it has been exposed, and 44. PREPARATION AND MOUNTING the amount of heat to which it has been subjected. On account of this property it is often used with chloroform : the balsam is exposed to heat until, on cooling, it assumes a glassy appearance. This will be most readily done by baking it in what we should call a “cool oven.” The time required will most likely be 20 or 30 hours. Care must be taken that the heat is not too great, else the balsam will be discoloured. It must then be dissolved in pure chloroform or benzole (the latter is preferable) until it becomes of the consistence of thick varnish. This liquid is very convenient in some cases, as air-bubbles are much more easily dispelled than when undiluted Canada balsam is used. It also dries readily, as the chloroform evaporates very quickly, for which reason it must be preserved in a closely-stoppered bottle. It has been said that this mixture becomes cloudy with long keeping, but I have not found it so in any cases where I have used it. Cloudiness is most frequently, if not always, caused by dampness in the object, as mentioned in Chapter IV. Should it, however, become so, a little heat will gene- rally dispel the opacity. The ordinary balsam, if exposed much to the air whilst being used, becomes thicker, as has been already stated. It may be reduced to the required consistency with common turpentine; but I have often found this in some degree injurious to the transparency of the balsam, and the amalgamation of the two by no means perfect. (See also Chapter IV.) Its cheapness renders it no extravagance to use it always undiluted; and when pre- served in a bottle with a hollow cover fitting tightly around the neck, both surfaces being finely ground, it remains fit for use much longer than in the ordinary jar. Canada bal- sam may now be procured in collapsible tin tubes, like those used by artists; and its manipulation is thus rendered much more easy, cleanly, and convenient, as well as economical. Chloroform is, however, frequently used for dilution, and is perhaps the safest solvent we can employ. Dammar VarnisH.—Some complain that this varnish is not easily procurable in a pure transparent state. It is often used by our American friends in mounting diatoms OF MICROSCOPIC OBJECTS. 45 and other fine work. It is very liquid, and is thought by some to be more easily worked than Canada balsam. -Dammar may be easily dissolved in benzole to any extent. The lumps should previously be scraped until they are freed from dust and other impurities, and then roughly crushed. AspHALTuM.—This substance is dissolved in linseed oil, turpentine, or naphtha, and is often termed “ Brunswick black.” It is easily worked, but is not generally deemed a trustworthy cement, as after a time it is readily loosened from its ground. It is, however, very useful for some pur- poses (such as “ finishing” the slides), as it dries quickly. I shall, however, mention a modification of this cement a little farther on. Marine Guiuz.—No cement is more useful or trustworthy for certain purposes than this. It is made in various pro- portions; but one really good mixture is—equal parts of india-rubber and gum shellac: these are dissolved in mineral naphtha with heat. It is, however, much better to get it from the opticians or others who keep it. It requires heat in the application, as will be explained in Chapter V.; but is soluble in few, if any of the liquids used by the microscopist, and for that reason is serviceable in the manufacture of cells, &c. Where two pieces of glass are to be firmly cemented together, it is almost always employed; and in all glass troughs, plates with ledges, &c., the beginner may find examples of its use. Gotp Sizz.—This substance may always be procured at any colourman’s shop. ‘The process of its preparation is long and tedious. It is therefore not necessary to describe it here. Dr. Carpenter says that it is very durable, and may be used with almost any preservative liquids, as it is acted upon by very few of them, turpentine being its only true solvent. If too thin, it may be exposed for awhile to the open air, which by evaporation gradually thickens it. Care must be taken, however, not to render it too thick, as it will then be useless. A small quantity should be kept on hand, as it is much more adhesive when old. Gum Dammar Crement.—An excellent cement may be 46 PREPARATION AND MOUNTING made by dissolving gum dammar in benzole, and adding about one-third of gold size: it dries very readily, and is especially useful when mounting objects in fluid, taking care that no moisture extends beyond the covering glass, which would prevent the complete adhesion of the cement. In those cases where glycerine is employed as the mounting medium, a ring of liquid glue put round the cover first, and when that is dry, a second coat of gum dammar will keep the cover very secure, and no leakage take place. Liquip GLvE is another of these cements, which is made by dissolving gum shellac in naphtha in such quantity that it may be of the required consistency. This cement appears to me almost worthless in ordinary work, as its adherence can never be relied upon; but it is so often used and recom- mended that an enumeration of cements might be deemed incomplete without it. Hven when employed simply for varnishing the outside of the glass covers, for appearance’s sake alone, it invariably chips. Where, however, oil is used as a preservative liquid, it serves very well to attach the thin glass; but when this is accomplished, another varnish less liable to chip must always be laid upon it. (See Chapter V.) Yet it makes excellent cells. Buiack Japan.—This is prepared from oil of turpentine, linseed oil, amber, gum anime, and asphalt. It is trouble- some to make, and therefore it is much better to procure it at the shops. It is a really good cement, and serves very well to make shallow cells for liquids, as will be described in Chapter IV. ‘The finished cell should be exposed for a short time to the heat of what is usually termed a “cool oven.” This renders it very durable, and many very careful manipulators use it for their preparations. ELECTRICAL CEMENT.—This will be found very good for some purposes hereinafter described. To make it, melt together— 5 parts of resin. 1 » beeswax. 1 » red ochre. OF MICROSCOPIC OBJECTS. 47 It must be used whilst hot, and as long as it retains even slight warmth can be readily moulded into any form. It is often employed in making shallow cells for liquids, as before ‘mentioned. GuM-WatTER is an article which nobody should ever be without; but labels, or indeed any substance, affixed to glass with common gum, are so liable to leave it spon- taneously, especially when kept very dry, that I have lately added five or six drops of glycerine to an ounce of the gum solution. This addition has rendered it very trustworthy even on glass, and now I never use it without. Ten grains of moist sugar to each ounce of gum solution will also answer equally well. This solution cannot be kept long without undergoing fermentation, to -prevent which the addition of a small quantity of any essential oil (as oil of cloves, &c.), or one-fourth of its volume of alcohol, may be made, which will not interfere in any way with its use. There is what is sometimes termed an ewtra adhesive . gum-water, which is made with the addition of isinglass, thus :—Dissolve two drachms of isinglass in four ounces of distilled vinegar; add as much eum arabic as will give it the required consistency. ‘This will keep very well, but is apt to become thinner, when a little more gum may be added. ne I may here mention that Messrs. Marion have lately brought out a cement for the purpose of mounting photo- graphs, which is very adhesive, even to glass. I find it useful in all cases where certainty is requisite; as gummed paper is liable in a dry place to curl from the slides, as before mentioned. All these, except one or two, are liquid, and must be kept in stoppered bottles, or, at least, as free from the action of the air as possible. When any two substances are to be united firmly, I have termed the medium employed “a cement ;” but often the appearance of the slides is thought to be improved by drawing a coloured ring upon them, extending partly on the 48 PREPARATION AND MOUNTING cover and partly on the slide, hiding the junction of the two. The medium used in these cases I term A VARNISH, and hereinafter mention one or two. Of course, the tenacity is not required to be so perfect as in the cements. Sratinc-Wax VARNISH is prepared by coarsely powdering sealing-wax, and adding spirits of wine; it is then digested at a gentle heat to the required thickness. This is very frequently used to finish the slides, as before mentioned, and can easily be made of any colour by employing different - kinds of sealing-wax; but is very liable to chip and leave the glass. The best qualities, however, will be less hable. Buack VarnisH—Is readily prepared by adding a small quantity of lampblack to gold-size and mixing intimately. Dr. Carpenter recommends this as a good finishing varnish, drying quickly and being free from that brittleness which renders some of the others almost worthless; but it should not be used in the first process when mounting objects in fluid. Amongst these different cements and varnishes I worked a long time without coming to any decision as to their comparative qualities, though making innumerable experi- ments. The harder kinds were continually cracking, and the softer possessed but little adhesive power. To find hardness and adhesiveness united was my object, and the following possesses these qualities in a great degree :— India-tabber : A/\aeante eee 99 Thus he now omits the arsenious acid, but places in the solution (which should be kept in a bottle with glass stop- * Dr. Carpenter says :—‘‘Glycerine has a solvent power for carbo- nate of lime;and should not be employed when the object contains any calcareous structure. In ignorance of this fact, the author (Dr. C.) employed glycerine to preserve a number of remarkably fine speci- mens of the pentacrinoid larva of the Comatula, whose colours he was anxious to retain; and was extremely vexed to find, when about to mount them, that their calcareous skeletons had so entirely disap- peared, that the specimens were completely ruined.” OF MICROSCOPIC OBJECTS. 125 per) a small piece of camphor. This requires no cell, as the adhesive power is sufficient. Derane’s CompounpD.—This is usually deemed about the best medium for preserving Alga, mosses, &c., and is thus prepared :—Soak 1 oz. of best gelatine in 4 oz. of water until the gelatine becomes soft, when 5 oz. of honey heated to boiling point are added; boil the mixture, and when it has eooled, but not enough to become stiff, add 4 oz. recti- fied spirit with which 5 or 6 drops of creosote have been well mixed, and filter the whole through fine flannel. This compound when cold forms a stiff jelly, the use of which will be described elsewhere. GLYCERINE JELLY.—This mixture closely resembles the above, but as the composition differs a little it may be men- tioned here. It is strongly recommended by Mr. Lawrance in the Microscopic Journal, where he states “that the beautiful green of some mosses mounted two years ago, is still as fresh as on the day they were gathered ;” and that this is the only medium he knows which will preserve the natural colour of vegetable substances. He takes a quantity of Nelson’s gelatine, soaks it for two or three hours in cold water, pours off the superfluous water, and heats the soaked gelatine until melted. To each fluid ounce of the gelatine, whilst it is fluid but cool, he adds a fluid drachm of the white of an ege. He then boils this until the albumen coagulates and the gelatine is quite clear, when it is to be filtered through fine flannel, and to each ounce of the clari- fied solution add 6 drachms of a mixture composed one part ot glycerine to two parts of camphor-water. At the Academy of Natural Sciences of Philadelphia, Mr. W. H. Walmsley stated, that, owing to the heat of that climate, the above formula for glycerine jelly was not satis- factory, and recommended the following :—Take one pack- age of Cox’s gelatine, wash repeatedly in cold water; allow it to soak in water sufficient to cover it for an hour or two, add one pint of boiling water, and boil ten or fifteen minutes. Remove, and when cool but still fluid, add the 126 PREPARATION AND MOUNTING white of an egg, well beaten, and again boil, until the albu- men coagulates. Strain whilst hot through flannel, and add an equal portion by measurement of Bowyer’s pure glycerine, and fifty drops of carbolic acid in solution; boil again for ten or fifteen minutes, and again strain through flannel, place in a water-bath and evaporate to about one- half, then filter into two or more broad-mouthed vials. (Cotton is the best filtering medium.) The use of this in mounting is the same as Mr. Lawrance’s, elsewhere described. GoapBy’s Fiuip.—This is much used in the preservation of animal objects; and seldom, if ever, acts upon the colours. It is thus prepared :—Bay salt, 4 oz.; alum, 2 oz.; corrosive sublimate, 4 grains. Dissolve these in two quarts of boiling water, and filter. For delicate preparations some recom- mend that this mixture be reduced by the addition of an equal quantity of water; but where there is bone or shell in the object, the above acts injuriously upon it, in which case this fluid may be used:—Bay salt, 8 oz.; corrosive subli- mate, 2 grains; water, 1 quart. Tuwaites’s Liquiv.—This is recommended for the preser- vation of Alge, &c., as having little or no action on the colour, and is thus prepared:—Take one part of rectified spirit, add drops of creosote enough to saturate it; to this add sixteen parts of distilled water and a little prepared chalk, and filter. When filtered, mix with an equal quantity of camphor-water (as before mentioned), and strain through fine muslin before using. CHLORIDE or Zinc Sotvution.—In the Micrographic Dic- tionary this is stated to be “ perhaps the best preservative known for animal tissues.” Persons of great experience, however, have given a very different opinion; but it is cer- tainly very useful in many cases where a small degree of coagulating action is not injurious. It is used of strengths varying according to the softness of the parts to be pre- served; the average being 20 grains of the fused chloride to 1 oz. of distilled water. To keep this liquid, a lump of cam- phor may be left floating in the bottle. I have heard com- OF MICROSCOPIC OBJECTS. 128 plamts that this mixture becomes turbid with keepiné; but I think this must only be the case when some impurity has got into the bottle. Carzporic Actp.*—The addition of a few drops of this to distilled water prevents the growth of interfering substances which would take place if pure water alone were used, and is therefore valuable as a preservative fluid. The same solution also is convenient, as it instantly kills infusoria, and almost everything that has life; and, indeed, is useful in the student’s gathering-bottles for the same reason. It is very highly spoken of as forming one of the constituents in the following formula for use in mounting soft animal textures :— Argenious acid, 20 parts. Crystallized carbolic acid, 10 parts. Alcohol, 300 parts. Distilled water, 700 parts. The Rev. W. W. Spicer, in his translation of Johann Nave’s work on Algz, recommends the following fluid for their preservation :—Pure alcohol, 3 parts; distilled water, 2 parts; glycerine, 1 part. If the desmid or other alga be placed in this fluid in a cell, and not covered by a glass for a time, the water and alcohol will evaporate slowly, and the mixture will become more dense in proportion, but quite gradually, and therefore without any destructive influence on the object. During this operation, water is withdrawn from the frustule, and the glycerine, which is not volatile, takes its place without causing any distortion of the object. Castor Om.—This is a very useful preservative for crystals and other objects. Many salts are quite destroyed when Canada balsam is used with them; but very few are acted upon by this oil. To use it, it must be dropped in sufficient quantity to cover the crystal or object to be * Dr. Crace Calvert stated at a meeting of the British Association, that after careful experiments he finds carbolic acid “ prevents the development of protoplasmic and fungoid life.” 128 PREPARATION AND MOUNTING preserved with a thin coating of oil. It may be necessary sometimes to spread it with a needle or other instrument. The thin glass should then be carefully placed upon it, so that all air may be excluded; and should any oil be forced out, owing to the quantity used being too great, it must be removed with blotting-paper. When the edge of the thin glass cover and the surrounding parts_of the slide are as clean as possible, a coating of sealing-wax varnish or liquid glue must be applied and allowed to dry. A second or even a third coating may be required, but not before the previous cover is quite dry. These varnishes, however, are very brittle, and it is much safer, as a finish, to use one of the tougher cements—gold-size, for instance—which will render it doubly secure. The above are the principal liquids, &., used for pre- serving objects in cells. The different cells may be here mentioned ; and it is recommended that these should always be kept some time before use in order that the cement may become perfectly dry; and care must be taken that no cement be used on which the preservative liquid employed has any action whatever. Cement Crtis.—Where the object is not very thick, this kind of cell is generally used. They are easily made with the turntable before described ; but when the objects to be preserved are very minute, these cells need not be much deeper than the ordinary circle of cement on the slide. When, however, a comparatively great depth is required, it is sometimes necessary to make the wall of the cell as deep as possible, then allow it to dry and make another addition. Of these cements gold-size is one of the most trustworthy, and may be readily used for the shallow cells. The as- phaltum and india-rubber, before noticed, I have found very durable when well baked, and exceedingly pleasant to work with. It may be used of such a thickness as to give space for tolerably large objects. Black japan also is much used. Many cements, however, which are recommended by some writers, are worse than useless, owing to the brittleness which OF MICROSCOPIC OBJECTS. 129 renders their durability uncertain, as sealing-wax, varnish, liquid-glue, &c. Dr. Bastian says the best cement for liquid cells is one, much used in Germany, made by adding a considerable quantity of nitrate of bismuth to a solution of gum mastic in chloroform. It can be procured at almost any optician’s. The student may feel himself at a loss in choosing the cement which will give him the safest cells, many of them becoming partially or wholly dry in a year or two, as stated in another place. I can only give him a few general direc- tions, and he must then use his own judgment. Of course it would be lost labour to employ any cement upon which the preservative liquid has any action whatever. It is also a good rule to avoid those in whose composition there are any particles which do not become a thorough and intimate portion, as these unreduced fragments will almost certainly, sooner or later, prepare a road by which the liquid will escape; and, lastly, whatever cement he uses, the cells are always better when they have been kept a short time before use. GuTTa-PERCHA Rives have been recommended by some, as affording every facility for the manufacture of cells for liquids; but they cannot be recommended, as, after a certain length of time, they become so brittle as to afford no safe- guard against ordinary accidents. Some have also used india-rubber bands thickly coated with various varnishes; but these I consider less trust-worthy than gutta- percha, as they become thoroughly rotten in ordinary use after a short probation. Often the cells must necessarily be of a large size, and for this reason are made by taking four strips of glass of the thickness and depth required, and grinding the places where these are to meet with emery, so as to form a slightly roughened but flat edge. The glass strip must also be ground on the side where it meets the plate, and each piece cemented with the marine-glue mentioned in Chapter II. in the following manner :—On that part of the glass to which K 130 PREPARATION AND MOUNTING another piece is to be attached should be laid thin strips of the glue; both pieces must then be heated upon a small brass table, with the aid of the spirit-lamp, until the strips become melted; the small piece is then to be taken up and placed upon the spot to which it is to be attached, and so on until the cell is completed. . It will be found necessary to spread the glue over the surface required with a needle or some other instrument, so that an unbroken line may be presented to the wall of the cell, and no bubbles formed. Too great a heat will burn the marine-glue, and render it brittle ; care must be therefore taken to avoid this. When shallow cells are required, those which are made by grinding a concavity in the middle of an ordinary slide will be found very convenient. The concavities are cut both circular and oblong; and the surface being flat, the cover is easily fastened upon it. These are now cheap, and are very safe as to leakage. It is a very great improvement, where it can be done, to turn a shallow ring outside the concavity of the slide, but close to it. This prevents the cement with which the cover is fastened from running in. Circular cells with a flat bottom used to be made by drilling a hole through glass of the required thickness, and fixing this upon an ordinary slide with marine-glue; but ' the danger of breakage and the labour were so great that this method is seldom used now, and, indeed, the rings about to be mentioned do away with all necessity for it. Guass Rines.—Where any depth is required, no method of making a cell for liquids is so convenient as the use of glass rings, which are now easily and cheaply procurable. They are made of almost every size and depth, and, except in very extraordinary cases, the necessity for building cells is completely obviated. These rings have both edges left — rough, and consequently adhere very well to the slide, this adherence being generally accomplished by the aid of marine-glue, as before noticed with the glass cells. Gold- size has been occasionally used for this purpose; and the adherence, even with liquid in the cell, I have always found OF MICROSCOPIC OBJECTS. 131] to be perfect. This method has the advantage of requiring no heat, but the gold-size must be perfectly dry, and the ring must have been fixed upon the slide some time before use. Canada balsam has also been used for the same purpose, but cannot be recommended, as, when it is perfectly dry, it becomes so brittle as to bear no shock to which the slide may be ordinarily exposed. Iron Rines——Many have worked with these, having taken care to varnish thoroughly before using with any preservative liquid; but they are always untrustworthy, as they can never be guaranteed against the action of some salt in the liquid used. They can be procured beautifully made, and for dry cells cannot be surpassed. Zine and pure tin rings ray also be procured, and are excellent, especially the latter. Vuucanite.—This substance is a great favourite with some of our working microscopists, as it is very slightly in- fluenced by change of temperature. But my own opinion is that a glass cell is the safest and most satisfactory re- ceptacle for any object in liquid, and if carefully prepared will not deceive the operator. These are the cells which are mostly used in this branch of microscopic mounting. The mode of using them, and the different treatment which certain objects require when in- tended to be preserved in the before-mentioned liquids, may now be inquired into. I may mention, however, that this class of objects is looked upon by many with great mistrust, owing to the danger there is of bubbles arising in the cells after the mounting has been completed, even for years. I know some excellent microscopists who exclude all objects in cells and preservative liquids from their cabinets, because they say that eventually almost all become dry and worthless; and this is no matter of surprise, for many of them do really become so. Perhaps this is owing to the slides being sold before they could possibly be thoroughly dry. As to the air-bubbles, I shall have. something to say presently. ee 132 PREPARATION AND MOUNTING We will now suppose the cell employed has been made by placing a glass ring upon the slide with marine-glue or gold-size, and is quite dry. Around the edge of the cleaned thin glass which is to cover it, I trace with a camel-hair pencil a ring of gold-size, and also around the edge of the cell to which it is to adhere. Dr. Carpenter objects to this, as rendering the later applications of the gold-size liable to “run in.” All danger of this, however, is completely ob- viated by leaving the slide and cover for awhile until the cement becomes partially fixed, but still adhesive enough to perform its function (Chapter III.). With many slides this is not accomplished in less than twenty-four hours, even if left two or three days no injury whatever ensues; but with other kinds an hour is too long to leave the exposed cement, so that the operator must use his own discretion. It is not always necessary to size the edge of the cover, since perfect adhesion may in many cases be secured with- out it, and it is always best to use the least quantity of cement that will answer, as it will then be less likely to run in. The liquid required may be drawn up by the mouth into the pointed tube mentioned in Chapter II., and then transferred to the cell. In the various books of instruction, the object is now to be placed in the cell; this, however, I think a great mistake, as another process is absolutely necessary before we advance so far. The cell, full of liquid, must be placed ander the receiver of an air-pump, and the air withdrawn. Almost immediately it will be perceived that the bottom and sides of the cell are covered with minute bubbles, which are formed by the air that is held in sus- pension by the liquid. The slide may now be removed, and the bubbles may require the aid of a needle or other point to displace them, so obstinately do they adhere to the surface of the glass. This process may then be repeated, and one cause, at least, of the appearance of bubbles in cells of liquid will be removed. The object to be mounted should also be soaked in one or two changes of the preservative liquid employed, and, during the soaking, be placed under OF MICROSCOPIC OBJECTS. 188 the air-pump and exhausted, It may then be transferred to the ceil, and will probably cause the liquid to overflow a little. The cover with the gold-size applied to the edge must then be carefully laid upon the cell, and slightly pressed down, so that all air-bubbles may be displaced. The two portions of gold-size will now be found to adhere wherever the liquid does not remain, although the whole ring may have been previously wet. The outer edge of the thin glass and cell must now be perfectly dried, and a coating of gold-size applied. When this is dry, the process must be repeated until the cement has body enough to pro- tect the cell from all danger of leakage. When some pre- servative liquids are used, a scum is frequently found upon the surface when it is placed in the cell, and this must be removed immediately before the cover is laid upon it. I believe this method to be perfectly secure against leakage when carefally performed; and some of my friends have told me that their experience (that of some years) has been equally satisfactory. In using some of the particular kinds of preservative liquids, it will be found necessary to make a slight change in the manipulation. This will be best explained by mention- ing a few objects, and the treatment they require. For the preservation of the Mosses, Algw, &c., Deane’s compound is much used, and considered one of the best media. ‘T'he specimen to be mounted should be immersed in the compound, which must be kept fluid by the vessel containing it being placed in hot water. In this state the whole should be submitted to the action of the air-pump, as it is not an easy matter to get rid of the bubbles which form in and around the objects. The cell and slide must be warmed; and heat will also be necessary to render the gelatine, &c., fluid enough to flow from the stock-bottle. The cell may then be filled with the compound, and the Specimen immersed in it. A thin glass cover must then be warmed, or gently breathed upon, and gradually lowered upon the cell, taking care, as with all liquids, that no 134 PREPARATION AND MOUNTING bubbles are formed by the operation. The cover may be fixed by the aid of gold-size, Japan, or any of the usual varnishes, care being taken, as before, that all the compound is removed from the parts to which the varnish is intended to adhere. The glycerine jelly of Mr. Lawrance, before mentioned, requires almost a similar treatment. ‘The objects to be mounted in this medium should be immersed for some time in a mixture of equal parts of glycerine and dilute alcohol: (six of water to one of alcohol). The bottle of glycerine jelly must be placed in a cup of hot water until liquefied, when it must be used like Canada balsam, except that it requires less heat. A ring of asphaltum varnish round the thin glass cover completes the mounting.” The Infusoria (see Chapter IV.) are sometimes preserved in liquid; but present many difficulties to the student. Different kinds require different treatment, and consequently it is well, when practicable, to mount similar objects in two or more liquids. Some are best preserved in a strong solution of chloride of calcium, others in Thwaites’ liquid, whilst a few keep their colour most perfectly when in glycerine alone. ‘There can be little doubt that light is the bleaching agent in most cases. Many of them, however, are so very transparent that they present but faint objects for ordinary observation. For this reason, however, they are sometimes dyed in solution of magenta or other colour, as elsewhere noticed. The Desmidiaceze require somewhat similar treatment, and may be mentioned here. The solution of chloride of calcium has been strongly recommended; but no preservative liquid seems to be without some action upon them. Both of the above classes of objects should be mounted in shallow cells, so as to allow as high a microscopic power as possible to be used with them. ENTOMOSTRACA.—In every ditch or place where vegetable matter exists, these little active, jerking pieces of life are certain to be found. They are covered with a horny trans- parent shell, and are various in form. Mr. Tatem gives the : OF MICROSCOPIC OBJECTS. 185 following, as the best way of preserving them :—When caught transfer to filtered water in watch-glasses for twenty- four hours, in order that the contents of the laden intestine may be discharged. Draw off the water and add a little spirit of wine, which quickly destroys life. Remove all dirt by aid of a camel-hair pencil, and place in a few drops of the medium used and water (half of each) until saturation is complete, and then put up in the medium in shallow cells. The medium advised is Mr. Farrant’s, which will be found amongst those recommended. Many of the Zoopuytzs which are obtained on our sea- coasts are well preserved by mounting in cells, in the manner before mentioned, with Goadby’s fluid, or distilled water with one of the additions noticed amongst the preservative liquids. For examination by polarized light, however, they are usually mounted in balsam (see Chapter IV.), whilst those in cells present a more natural appearance as to position, &c., for common study. The Potyzoa, also, are exquisitely beautiful objects for the microscope, but require some little care. They should be kept in sea-water until their tentacula are expanded, and may then be readily killed by plunging in cold fresh water. Thus all their beauty will be preserved, and they may be then mounted in one of the preservative liquids. Many operators speak well of distilled water well shaken with a few drops of creosote, as before mentioned. As to the use of preservative liquids with the Diatemacesz there are varieus opinions. Some experienced microscopists say that there is little or no satisfaction in mounting them in this way. Dr. Carpenter, however, explains this differ- ence by his instructions as to what method should be used when certain ends are desired. He says: “If they can be obtained quite fresh, and it be desired that they should exhibit as closely as possible the appearance presented by the living plants, they should be put up in distilled water within cement cells; butif they are not thus mounted within a short time after they have been gathered, about a sixth 136 PREPARATION AND MOUNTING part of alcohol should be added to the water. If it be desired to exhibit the stipitate forms in their natural para- sitism upon other aquatic plants, the entire mass may be mounted in Deane’s gelatine in a deeper cell; and such a preparation is a very beautiful object for black-ground illumination. If, on the other hand, the minute structure of the silicious envelopes is the feature to be brought into view, the fresh diatoms must be boiled in nitric or hydro- chloric acid” (which process is fully described in Chapter III.). It is very convenient to have many of these objects mounted by two or more of the above methods; and if they are to be studied, this is indispensable. Mr. Hepworth once showed me about one hundred slides which he had mounted in various ways, for no other purpose than the study of the fly’s foot. My friend, Mr. Rylands, successfully mounts the diatoms in the state in which he finds them, and gave me the follow- ing method as that which he always employs. He says that he has had no failures, and hitherto has found his specimens unchanged. Take a shallow ring cell of asphalt or black varnish (which must be at least three weeks old), and on the cell, whilst revolving, add a ring of benzole and gold-size mixed in equal proportions. In a minute or two pure distilled water is put in the cell until the surface is slightly convex. The object having been already floated on to the cover (the vessel used for this purpose being an ordinary indian-ink pallet), is now inverted and laid carefully upon the water in the cell. By these means the object may be laid down without being removed. The superfluous moisture must not be ejected by pressure, but a wetted camel-hair pencil, the size made in an ordinary quill, being partially dried by drawing through the lips, must be used repeatedly to absorb it, which the pencil will draw by capillary attraction as it is very slowly turned round. When the cover comes in contact with the benzole and gold-size ring, there is no longer any fear of the object being removed, and a slight pressure with the end of the cedar stick of the OF MICROSCOPIC OBJECTS. 137 pencil will render the adhesion complete, and cement the cover closely and firmly to the cell. When dry, an outer ring of asphalt makes the mounting neat and complete. The Fungi have been before mentioned; but it may be here stated that some few of the minute forms are best pre- served in a very shallow cell of liquid. For this purpose creosote-water may be advantageously used, The antenne of insects have been before noticed as being very beautiful when mounted in balsam. This is readily accomplished when they are large; but those of the most minute insects are much more difficult to deal with, and are less liable to injury when put up in fluid. Goadby’s Fluid serves this purpose very well; but, of course, the object must be thoroughly steeped in the liquid before it is mounted, for a longer or shorter time according to the thickness. The eggs of insects afford some worthy objects for the microscope, amongst which may be mentioned those of the common cabbage butterflies (small and great), the meadow- brown, the puss-moth, the tortoiseshell butterfly, the bug, the cow-dung fly, &c. These, however, shrivel up on be- coming dry, and must, therefore, be preserved in some of the fluids before mentioned. To accomplish this no parti- cular directions are required; but the soaking in the liquid about to be employed, &c., must be attended to as with other objects. Glycerine may be advantageously used for the preserva- tion of various insects. These should first be cleaned with alcohol to get rid of all extraneous matter, and then, after soaking in glycerine, be mounted with it like other objects. There is, however, a difficulty in clearing glycerine from the edge of the thin glass cover; but Mr. Whalley told me he met with no annoyance, After laying the cover upon the object with the glycerine, he took away all the superfluous liquid with a small piece of linen, cleaning it at last with a damped piece of the same. The small quantity of water which gets mingled with the glycerine does no injury, and 138 PREPARATION AND MOUNTING the edges can be thus cleaned perfectly enough for any cement to adhere. Mr. Suffolk, at the Quekett Club, said: —When the cell was closed he varnished it with a coating of common liquid-glue, and when this was dry he put it under the tap and thoroughly washed it, in order to remove any glycerine which might remain outside. After carefully drying the slide with blotting-paper, he gave it another coating of the liquid-glue, and when dry repeated the wash- ing process, and after having given it a third coating of liquid-glue in the same manner, he gave it a final coat of gold-size, and he never had any trouble with cells closed in this manner. Mr. Hislop, at the same place, said :—His plan was, to make a good seat for the cover first by a thick ring of gum dammar—allow this to become sticky; next put in the glycerine, lay on the cover, and then carefully wash off all superfluous glycerine. When perfectly well- washed and dried lay on two or three coats of gum dammar to finish it. Some insects, such as May-flies, &c., are, however, often preserved by immersion in a solution of one part of chloride of calcium in three or four parts of water; but this has not been recommended amongst the preservative liquids, as the colour, which is often an attractive quality of this class of objects, is thereby destroyed. We have now noticed the treatment which must be ap- plied to those objects which are to be preserved in liquids and cells. We may here state that all slides of this kind should be examined at short intervals, as they will be found now and then to require another coating of varnish round the edge of the thin glass cover to prevent all danger of leakage. The use of the air-pump, in the first instance (as before recommended), and this precaution as to the varnish, will render the slides less liable to leakage and air- bubbles, which so very frequently render them almost worthless. OF MICROSCOPIC OBJECTS. 139 CHAPTER VI. SECTIONS AND HOW TO CUT THEM, WITH SOME REMARKS ON DISSECTION.* Many objects are almost worthless to the microscopist until extraneous matter is removed from them; and this is fre- quently difficult in the extreme to perform satisfactorily. As an instance, certain Foraminifera may be mentioned in which _ the cells are placed one upon another, consequently the ob- ject must be reduced to a certain degree of thinness before a single uniform layer of these cells can be obtained to show something of the internal arrangements. Most animal and vegetable forms require an examination of the separate parts before much can be known about them. The mass must be divided into separate portions, each part intended to be preserved being cleaned from the useless matter with which it is surrounded. It will frequently be found necessary to make thin sections, which from a very tender substance is no easy matter; and much patience will be necessary to attain anything like proficiency. This making of sections was not until very recently undertaken by many except those belonging to the medical profession, but I do not see why this should be so, as much may be accomplished by a persevering and interested mind where there is time for entering into the subject. I will therefore make an attempt to give some instructions on this subject also. We will first consider the cutting of sections from hard substances, in which the ordinary knife, chisel, * As some knowledge of dissection is necessary to success in injec- tion, additional matter on this subject will be found in Chapter VII. 140 PREPARATION AND MOUNTING &c., are of no avail. Most of these require no particular care in mounting, but are placed in balsam like the other objects noticed in Chapter IV.: where, however, any special treatment is necessary it will be commented upon as we proceed. Suetis, &c.—It is seldom, if ever, necessary to possess apparatus for this process except a small thin saw made with a steel blade, for which a piece of watch-spring serves very well; a fine stone such as is used for sharpening pen- knives; and two smooth leather strops, one of which is to be used with putty-powder to polish the section after grind- ing, and the other dry, to give the final surface. It is, how- ever, very convenient to have three or four files of different degrees of fineness. A very useful implement in this process is, the Corundum file or rubber, sold by most dealers in watchmakers’ tools. It may be procured of almost any size or grain, either circular or flat, and will cut almost any- thing. They possess the very great advantage of not carry- ing much, if any, impurity into the texture of the object upon which they are used. The shell, if very thick, may be divided by using the watch-spring saw; and this section may then with ordinary care be rubbed down with water on the stone until one side of it is perfectly flat. When this is accomplished it must be rubbed with putty-powder upon the strop, and finally upon the other strop without the powder. This surface will then be finished, and must be firmly united to the slide in the position it is intended to occupy. To do this a small quantity of Canada balsam may be dropped upon the middle of the slide and heated over the lamp until on cooling it becomes hard; but this must be stopped before it is rendered brittle. Upon this the polished surface must be laid, and sufficient heat applied to allow the object to fall closely upon the slide, when slight pressure may be used to force aside all bubbles, &e. On cooling, the adherence will be complete enough to allow the same grinding and polishing upon the upper surface which the lower received. Whilst undergoing this, the section OF MICROSCOPIC OBJECTS. 141 must be examined from time to time to ascertain whether the necessary degree of thinness has been reached. When this is the case the section should be washed thoroughly and dried. It must then be covered, which is best done by using ordinary Canada balsam, as recommended in Chapter [V.; or, if the section is to be mounted dry, it must be freed from balsam by washing, or soaking if necessary, in turpentine or other solvents. Sections of some exquisitely beautiful objects are cut with much less trouble than the above. The Orbitolite, for instance, may be prepared in this manner. Take the object and by pressure with the finger rub the side upon a flat and smooth sharpening stone with water until the portion is reached which it is wished to show. The strength of the object will easily allow this to be accomplished with ordinary care. This side may then be attached to the glass slide _ with heated balsam, as above described, and the object may then be gently rubbed down to the degree of thinness re- quired to show it to the best advantage. After removing all disengaged matter from the object by washing and thoroughly drying, it may be mounted in balsam in the usual manner, when it is equally beautiful as a transparent or opaque object. From this it will be seen that in many instances where a smooth stone is found sufficient for the work (which is often the case when the section is mounted in balsam) the final process of polishing advised above may be dispensed with, as in the Orbitolite, Nummulite, &., &c. It is quite necessary that the stones on which the objects are rubbed be perfectly flat, otherwise one side must be acted upon before the other, and it will be found impos- sible to attain anything like uniformity. Where it is not practicable to cut a section, and the object is very thick, a coarse stone may be first used to reduce it and the smoother afterwards. The consideration of the cutting of sections from shells would scarcely bedeemed complete without some mention of what Dr. Carpenter terms the decalcifying process. Muriatic 142 PREPARATION AND MOUNTING acid is diluted with twenty times its volume of water, and in this the shell is immersed. After a period, differing ac- cording to the thickness of the shell, the carbonate of lime will be dissolved away, and a peculiar membrane left, show- ing the structure of the shell very perfectly. This may be mounted dry, in balsam, or sometimes in liquid, according to the appearance of the object; but no rule can be given. The discretion of the student, however, will enable him to choose the most suitable method. From some shells it is easy to divide thin plates, or laming, which require nothing but mounting in Canada balsam to show the texture very well. In working, however, with those which are pearly, it will be found that expe- rience and patience are needed, as they are very brittle and peculiarly hard; but a little practice will overcome these difficulties. Amongst the Hchinodermata, which include the star- fishes, sea-hedgehogs, &c., there are many whose outer sur- face is covered with spines, or thin projections. Some of these are sharp and thorn-like, others blunt, longer or shorter, and, indeed, of endless variety. In many of these, ~ when a section is made, rings are seen which have a common centre, with radiating supports, resembling sections of some of the woods. These are very beautiful objects, and methods of procuring them may now be considered. It is the best to cut as thin a section as can safely be got with the watch- spring saw first, when the smooth sharpening stone may be used to polish one side, which is easily accomplished with water only. When this is effected, it must be washed clean, and thoroughly dried, and then may be united to the slide in the same manner as before recommended for the Orbito- lite, &c. If it is ever necessary to displace it on account of inequalities, bubbles, or other remediable fault, this may be done by warming the slide; though too much heat must be avoided, otherwise fresh bubbles will certainly be pro- - duced. The covering with thin glass, balsam, &., will present no difficulty to the student; but he must remember OF MICROSCOPIC OBJECTS. 143. that the transparency is somewhat increased by this last operation. Corals are often treated in this way, in order to reveal their structure. Except, however, the student has had much practice, he will often find this a most difficult task, as many of them are exceedingly brittle and hard. THe wil! find the method before described equally applicable here, and should take both horizontal and vertical sections. Coat.—This substance is one of the most interesting objects to the microscopist. It is, of course, of vegetable origin; and though it is in many cases in such minute separate portions as to have lost all appearance of vegeta- tion, yet it is very frequently met with in masses, bearing the form, even to the minute markings, of wood, in various directions. To see this and prepare it for microscopic re- search, a suitable piece of coal must be obtained; but in every case the cutting and preparation of these sections require great care and skill. Sometimes the coal is first made smooth on one side, fastened to the glass, reduced to the requisite degree of thinness, and finished in the method before described. This mode of treating it is sometimes, however, very tantalizing, as, at the last moment, when the section is about thin enough, it often breaks up, and so renders the trouble bestowed upon it fruitless. The dark colour and opacity of coal render an extraordinary thinness necessary, and so increase the liability to this accident. Mr. Slade recommends that the piece of coal, having been smoothed on one side, be cemented on that side to a glass slip by marine-glue of the best quality, quite free from undissolved or foreign matter. Great care must be taken to press out all air-bubbles, the coal breaking up at such places as it gets thin, a hole resulting. It may then be reduced in the usual way, and when thin enough mounted in Canada balsam and covered by thin glass. Perhaps the best method which can be pursued is that recommended in the Micrographic Dictionary, which is as follows :—% The coal is macerated for absut a week in a 144 PREPARATION AND MOUNTING solution of carbonate of potash; at the end of that time it is possible to cut tolerably thin slices with a razor. These slices are then placed in a watch-glass with strong nitric acid, covered, and gently heated; they soon turn brownish, then yellow, when the process must be arrested by dropping the whole into a saucer of cold water, else the coal would be dissolved. The slices thus treated appear of a darkish amber colour, very transparent, and exhibit the structure, when existing, most clearly. We have obtained longitudinal and transverse sections of coniferous wood from various coals in this way. The specimens are best preserved in glycerine in cells; we find that spirit renders them opaque, and even Canada balsam has the same defect. Schultze states that he has brought out the cellulose reaction with iodine in coal treated with nitric acid and chlorate of potash.” Now and then in coal we meet with a half- formed carbon-looking substance which is no more difficnlt to work with than ordinary charcoal. From this it is an easy thing to procure interesting slides. Cannel-coal is so close and firm in its structure as to be much used instead of jet in the manufacture of ornaments: it takes a beautiful polish, and consequently presents the student with none but ordinary difficulties in getting sections of it. Its formation is somewhat different from that of coal, sometimes showing the transition very clearly. Fossil Wood.—This is very often brittle and requires great care in cutting. There are, however, different kinds of fossil wood, but to obtain anything like certainty and perform much work a lathe is necessary. I know of no method better than that given by Mr. Butterworth, and shall therefore make use of his words. First, I will begin with the cutting. To the framework of an ordinary foot- lathe I attach an upright spindle (see engraving). On this upright spindle I drive by a band passing over “ carry- pulleys” from the wheel below. On the top of this spindle I fix my cutting-disc, which is made from a very thin piece of sheet iron, and is about six inches in diameter. —— ———— OF MICROSCOPIC OBJECTS. 145 The edge of this saw I charge with diamond-powder. To the edge of the saw I hold my specimen, and as it cuts I lubricate the edge with a small brush dipped in turpentine. With this method I have cut sections of fossil wood so thin that all its structure has been well defined and required nothing but mounting in balsam; this has been silicated fossil wood. In cutting calcareous fossil wood, I have to cut the sections thicker and grind them down. My grinding apparatus is composed of leaden laps, which I make to revolve in a horizontal position on the same upright spindle L 146 PREPARATION AND MOUNTING on which I fix my cutting saw. I use two laps, one for rough grinding, the other for smoothing. I use No, 1. emery and a little water with the first, and flour of emery with plenty of water on the second. In preparing a speci- men, I first grind a smooth surface on one side, and then fix it to a plate of glass, of such a size as will suit my specimen, with Canada balsam. I then reduce it in thickness on the rough lap till I begin to see the light through it. Then I begin with the smoothing lap, and reduce it with flour of emery until every part of its structure is distinct. If I choose to polish the specimen I do so on a lap made of plush cloth or cotton vevet and putty-powder. I then float them off the slide on which they have been ground, and fix them on another with Canada balsam. I prefer, where it is practicable, to mount them in balsam under a thin cover in the usual way, as I am. satisfied that the structure is better brought out. In flint there are often found remains of sponges, shells, Diatomaceze, &c.; but to show these well, sections must be cut and polished by the lathe and wheel of the lapidary, which the microscopic student seldom possesses. Thin chip- pings may, however, be made, which when steeped in turpen- tine and mounted in balsam, will frequently show these remains very well. Teeth are very interesting objects to all microscopists, more especially to those who give much study to them; as the class of animal may very frequently be known from one solitary remaining tooth. To examine them thoroughly, it is necessary to cut sections of them; but this is rather diffi- cult to perform well, and needs some experience. Some instructions, however, will at least lessen these difficulties, and we will now endeavour to give them. Sections of teeth and bone may be successfully made by rubbing slices cut with a saw between two plates of ground glass, with water and a little powdered pumice-stone, the old and partially worn glass being kept for the final polishing of the sections. OF MICROSCOPIC OBJECTS. 147 It is generally thought that Canada balsam injures the finer markings of these sections, consequently, they are almost invariably mounted dry. A thin piece is first cut from the tooth with the saw of watch-spring before mentioned, if possible; but should the substance be too hard for this, the wheel and lathe must be used with diamond dust. If this cannot be procured, there is no alternative but to rub down the whole substance as thin as practicable on some coarse stone or file, or best of all the corundum rubber. The surface will then be rough; but this may be much reduced by rubbing upon a flat sharp- ening stone with the finger, or a small piece of gutta-percha upon the object to keep it in contact. The scratches may ‘be much lessened by this, but not so thoroughly removed as microscopic examination requires in dry sections. It must, therefore, be polished with the putty-powder and dry strop, as recommended in the working of shell-sections. The other side of the section of the tooth may then be rubbed down to the vequisite thinness, and polished in the same manner, when the dust and other impurities must be re- moved by washing, after which the section must be carefully dried and mounted. Sometimes it may be deemed desirable to make a preparation of the teeth in situ; for this purpose © take the lower jaw of some animal like the rat, weasel, or guinea-pig, and soak it in absolute alcohol first, let that evapo- rate out, then soak in the solution of balsam and benzole ; when that has evaporated to hardness, grind down the jaw as a section, the teeth are fixed in by the balsam. Some of these sections are equally interesting as opaque or trans- parent objects. The dentine of the teeth may be decalcified by immersion of the section in dilute muriatic acid; after drying and mount- ing in Canada balsam it presents a new and interesting appearance, showing the enamel fibres very beautifully when magnified about three hundred diameters. A friend tells me that after submersion of the whole tooth in the acid he has been able to cut sections with a razor. i 2 148 PREPARATION AND MOUNTING Sections oF Bont.—With the aid of the microscope few fragmentary remains have proved so useful to the geologist and students of the fossil kingdom as these. From a single specimen many of our naturalists can tell with certainty to what class of animal it has once belonged. To arrive at this point of knowledge much study is necessary, and sections of various kinds should be cut in such a manner as will best exhibit the peculiarities of formation. The methods of accomplishing this will now be considered. It may, how- ever, be first mentioned that the chippings of some bones will be found useful now and then, as before stated with flint, though this is by no means a satisfactory way of pro- ceeding. Sometimes the bones may be procured naturally so thin that they may be examined without any cutting; and only require mounting dry, or in fluid, as may be found the best. When commencing operations we must provide the same apparatus as is needed in cutting sections of teeth, before described. A fine saw, like those used for cutting brass, &c., two or three flat files of different degrees of coarseness ; two flat “sharpening” stones; and a leather strop with putty-powder for polishing. As thin a section as possible © should first be cut from the part required by the aid of the fine saw; and it is better when in this state to soak it for some short time in camphine, ether, or some other spirit to free it from all grease. With the aid of a file we may now reduce it almost to the necessary degree of thinness, and proceed as before recommended with teeth. The “ sharpen- ing ” stone will remove all scratches and marks sufficiently to allow it to be examined with the microscope to see if it is ground thin enough; and if it is to be mounted dry we must polish it with putty-powder and water upon the strop to as high a degree as possible, and having washed all remains of polishing powder, &c., from the section we must place it upon the slide and finish it as described in Chapter III. But where these sections are required for mounting in balsam a less amount of polish is necessary; thus.rendering the whole process much more readily completed. OF MICROSCOPIC OBJECTS. 149 If the bone is not sufficiently hard in its nature to bear the above method of handling whilst grinding and polishing —as some are far more brittle than others—as thin a section as possible must first be cut with the saw, and one surface eround and polished. The piece must then be dried and united to the glass by heated balsam in the same manner as shells, &c. After which the superabundance of balsam must be removed from the glass; then rub downuponthe stone and strop as before. Great care must be taken that the canals be not filled during the process with the dust of the bone, or of the polishing material. Dr. Beale, in the journal of _ the Q. M. C. takes occasion to say “that he cannot admit that the best way of preparing such sections is by grinding down, since it is too liable to fill the canals with débris.” He recommends that a fresh bone be taken—and a small slice cut off by a strong sharp knife. This is then to be im- mersed in carmine dissolved in ammonia—the ammonia being first neutralized by acetic acid. The walls of the vessels which penetrate the lacwne and canaliculi are by this means stained crimson, and thus the true structure of bone is rendered visible. When the polishing is completed the whole slide must be immersed in chloroform, ether, or some other spirit, to release and cleanse the section, when it may be mounted as the one above mentioned. Some have recommended a strong solution of isinglass to affix the half-ground teeth or bones to the glass as causing them to adhere very firmly and requiring no heat, and also being readily detached when finished. The reason why the sections of bone are usually mounted dry is that the lacune, bone cells, and canaliculi (re- sembling minute canals) show their forms, &c., very per- fectly in this state, as they are hollow and contain air, whereas if they become filled with liquid or balsam—which does sometimes occur—they become almost indistinguishable. There are some dark specimens, however, where the cells are already filled with other matter, and it is well to mount these with balsam and so gain a greater degree of transparency. 150 PREPARATION AND MOUNTING To obtain a true knowledge of the structure of bone, sections must be cut as in wood, both transversely and longitudinally ; but with fossil bones, without the lapidary’s wheel, it is a laborious task, and indeed can seldom be properly accomplished. In this place, also, it may be mentioned that by submitting bone to the action of muriatic acid diluted ten or fifteen times with water, the lime, &, is dissolved away and the cartilage is left, which may be cut into sections: in caustic potash the animal matter is got rid of. Both of these preparations may be mounted in fluid. The method of cutting thin sections of bone may be also employed with the stones of fruit, vegetable ivory, and such like substances; many of which show a most interesting arrangement of cells, especially when the sections are trans- verse. Most of these objects present a different appearance when mounted dry to that which they bear when i balsam, owing to the cells becoming filled; and to arrive at a true knowledge of them we must have a specimen mounted in both ways. Some will perhaps remark that most of the directions for section cutting are given to those who are totally without artificial power, and must rely upon their own manual exer- tions. I reply that these hints are mostly given to such ; but Mr. Butterworth’s directions to use the lathe are so ample, that a repetition of them at the mention of each class of “ sectional” substance would be mere tautology. To those who study polarized light, few objects are more beautiful than sections of the different kinds of horn. We will briefly inquire into the best method of cutting these. There are three kinds of horn, the first of which is hard, as the stag’s, and must be cut in the same manner as bone. The second is somewhat softer, as the cow’s. The third is another and still softer formation, as the “horn” (as it is termed) of the rhinoceros. In cutting sections of the two last we should succeed best by using the machine invented for these purposes,. which I shall shortly describe when the OF MICROSCOPIC OBJECTS. 151 method of cutting wood is considered. To aid us in this - when the horn is hard it must be boiled for a short time in water, after which the cutting will be more easily affected. The sections should be both transverse and longitudinal, those of the former often showing cells with beautiful crosses, the colours with the selenite plate being truly splendid. Of this class the rhinoceros horn is one of the best; but the buffalo also affords a very handsome object. The cow’s, and indeed almost every different kind of horn, well deserves the trouble of mounting. Whalebone, when cut transversely, strongly resembles those of the third and softer formation. All these are best seen when mounted in Canada balsam, but care must be taken that they have been thoroughly dried after cutting, and then steeped in turpentine. An interesting object may 2lso be procured from whale- bone by cutting long sections of the hairs of which it is composed. Down the centre of each hair we shall find a line of cells divided from one another very distinctly. And (as recommended in the Micrographie Dictionary) if whalebone be macerated twenty-four hours in a solution of ' caustic potash it will be softened, and by afterwards digest- ing in water, the outer part will be resolved into numerous transparent cells, which will show more plainly the structure of this curious substance. An object which frequently comes to the hand of any man who moves about in the world is a porcupine quill. This is a really valuable object for the microscopist. Trans- verse and longitudinal sections possess their respective beauty; and their appearance varies somewhat as to the distance from the point at which the section is made. Soaking in hot water for ashort time renders it easy enough to cut, and when dry and mounted in balsam the student is well repaid. In a former chapter, hairs were mentioned, their many and interesting forms, and their beauty when used with polarized light. The sections of them, however, are no less a matter of study, as this mode of treatment opens to sight 152 PREPARATION AND MOUNTING the outer “ casing,” and the inner substance somewhat re- sembling the pith of plants. | It would be out of place to enter into the description of the different forms met with; but the ways in which sections are to be procured may be noticed. If transverse sections are required, some place a quantity of hairs betwixt two flat pieces of cork, which by pressure hold them firmly enough together to allow the required portions to be cut with a razor. Others take a bundle of the hairs and dip it into gum or glue, which gives it when dry a solidity equal to wood. Sections of this are then cut with the machine mentioned a little further on, and these may be mounted in balsam. The human hair is easily procured in the desired sections by shaving as closely as possible a second time and cleansing from the lather, &c., by carefully washing. Most hairs, however, should be examined both transversely and longitudinally. It is not difficult to procure the latter, as we may generally split them with the aid of a sharp razor. In a great number of hairs there is a quantity of greasy matter which must be removed by soaking in ether or some other solvent before mounting. We may next consider the best method of procuring sections of wood, which must be cut of such a degree of thinness as to form transparent objects, and so display all the secrets of their structure. There is no monotony in this study, as the forms are so various, and the arrange- ment of the cells and woody fibre so different, that the microscopist may find endless amusement or study in it. From a single section the class of trees to which it has belonged may be known, often even when the wood is fossil. The apparatus best adapted for cutting these sections is made as follows:—A flat piece of hard wood, about six inches long, four wide, and one thick, is chosen, to which another of the same size is firmly fixed, so as to form, in a side view, the letter T. On one end of the upper surface is fastened a brass plate, perfectly flat, in the centre of which a circular opering is cut about half an inch in diameter. OF MICROSCOPIC OBJECTS. 153 Coinciding with this opening is a brass tube, fixed in the under side of the table (if it may be termed so). This tube is so cut at the bottom as to take a fine screw. Another screw is also placed at the same end of the “ table,” which works at right angles to this, so that any substance in the tube may be wedged firmly by working this last screw. To use this instrument, the piece of wood or other object of which a section is required must be placed in the tube, when, by turning the screw underneath, the wood is raised above the brass plate more or less as wished, and by using the screw at the end, itis held firmly in the same position. With a flat chisel the portion of the object which projects above the surface of the brass plate may now be cut off, and by means of the bottom screw another portion may be raised and treated in the same manner. As to the thickness of which objects should be cut, no proper directions can be given, as this differs so greatly that nothing but experience can be any guide. The same thickness can be obtained by working the screw underneath in uniform degrees, the head being marked for this purpose; and where the substance to be cut is very much smaller than the hole in the brass plate, it may be wedged with cork. *As this instrument is peculiarly adapted for cutting wood (though used for other substances, as before men- tioned), I shall notice a few particulars concerning this branch of sections. It may here be remarked, that to obtain anything like a true knowledge of the nature of wood, it should be cut and examined in at least two direc- tions, across and along. ‘The piece of wood is often placed in spirits for a day or two, so that all resinous matter may be dissolved out of it; it must then be soaked in water for * M. Mouchet, in order to avoid all danger of ‘‘ beards” in cutting wood sections, procured a knife with a semicircular blade. This was fastened at the end upon a flat plate, in order to revolve, as we may call it, the handle being long enough to give leverage for any required power. The wood supporter being placed in a favourable position, the knife is easily brought round, and the section cus by a circular action. 154 PREPARATION AND MOUNTING the same length of time, so as to soften and render it easy to cut. Sections may then be obtained in the manner just described, but they often curl to such a degree from their previous immersion in water as to render pressure necessary to flatten ‘them until dry. They are often mounted dry, and require no care beyond other objects, as in Chapter III. Some, however, are best mounted in balsam, par- ticularly the long sections when used for the polariscope; these must be soaked in turpentine, and the greatest care taken that all air-bubbles are removed. Others are thought to be most useful when mounted in shallow cells with some of the preservative liquids mentioned in Chapter V.—weak spirit and water, chloride of calcium solution of the strength of one part of the salt to three parts of distilled water, &c. The above “ section-cutter”’ may not be within the reach © of every student, nor is it absolutely necessary ; though where any great nwmber of specimens is required it is very useful, and insures greater uniformity in the thickness. Many employ a razor for the purpose, which must always be kept sharp by frequent stropping. Sections of leaves also may be procured by the same means, though, as before mentioned, they are sometimes divided by stripping the coatings off with the fingers. The cells which come to sight by cutting some of the orchideous plants are most interesting. Tocut — these leaves they may be laid upon a flat piece of cork, thus exposing the razor to no danger of injury by coming in contact with the support. It may be mentioned here that the razor may also be used in cutting sections of the rush, than which a more beautiful object can scarcely be found when viewed transversely, as it shows the stellate arrange- ments of the parenchyma. This should be mounted dry. In the same way sections of the leaf-stalks of ferns may also be cut, some of which, as Dr. Carpenter states, show the curious ducts very beautifully, especially when cut rather obliquely. It has been found a ready method of cutting sections of the rush and such like plants, to suck a solution of gum up — OF MICROSCOPIC OBJECTS. 155 into the pith, and when this is dry thin sections could be cut and the gum washed out again, and these could be mounted in balsam. The plan adopted by most practical histologists for cut- ting sections of soft tissues is as follows :—The tissue to be cut is first hardened by immersion in a chromic acid solu- tion varying in strength from 0°25 to 2 per cent., or by im- mersion in alcohol. The substance to be cut may then be embedded in melted wax and spermaceti, in proportions suitable to the nature of the substance to be cut; when this is cold the section may be cut with a razor ground flat on one side, and may then be floated off in spirits of wine. These sections mount very well in Canada balsam, if after being removed from the spirit they are immersed in oil of cloves till they become clear, then put into turpentine before the balsam. The thinness of the section will depend very much on the dexterity of the operator, but section- cutting instruments for soft tissues can now be obtained at most scientific instrument shops. When sections of softer substances are required, no instru- ment can be compared with “ Valentin’s knife,” which con- ‘sists of two steel blades lying parallel with each other and attached at the lower end. The distance of separation may be regulated at will by a small screw near the handle. When, therefore, a section is wanted, the substance must be cut through, and betwixt the blades a thin strip will be found, which may be made of any thickness, according to the distance of their separation. By loosening the screw the blades may be extended, and the section may be floated out in water if the damp will not injure it. The knife cuts much better if dipped in water or glycerine immediately be- fore use, and also when the substance to be operated upon is wet, or even under water altogether; but care must be taken, after use, to clean the blades thoroughly and oil them before laying by, if the place is at all damp. This instru- ment is most useful in such subjects as anatomical prepara- 156 PREPARATION AND MOUNTING tions where the sections are required to show the position of the different vessels, &c.; but, as before stated, is very valu- able for all soft substances. As an instance of this, it may be mentioned, that it is frequently used in cutting sections of sponges; but as these are often very full of spicula, it is much better to press the sponge flat until dry, and then cut off thin shavings with a very sharp. knife; these shay- ings will expand when placed in water. After this they may be laid betwixt two flat surfaces and dried, when they may be mounted as other dry objects, or, when desirable, in balsam. Valentin’s knife is very much used in taking sections of skin, which are afterwards treated with potash solution, acids, &c., to bring out in the best way the different por- tions. Dr. Lister’s mode, however, of getting these‘is thus given in the Microscopic Journal :—“ But I afterwards found that much better sections could be obtained from dried specimens. A portion of shaved scalp being placed between two thin slips of deal, a piece of string is tied round them so as to exercise a slight degree of compression ; the preparation is now laid aside for twenty-four hours, when it is found to be dried to an almost horny condition. It then adheres firmly by its lower surface to one of the slips, and thus it can be held securely, while extremely thin and equable sections are cut with great facility in any plane that may be desired. These sections, when moistened with a drop of water and treated with acetic acid, are as well suited for the investigation of the muscular tissue as if they had not been dried.” There are many who almost confine their attention to polarized light and its beautiful effects. Such would not deem these efforts to aid the student in cutting sections complete, without some notice of those which are taken from various crystals, in order to display that curious and beautiful phenomenon, the rings with a cross, The arrange- ment of these is somewhat changed by the crystal which affords the section; but nitrate of potash gives two sets of OF MICROSCOPIC OBJECTS. PAZ rings with a cross, the long line of which passes through both, the short line dividing it in the middle. The process of cutting these sections is rather difficult, but a little care and perseverance will conquer all this. The following is extracted from the Hncyclopedia Metropolitana : “ Nitre crystallizes in long six-sided prisms whose section, perpendicular to their sides, is the regular hexagon. They are generally very much interrupted in their structure; but by turning over a considerable quantity of the ordinary saltpetre* of the shops specimens are readily found which have perfectly transparent portions of some extent. Select- ing one of these, cut it with a knife into a plate above a quarter of an inch thick, directly across the axis of the prism, and then grind it down on a broad wet file till it is reduced to about one quarter or a sixth of an inch thick, smooth the surface on a wet piece of emeried glass, and polish on a piece of silk strained very tight over a strip of plate-glass, and rubbed with a mixture of tallow and colco- thar of vitriol. This operation requires practice. It cannot be effected unless the nitre be applied wet and rubbed till quite dry, increasing the rapidity of the friction as the moisture evaporates. It must be performed in gloves, as the vapour from the fingers, as well as the slightest breath, dims the polished surface effectually. With these precau- tions a perfect vitreous polish is easily obtained. We may here remark, that hardly any two salts can be polished by the same process. Thus, Rochelle salt must be finished wet on the silk, and instantly transferred to soft bibulous linen and rapidly rubbed dry. Experience alone can teach these peculiarities, and it is necessary to resort to contrivances (sometimes very strange ones) for the purpose of obtaining good polished sections of soft crystals, especially of those easily soluble in water. * Sometimes the saltpetre of the shops is nitrate of soda, and as this is slightly deliquescent, it is well to be certain that we have the nitrate of potash, which is free from this defect. 158 PREPARATION AND MOUNTING “The nitre is thus polished on both its surfaces, which should be brought as near as possible to parallelism.” Some sections of the naturally formed crystals also show the “rings” very well,—as Iceland spar, which gives a single ring and cross; but the difficulty of cutting and polishing them is almost too great for the amateur, and must be left to the lapidary. This curious phenomenon, however, may be seen by using a plate of ice smmmavine"s: formed of about one inch in thickness. Before concluding these remarks on sections, I must men- tion a few difficulties which may be met with, and their remedies. The foremost of these is the softness of some objects, which have not resistance enough in themselves to bear cutting even with the sharpest instruments. This may often be removed by soaking in a solution of gum, and then drying, which will render the substance firm enough to be cut, when the sections must be steeped in water, and the gum thus removed. Small seeds, &c., may be placed in wax when warmed, and will be held firmly enough when it is again cold to allow of them being cut into sections.* And, lastly, where a substitute for a microscopist’s hand- vice is required, a cork which fits any tube large enough may be taken and split, the object being then placed between the two parts, and the cork thrast into the tube, a sufficient degree of firmness will be obtained to resist any necessary cutting. * Mr. T. K. Parker informs me that he uses paraffine as an ‘‘ ob- ject-support ” when sections are required, as follows :—‘‘ The mixture use for embedding objects consists of solid paraffine (ordinary paraf- fine candles will do very well) melted down and mixed with a little paraffine oil, without which the paraffine is too hard to be easily cut. The mixture when cold is cut into suitable pieces, a hole is scooped out in the centre, the object to be cut placed in it, and a little of the melted mixture poured round it. Thé sections are cut with an ordi- nary razor, which, as well as the object, must be continually wetted with spirit. This method is useful for all objects which are either too small for the hand or too soft or brittle to be cut in the ordinary way. is especially useful for histological specimens, leaves, embryos, re, ” OF MICROSCOPIC OBJECTS. 159 The ether process of drying tissues has been described by Mr. Suffolk, at a meeting of the Quekett Microscopical Club, and was communicated by Mr. Crooker to him—it is as follows :—A wide-mouthed well-stoppered bottle must be selected. At the bottom is placed a slice from the bowl of a tobacco-pipe, forming a support for a Berlin crucible with its cover. A quantity of fused chloride of calcium in frag- ments is placed at the bottom of the bottle, which is nearly filled with pure ether, so that the crucible may be covered. The tissue to be dried is placed in the crucible, and is covered, if necessary to keep it from floating, by a piece of glass. The ether takes water from the tissue, and the chloride again takes it from the ether; so that the section is thus “gradually dried, and with as little shrinking as possible, however delicate it may be. This process is most fitted for the preparation of succulent roots, tubers, or stems, and in- deed is only fit for those tissues which are not injured by immersion in ether, or dissolved by it, such as fat, &c., or colouring matter. Dissection.—As I stated at the commencement of this chapter, no written instructions can enable any student to become an adept in this branch without much experience and no little study. I will, however, describe the necessary apparatus, and afterwards mention the mode of treatment which certain objects require. A different microscope is manufactured for the purpose of dissection, most first-rate makers having their own model. The object-glasses of many of these are simple, and conse- quently not expensive; but one of the great requisites is a stage large enough to hold the trough, in which the opera- tion is often performed. Where this is the case it would scarcely be worth the expense of getting a dissecting micro- scope if the student were pursuing no particular study, but merely used the instrument when an object to be operated upon turned up accidentally. The ordinary form is much improved for this purpose, by having two wooden rests placed at the sides of the microscope, upon which the 160 PREPARATION AND MOUNTING hands may be supported when working upon the stage. They should be weighty enough to be free from danger of moving. These supports will also be found to remedy much of the weariness which inevitably arises from having to sustain the hands as well as work with them. The erector, as I before observed, is necessary to a young student; but with a little practice he may work-very well without it. We will now notice some of the instruments which are most useful in dissection. Two or three different sizes of ordinary scissors should be possessed, but the shapes must be as modified in others for many purposes, as those used by surgeons; a pair with the cutting parts bent in a hori- zontal direction, and another pair slightly curved in a perpendicular; so that parts of the substance operated upon may be reached, which it would be impossible to touch with straight scissors. One point of these is sometimes blunt, and the other acute, being thus made very useful in opening tubular formations. Another form of these is made, where the blades of the scissors are kept open by a spring, the handles being pressed together by the fingers. Where it is desirable, one or both of these handles may be lengthened to any degree by the addition of small pieces of wood. Tue Knives which are most useful are those of the smallest kind which surgeons employ in very delicate opera- tions. These are made about the length of an ordinary. pen-knife, and are fixed in rather long flattish handles; some are curved inwards, like the blade of a scythe, others backwards ; some taper to a point, whilst others again are broad and very much rounded. Complete boxes are now fitted up by the cutlers, of excellent quality and sur- prisingly cheap. Nerpizs.—These are very useful and should be firmly fixed in handles as recommended in Chapter II. It is con- venient to have them of various lengths and thicknesses. If curved by heating and bending to any required shape they may be re-hardened by putting them whilst hot into cold water. Dr. Carpenter also makes edged instruments by + OF MICROSCOPIC OBJECTS. 163 rubbing down needles upon ahone. They are more pleasant to work with when short, as the spring they have whilst long robs them of much of their firmness. Glass points made by drawing out glass rods to a point will be found useful in manipulating with acids. A glass syringe is also useful in many operations, serving not only to cleanse the objects but to add to, or withdraw liquids from, the dissecting-trough. This trough will now be described, as many substances are so changed by becoming dry that it is impossible to dissect them unless they are immersed in water during the operation. If the object be opaque and must be worked by reflected light, a small square trough may be made to the required size, of gutta-percha, which substance will not injure the edge of the knives, &c.; but where transparency is necessary, a piece of thin plate- glass must be taken, and by the aid of marine-glue (as explained in Chapter V.) sides affixed of the required depth. As pins, &c., cannot be used with the glass troughs and the substance must be kept extended, a thin sheet of cork loaded with lead in order to keep it under water may be used; but this, of course, renders the bottom opaque. When working with many thin substances, a plate of glass three or four inches long and two wide will serve every purpose, and be more pleasant to use than the trough. A drop or two of water will be as much liquid as is needed, and this will lie very well upon the flat surface. As these are the principal apparatus and arrangements which are requisite in dissection, the method of proceeding in a few cases may now be noticed. V2EGETABLES.—The dissection of vegetable matter is much less complicated than that of animal; maceration in water being a great assistant, and in many cases removing all necessity for the use of the knife, especially if hot water can be used without injury to the objects, as is the case with many. This maceration may be assisted by needles, and portions of the matter which are not required may be re- moved by them. When, for instance, the spiral vessels M 162 PREPARATION AND MOUNTING which are found in rhubarb are wanted, some parts con- taining these are chosen and left in a small quantity of water until the mass becomes soft, and this is more quickly effected when the water is not changed. ‘The mass must be then placed upon a glass plate when practicable, or in the trough when large, and with the aid of two needles the matter may be removed from the spiral vessels, which are plainly seen with a comparatively low power; and by con- veying these to a clean slip of glass, repeating the process, and at last washing well, good specimens may be procured. Most of these should be mounted in some of the preservative liquids in the manner described in Chapter V. Many, however, may be dried on the slide, immersed in turpentine, and then mounted in balsam ; but liquid is preferable, as it best preserves their natural appearance. Certain kinds of vegetables require a different treatment to separate these spiral vessels. Asparagus is composed of very hard vegetable matter, and some have recommended the stems to be first boiled, which will soften them to such a degree that they may easily be separated. Dilute acids are also occasionally used to effect this; and in some instances to obtain the vaphides caustic potash may be employed; but after any of these agents have been used, the objects must be thoroughly cleansed with water, else the dissecting instruments (and perhaps the cell) will be injured by the action of the re- maining portion of the softening agent. For the dissection of animal tissues it is necessary that the instruments be in the best order as to sharpness, &c. ; and as the rules to be observed must necessarily be some- what alike in many instances, the treatment required by some of the objects most frequently mounted will now be described. We may here remark that cartilage can be best examined by taking sections which will show the ar- rangement of the cells very perfectly. This, however, is plainly seen in the mouse’s ear without any section being ~ necessary. Glycerine, the preservative liquids before men- tioned, and. Canada balsam are all used to mount it OF MICROSCOPIC OBJECTS. 163 but perhaps the first-named may be preferred in many cases. Before treating of separate objects it will be well to notice what M. Brunetti has said on preparing anatomical speci- mens, The process consists of four stages—viz., washing, - divesting of fat, treating with tannin, and desicwation. A stream of pure water is injected through the blood-vessels and secretory ducts of the part to be preserved; the water is afterwards expelled by means of alcohol. ‘To remove the fat, the vessels are in like manner injected with ether, which penetrates the tissues and dissolves all the fatty matters. These operations occupy about two hours, and the object thus prepared may then be kept for a long time in ether, if desired. A solution of tannin is next injected in a similar manner, and the ether washed out by a stream of pure water. The preparation is then placed in a double- bottomed vessel containing boiling water—a sort of bain- marie—in order to displace the fluid previously used by dry heated air. Air compressed in a reservoir to about two atmospheres is forced into the vessels and ducts through heated tubes containing chloride of calcium: all moisture is thus expelled and the process is completed. The preparation thus treated is light, and retains its volume, its normal consistence, and all its histological elements. Musctz.—This is what is commonly called the flesh of animals. If a piece be laid upon the slide under the microscope, bundles of fibres will be perceived, which with needles and a little patience may be separated into portions, some of these being striated, or marked with alternate spaces of dark and light. Some of the non-striated or smooth class of muscle, such as is found in intestines, may be prepared for the microscope by immersing for a day or two in nitric acid diluted with three or four parts of water, and then separating with needles and mounting as soon as possible. Sometimes boiling is resorted to to facilitate the separa- tion, and occasions little or no alteration in the material. mM 2 164 FREPARATION AND MOUNTING Specimens are often taken from the frog and the pig, as being amongst the best, Goadby’s Solution being generally used in mounting them. The muscles of insects also show the striz very perfectly. NERVE-TIssvuE.—This is seldom mounted; as Dr. Carpenter observes, “no method of preserving the nerve-tissue has been devised which makes it worth while to mount pre- parations for the sake of displaying its minute characters,” but we will mention a few particulars to be observed in its treatment. The nerve should be taken from the animal as soon as possible after death, and laid upon a glass slide, with a drop or two of serum if possible. The needles may be used to clean it, but extreme delicacy is necessary. It will be found that the nerve is tubular and filled with a substance which is readily ejected by very slight pressure. When the nerve is submitted to the action of acetic acid, the outer covering, which is very thin, is considerably con- tracted, whilst the inner tube is left projecting; and thus is most distinctly shown the nature of the arrangement. Dr. Lockhart Clarke, who has made great researches into the structure of the spinal cord, gives a part of his experience as follows:—He takes a perfectly fresh spinal cord and submits it to the action of strong spirits of wine. This gives the substance such a degree of hardness that thin sections may be readily cut from it, which should be placed upon a glass in a liquid consisting of three parts of spirit and one of acetic acid, which renders them very distinct. M. Grandry has treated merve-tissue thus :—Taking portions of nervous tissue obtained from the frog and rabbit, he placed them in a one-fourth per cent. solution of nitrate of silver in pure water, macerating them for five days in the dark, and then exposing them for three days to bright light. If the surface of the cord thus treated be carefully teazed out with needles, the axis-cylinders are found to exhibit a very regular and sharply defined transverse striation—clear, unstained striz alternating with deeply tinted ones. Dr. Bastian recommends us to mount delicate specimens of OF MICROSCOPIC OBJECTS. 165 nerve-tissue in a mixture of glycerine and carbolic acid in the proportion of fifteen of the first to one of the second. To mount these sections, they must now be steeped in pure spirit for two hours, and afterwards in oil of turpentine, and lastly must be mounted in Canada balsam. Dr. Lionel Beale recommends the use of chloride of gold for colouring nerve fibres. A solution containing from 2 to 1 per cent. in distilled water should be made. The tissue having been soaked in it until it becomes straw-coloured, is to be washed, and then placed in very dilute acetic acid containing one per cent. or less. The nerves exhibit a blue or violet tinge on exposure to light for a few hours. He speaks also of solution of osmic acid for the same purpose, 1 part to 100 of water, but not with much approval. The aniline colours, such as magenta and solferino, may, accord- ing to the same authority, be also employed for most tissues. They are not very soluble in water, but are readily dissolved by alcohol. A grain of the colour, 10 to 15 minims of alcohol, and an ounce of distilled water, make a dark red or blue (purple) solution which colours tissues very readily. For these and many other useful formule for the same pur- pose, the reader may consult “ Beale on the Microscope.” Dr. Klein, in No. 40 of the Monthly Microscopical Journal, in order to demonstrate the nerves of the cornea, takes that of the rabbit or guinea-pig, a quarter or half an hour after death, and places it in a half per cent. solution of chloride of gold, for from one and a half to two hours—that of the guinea-pig for an hour to an hour and a quarter. After that, the cornea is washed in distilled water, and exposed to the light in distilled water for from 24 to 36 hours (the water being changed twice, or oftener). After this time has elapsed, the cornea is transferred into a mixture of one part pure glycerine and two of distilled water, where it remains for two or three days. Up to this time the cornea has not assumed a darker colour than ash-grey, perhaps having a violet tint; at all events the whole of the cornea is trans- parent. It is then brushed over on its anterior surface 166 PREPARATION AND MOUNTING under water with a fine camel-hair brush very gently, so as to remove the precipitates of the gold salt. Sections of a cornea so prepared may be made on the finger by a sharp razor, and must be examined and kept in glycerine. Liver, Kinney, Spiteen, Lune, &c.—Some parts which are too soft to be cut into sections in their ordinary state, are usually hardened by being steeped in a solution of chromic acid, about two grains to an ounce of water. This will take some weeks according to the substance, and the solution should be changed now and then. Dr. Bastian, for mounting, uses Canada balsam partially dried to dispel the turpentine, and then diluted to necessary consistence with benzole. The section being cut from the hardened organ is washed in spirits of wine for some minutes, then a drop of liquid carbolic acid is placed on the slide where the speci- men is to be mounted. Take the specimen and let its edge touch’ a piece of blotting-paper, and place it upon the carbolic acid, which will render a thin section transparent in about a minute. Remove the superfluous acid with blotting-paper, when two or three drops of chloroform must be poured upon the section and remain one minute. Drain off and place upon the object the solution of Canada balsam in benzole, and apply the thin glass cover. Or place the object in ordinary spirits of wine for about a minute to wash it, then remove into absolute alcohol for five minutes. Lay it upon the slide and drain, cover with one or two drops of benzole for about a minute, tilt to drain off, and proceed as above. Both these methods are good, but the first does not always answer for sections of liver, as they generally are acted upon by carbolic acid; but few other tissues are thus affected. Tinted specimens seem equally safe when mounted in this way. Sections oF Brain anp Sprnan Corp.—Dr. Bastian gives his experience of these tissues as follows :—I immerse the section for about ten minutes in absolute alcohol diluted with eight per cent. of water, then place upon the glass OF MICROSCOPIC OBJECTS. 167 slide, and before it becomes dry pour over it two or three drops of pyroacetic acid for about half a minute. Tilt this off and replace by chloroform. Watch the effects, as before, under the microscope, and then cover with the Canada balsam solution and finish. These specimens, however, are not always permanent in their appearance, according to the results of some. Mr. Alfred Sanders gives his experience as differing some- what from this. He says—The brain, or other structure, being, as usual, hardened in chromic acid, the section is put for a short time in spirits of wine, and thence transferred to the creosote, which makes it transparent in a few minutes, when it is placed in Canada balsam. The balsam will mix easily with the creosote, or the solution in benzole may be employed. TRACHE® oF InsEcts, &c.—The nature of these was de- scribed in Chapter IV., but the method of procuring them was not explained, as this clearly belongs to dissection. The larger tubes are readily separated by placing the insect in water, and fixing as firmly as possible, when the body must be opened and the viscera removed. The tracheze may then be cleaned by the aid of a carmel-hair pencil, and floated upon a glass, where they must first be allowed to dry, and then be mounted in balsam. Mr. Quekett gives the following method of removing the trachez from the larva of an insect :—* Make a small opening in its body, and then place it in strong acetic acid. This will soften or decompose all the viscera, and the trachez may then be well washed with the syringe, and removed from the body with the greatest facility, by cutting away the connections of the main tubes with the spiracles by means of fine-pointed scissors. In order to get them upon the slide, it must be put into the fluid, and the trachez floated upon it; after which they may be laid out in their proper position, then dried and mounted in balsam.” If we wish them to bear their natural appearance, they must be mounted in a cell with Goadby’s fluid; but the structure is sometimes well 368 PREPARATION AND MOUNTING shown in specimens mounted dry. As before mentioned, these tracheze terminate on the outside in openings termed spiracles, which are round, oblong, and of various shapes. Over these are generally a quantity of minute hairs, forming a guard against the entrance of dust. The forms of these are seldom alike in two different kinds of insects, so that there is here a wide field for the student. ‘Phe dissection, more- over, is very easy, as they may be cut from the body with a sharp knife or scissors, and mounted in balsam or fluid. Many of the larvze afford good specimens, as do also some of the common Coleopterous insects. Perhaps, no more satisfactory object can be met with to give the student good examples of spiracles than the water-beetle Dytiscus, before mentioned, as affording such perfectly beautiful suckers. They will be found to vary in appearance according to the part of the body from which they are taken; but all are equally interesting. Mr. Lewis G. Mills, LU.B., gives the following account of his extracting the poison glands from a spider :—Having killed a large spider with chloroform, I left it in water for seven or eight days. This treatment usually softens the outer skin of insects and causes the viscera to swell, so as to burst through the outer integument, and it is in this state, perhaps, that the poison glands are most easily dis- covered and traced to their points of attachment. I then drew the mandibles from the body, and, having placed them with a little water on a slide and covered them with a piece’ of thin glass, I found that, upon the application of | pressure, the two glands shot out and protruded from the cases of the mandibles. I tore open one of the mandibles with needles, so as to disturb the gland as little as possible. The gland then appeared as a closed sac, attached by a hollow cord, about the length of the gland itself, to the base of the fang, where also was a large bundle of muscular fibre. Fiso.—The most interesting part of fish to the micro- scopic anatomist is undoubtedly the breathing apparatus. OF MICROSCOPIC OBJECTS. 169 It is not a very difficult matter to open the head and remove the gills, which are very beautiful. Under the outer covers lie a quantity of thin plates or leaves (as of a book) which in different fishes are of various shapes, but are made like net-work by the numerous veins and arteries which convey the blood to be acted upon by the air and gases in the water, as is done in the lungs of a man. ‘These plates are of such numbers that in a good-sized salmon the surface exposed has been estimated at two thousand square inches, ¢.e., about fourteen square feet. The beauty of these is, of course, not perfectly shown until they are injected, which will be noticed elsewhere. Toncuzs, OR Patates, oF Motztuscs.—Of the nature of these, Dr. Carpenter gives the following description :—‘ The organ which is commonly known under this designation is one of a very singular nature; and we should be altogether wrong in conceiving of it as having any likeness to that on which our ordinary ideas of such an organ are founded. For, instead of being a projecting body, lying in the cavity of the’ mouth, it is a tube that passes backwards and down- wards beneath the mouth, its higher end being closed, whilst in front it opens obliquely upon the floor of the mouth, being, as it were, slit up and spread out so as to form a nearly flat surface. Ox the interior of the tube, as well as on the flat expansion of it, we find numerous trans- verse rows of minute teeth, which are set upon flattened plates ; each principal tooth sometimes having a basal plate of its own, whilst in other instances one plate carries several teeth.” These palates, or tongues, differ much amongst the Gasteropods in form and size, some of them being comparatively of an immense length. Many are amongst the most beautiful objects when examined with polarized light. They must, however, be procured by dis- section, which is usually performed as follows :—Tbe animal is placed on the cork in the dissecting-trough before men- tioned, and the head and forepart cut open, spread out, and firmly pinned down. With the aid of fine scissors or knife, 170 PREPARATION AND MOUNTING the tongue must be then detached from its fastenings, and placed in water for a day or two, when all foreign matter may with a little care be removed. In what way it should be — mounted will depend on the purpose for which it is intended. If for examination as an ordinary object, it may be laid upon the slide and allowed to dry, which arrangement will show the teeth very well. If we wish to see it as it is natu- rally, it must be mounted in a cell with Goadby’s fluid; but if it is wanted as a polarizing object, it must be floated upon a slide, allowed to dry thoroughly, and then Canada balsam added in the usual manner. In the stomach, also, of some of these molluses teeth are found, which are very interesting objects to examine, and must be dissected out in the same manner as the tongues. Since writing the above, Dr. Alcock (whose very beau- tifal specimens prove him to be a great authority in this branch) has published some of his experience in the second volume of the third series of “ Memoirs of the Literary and Philosophical Society of Manchester.” By his permission IL make the following extract :— “This closes my present communication on the tongues of mollusca; but as some members may possibly feel inclined to enter upon the inquiry themselves, I think it will not be amiss to add a few remarks on the manner in which they are to be obtained. “ First, as to the kinds best worth the trouble of pre- paration. Whelks, Limpets, and Trochuses should be taken first. Land and fresh-water snails can scarcely be recom- mended, except as a special study,—their tongues being rather more difficult to find, and the teeth so small that they require a high power to show them properly. It would appear, from Spallanzani’s description of the anatomy of the head of the snail, that even he did not make out this part, although, in his curious observations on the reproduc- tion of lost parts, he must have carefully dissected more snails than any other man. OF MICROSCOPIC OBJECTS. 17P “ As to preserving the animals till wanted, they should simply be dropped alive into glycerine or alcohol. Glycerine is perhaps best where only the tongues are wanted ; but it leaves the animals very soft; and as it does not harden their mucus at all, they are very slippery and difficult to work upon when so preserved. “Then as to the apparatus required for dissection. In the first place, all the work is to be done under water, and a common saucer is generally the most convenient vessel to use. No kind of fastening down or pinning out of the animal is needed; and, in fact, it is much better to have it quite free, that you may turn it about any way you wish. The necessary instruments are a needle-point, a pair of fine-pointed scissors, and small forceps; the forceps should have their points slightly turned in towards each other. « A word or two on the lingual apparatus generally, and on its special characters in # few different animals, will con- clude what I have to say. “The mode of using the tongue can be easily seen in any of the common water-snails, when they are crawling on the glass sides of an aquarium; it may then be observed that from between the fleshy lips a thick mass is protruded, with a motion forwards and upwards, and afterwards withdrawn,, these movements being almost continually repeated. The action has the appearance of licking; but when the light falls suitably on the protruded structure, it is seen to be armed with a number of bright points, which are the lingual teeth, so arranged as to give the organ the character and action of a rasp. “Tf you proceed to dissection, and open the head of one of these mollusca (say, for instance, a common limpet), you will find the cavity of the mouth almost filled with the thick fleshy mass, the front of which is protruded in the act of feeding; and on its upper surface, extending along the middle line from back to front, is seen the strong membranous band upon which the teeth are set. The mass itself consists of a cartilaginous frame, surrounded by strong muscles ; and: 172 PREPARATION AND MOUNTING these structures constitute the whole of the active part of the lingual apparatus. “But the peculiarity of the toothed membrane, which makes its name of ribbon so appropriate, is, that there is always a considerable length of it behind the mouth, per- fectly formed, and ready to come forward and supply the place of that at the front, which is continually wearing away by use. “In the limpet this reserve ribbon is of great length, being nearly twice as long as the body, and the whole of it is exposed to view on simply removing the foot of the animal ; nothing, then, can be easier than to extract the tongue of the common limpet. But, unfortunately, what you find in one kind of mollusc is not at all what you find in another. In the Acmzeas, for instance, which are very closely related to the limpets, and have shells which cannot be distinguished, the reserve portion of the ribbon has to be dug out from the substance of the liver, in which it is imbedded, that organ being, as it were, stitched completely through by a long loop ef it. iy 23.62 It might be thought a comfortable reflection that, at all events, one end of the ribbon can always be found in the mouth; but in many cases this is about the worst place to look for it. Perhaps it may appear strange that in some of the smaller species, with a retractile trunk, a beginner may very likely fail altogether in his attempt to find the mouth; if, however, the skin of the back be removed, commencing just behind the tentacles, there will be very little difficulty in making out the trunk, which either contains the whole of the ribbon, as in the whelk, or the front part of it, as in Purpura and Murex, where a free coil is also seen to hang from its hinder extremity. ...... In the periwinkles the same plan of proceeding, by at once opening the back of the animal, is best: and on doing so, the long ribbon, coiled up like a watch-spring, cannot fail to be found. Tn the Trochuses, and indeed in all the Scutibranchiata, one point of the scissors should be introduced into the OF MICROSCOPIC OBJECTS. 173 mouth of the animal, and an incision made directly back- wards in the middle line above to some distance behind the tentacles; the tongue is then immediately brought into view, lying along the floor of the mouth.” Dr, Alcock’s method of dissection will be found to differ in some degree from the general rules before given; and when the tongue is dissected out he washes it for one hour (shaking it now and then) in a weak solution of potash. After cleaning thoroughly in water, it must be mounted by one of the methods before mentioned. Mr. Edwards, of New York, no mean authority, gives his experience as follows:—I use a rather strong solution of caustic potassa, the strength of which I cannot specify as it must differ with the species under manipulation, as some ribbons (or tongues) are injured much sooner than others. Plunge the whole animal in this solution; in the case of very small creatures shell and all. I have found it better to let the animal stand until it dies and begins to decom- pose, when it can readily be removed and falls in pieces. The lingual ribbon is not so easily decomposed. Now place and leave the animal in the potassa solution for some days, or boil at once. Almost everything is now dissolved but the shell, some few fragments, and the desired ribbon. Wash carefully with fresh water, and if it is to be preserved. before mounting, remove to alcohol. ‘To mount it, remove from the spirit and boil a short time in turpentine, when it can be put up in Canada balsam. Mr. May expresses him- self as “standing utterly aghast”? at any man so interfering with nature as to put up these objects in balsam, thus pressing and destroying their true forms. He recommends a cell and a weak form of Goadby’s solution. Amongst insects, especially the grasshopper tribe, are found many which possess a gizzard, armed with strong teeth, somewhat similar to those of the molluscs. It requires great nicety of manipulation to obtain these for the microscope; but Mr. L. G. Mills, before quoted, gives the following instructions :—Kill the insect with chloroform and 174 PREPARATION AND MOUNTING place it in a vessel of water. Hold it down firmly with a pair of tweezers, and with the back of a dissecting knife ‘draw the head steadily from the body. The head brings with it the stomach, gizzard, and chief portion of the digestive tubes. Place all these under a dissecting microscope, when the gizzard, being just below the stomach and darker in- colour, is easily distinguished, and may_be separated by two cuts with the knife. It then forms a short tube, the teeth being inside. The opening-out of this tube, especially if. it be small, requires delicate handling: if the point of a fine knife can be fairly inserted, then one firm cut downward upon the glass will lay open the gizzard. Here great care is needed; and sometimes it is well to put a fine needle up the tube, and cut down upon the needle. Among the small weevils the membrane is delicate, so that great care is necessary. We have now considered most of those objects which require any peculiar treatment in section-cutting, &.; but in no branch of microscopic manipulation is experience more necessary than in this. OF MICROSCOPIC OBJECTS. 175 CHAPTER VII. INJECTION. 1, Insection is the filling of the arteries, veins, or other vessels of animals with some coloured substance, in order that their natural arrangement may be made visible. This is, of course, a delicate operation, and needs special appa- ratus, which I will now attempt to describe. 2. Syringe-—This is usually made to contain about two ounces. On each side of the part next to the handle is a ring, so that a finger may be thrust through it, and the thumb may work the piston as in an ordinary syringe. The plug of the piston must be packed with soft leather well oiled or greased, in order to free it from all danger of any liquid penetrating it, and fit so closely as to be perfectly air-tight; and if, when it has been used awhile, it is found that some of the liquid escapes past the plug into the back part of the body, it must be repacked, which operation will be best understood by examining the part. These syringes are made of various sizes, but in ordinary operations the above will be all that is needed. The nozzle is about an inch long, and polished so aceurately that there is no escape when the pipes are tightly placed upon it dry. 3. The pipes are usually about an inch long, to their ends are affixed thicker tubes so as to fit the nozzle, as before mentioned, whilst a short arm projects from each side of these, so that the silk or thread which is used to tie the artery upon the thin pipe, may be carried round these arms, and all danger of slipping off prevented. The pipes are made of different sizes, from that which will admit only of a very fine needle (and this will need now and then to be cleaned, or to be freed from any chance obstruction), to 176 PREPARATION AND MOUNTING that which will take a large pin. These sizes must always be at hand, as the vessels of some subjects are exceedingly minute. A, Stopcock.—This is a short pipe like a small straight tap, which fits accurately upon the end of the syringe like the pipes, and also takes the pipes in the same manner. The use of this is absolutely necessary when the object is so large that one syringe full of liquid will not fillit. If no preventive were used, some part of the liquid would return whilst the syringe was being replenished, but the stopcock is then turned as in an ordinary tap, and all danger of this effectually removed. 5. Curved needles—These are easily made by heating common needles at the end where the eye is situated, and bending them with a small parr of pliers into a segment of a circle half an inch in diameter. . They are, perhaps, more convenient when the bent part is thrown slightly back where it commences. The pointed end is then thrust into a common penholder, and the needle needs no re-tempering, as the work for which it is wanted is simply to convey the thread or silk wnder any artery or vessel where it would be impossible to reach with unassisted fingers. 6. A kind of forceps, commonly known by the name of “bulidog forceps,” will be. constantly required during the process of injecting. These are short, usually very strong, but not heavy, and close very tightly by their own spring, which may be easily overcome and the artery so released by the pressure of the fingers. When any vessel has not been tied by the operator, and he finds the injected fluid escaping, one of these “ bulldogs” may be taken up and allowed to close upon the opening. This will cause very little in- terruption, and the stoppage will be almost as effectual as if it were tied. 7. When the ordinary mode of injection is employed, it is necessary that the preparations be kept warm during the time they are used, otherwise the gelatine or size which they contain becomes stiff, and will not allow of being worked by OF MICROSCOPIC OBJECTS. 177 the syringe. For this purpose we must procure small earthenware or tin pots of the size required, which will differ according to the kind of work to be done; and to each of these a loose lid should be adapted to protect it from dust, &c. These pots must be allowed to stand in a tin bath of water, under which a lamp or gas flame may be placed to keep the temperature sufficiently high to insure the perfect fluidity of the mixture. The tin bath is, perhaps, most convenient when made like a small shallow cistern; but some close it on the top to place the pots upon it, and alter the shape to their own convenience. 8. We will now inquire into some of the materials which are needed in this operation; the first of which is size. This substance is often used in the form of glue, but it must be of the very best and most transparent kind. To make the liquid which is to receive the colours for the usual mode of injecting, take of this glue seven ounces, and pour upon it one quart of clean water; allow this to stand a few hours, and then boil gently until it is thoroughly dissolved, stirring with a wooden or glass rod during the process. Take all impurities from the surface, and strain through flannel or other fine medium. The weather affects this a little as to its stiffness when cold, but this must be counteracted by adding a little more glue if found too liquid. 9. Instead of glue, gelatine is generally used, especially when the work to be accom)lished is of the finer kind. The proportions are very different in this case, one ounce of gelatine to about fourteen ounces of water being sufficient. This, like glue, must be soaked a few hours in a small part of the cold water, the remainder being boiled and added, when it must be stirred until dissolved. A good size may be made by boiling clean strips of parchment for awhile, and then straining the liquid whilst hot through flannel ; but when the injections are to be transparent, it is of the greatest importance that the size be as colourless as pos- sible. For this purpose good gelatine must be employed, N 178 PREPARATION AND MOUNTING as Nelson’s or Cox’s: some persons of experience prefer the latter. 10. Colowrs.—The size-solution above mentioned will need some colouring matter to render it visible when in- jected into the vessels of an animal, and different colours are used when two or more kinds of vessels are so treated, in order that each set may be easily distinguished by sight. The proportion in which these colours are spi to the size-solution may be given as follows :— 11. For BRE 6. show 8 parts of size-solution (by weight) to 1 part of vermilion. Yellow... 6 BS & 1,4, chrome yellow. White 5 Be “ 1 ., flake-white. Blue, ..... 3 e a 1 =, blue-smalt, fine. Black ...12 & m 1,4, ~—lampblack. Whichever of these colours is used must be levigated in a mortar with the addition of a very small quantity of water until every lump of colour or foreign matter is reduced to the finest state possible, otherwise in the process of injecting it will most likely be found that some of the small channels have been closed and the progress of the liquid stopped. When this fineness of particles is attained, warmth sufficient to render the size quite fluid must be used, and the colour added gradually, stirring all the time with arod. It may be here mentioned that where one colour only is required, vermilion is, perhaps, the best; and blue is seldom used for opaque objects, as it reflects very little more light than black. 12. When it is wished to fill the caollarea (the minute vessels connecting the arteries with the veins), the Micro- graphic Dictionary recommends the colouring matter to be made by doyble decomposition. As a professed handbook would be, perhaps, deemed incomplete without some direc- tions as to the mode of getting these colours, I will here OF MICROSCOPIC OBJECTS. 179 use those given in that work. For red, however, vermilion, as above stated, may be used; but it must be carefully examined by reflected light to see whether it be free from all colourless crystals or not. It must first be worked in a mortar, and then the whole thrown into a quantity of water and stirred about; after leaving it not longer than a quarter of a minute, the larger portions will settle to the bottom, and the liquid being poured off will contain the finer powder. This may then be dried slowly, or added to the size whilst wet in the manner before advised. 13. Yellow injection—To prepare this, take— Acetate (sugar) of lead ......... 380 grains. Bichromate of potash ............ 15 BC ee ae taiaaiche mann sicimsaiuecinn cs -e 8 ounces. -Dissolve the lead salt in the warm size, then add the bichro- mate of potash finely powdered. Some of the chromic acid remains free, and is wasted in this solution, so the following is given :— Aectate of lead... .aseeserescen ieee 190 grains. Chromate of potash (neutral) .. Se LOORS iw BIEZON Wed. ates Snipers aba sceanictebnnsabwiare 4, ounces. The first of these has the deepest colour, and is the most generally used. 14, White injection.—This is a carbonate of lead :— Pecobates OF lead | .....0.cc.ceceeeee 190 grains. Carbonate of potash....... “ARE Soe 855 i SE as ead ene Bip eae 4 ounces. Dissolve the acetate of lead in the warm size, and filter through flannel; dissolve the carbonate of potash in the smallest quantity of water, and add ito the size: 143 grains of carbonate of soda may be substituted for the cars bonate of potash. N 2 480 PREPARATION AND MOUNTING 15. For blue injection, which is not, however, much use: with reflected light, as before stated, take— Prussian Wiley... .\0c02 -ksagudeeeans 73 grains, nallie BOI, wea; ockeokaaeeeeeeeeeee j ss PIES WE ccGyeiescuckdncunbaeetbec- bese 4 ounces. The oxalic acid is first finely powdered in a mortar, the Prussian blue and a little water added, and the whole then thoroughly mixed with the size. 16. It may here be repeated, that it is only when the capillaries are to be filled that there is any need to be at the trouble to. prepare the colours by this double decomposition; and, indeed, colours ground so finely may be procured that the above instructions would have been omitted, had it not been supposed that some students might find a double plea- sure in performing as much of the work as possible by their own unaided labours. 17. The process of injection may now be considered; but it is impossible for written instructions to supply the place of experience. I will do my best, however, to set the novice at least in the right way. There are two kinds of injection —one where the object and colours are opaque, and conse- quently fit for examination by reflected light only; the other, where the vessels are filled with transparent colours, and must be viewed by transmitted light. The first of these is most frequently employed, so we will begin with it. In the object which is to be injected, a vessel of the kind which we wish to be filled must be found; an opening must then be made in it to allow one of the small pipes before men- tioned to be thrust some distance within it. When this is accomplished, thread the curved needle with a piece of silk thread; or very fine string, which some operators rub well with beeswax. This thread must not be too thin, else there is danger of cutting the vessel. The cord is then carried ander the inserted pipe, and the vessel bound tightly upon it, the ends being brought up round the transverse arms, OF MICROSCOPIC OBJECTS. 181 and there tied; so that all danger of accidentally with- drawing the pipe is obviated. Care must now be used im closing ali the vessels which communicate with that where the pipe is placed lest the injecting fluid escape; and this must be done by tying them with silk. Should, however, any of these be left open by accident, the bulldog forceps must be used, as before recommended. 18. The part to be injected must now be immersed in warm water, not, however, above 100° Fahrenheit, and left until the whole is thoroughly warmed. Whilst this is being done, the coloured size must be made ready by the pot being. placed in the tin bath of warm water, which must be of sufficient temperature (about 110° Fahrenheit) to keep it perfectly liquid.. For the same purpose, the syringe is often tightly covered with two or three folds of flannel; and; indeed, there is no part of the process which requires more attention. If the substance to be injected is too hot, it is injured; whilst, if any of the articles are too cold, the gela- tine, or size, loses a part of its fluidity, and consequently cannot enter the minute vessels. When all is prepared, the syringe, with the stopcock attached, should be warmed, and then filled and emptied with the injecting fluid two or three times, care being taken that the end of the syringe be kept beneath any bubbles which form upon the surface. The syringe may then be filled, and closely attached to the pipe which is tied in the vessel. With a firm and steady pressure the piston must be forced downwards, when the substance will be perceived to swell, and the colour show itself in places where the covering is thin. When the syringe is almost emptied of its contents, the stopcock must be turned to pre- vent any escape of the injection from the subject. It must then be refilled, as in the first instance, and the process repeated. I say almost emptied, because it is well not to force the piston of the syringe quite to the bottom, lest the small quantity of air which frequently remains be driven into some of the vessels, and the object be injured or quite ruined. As the injection is continued, it will be found that 182 PREPARATION AND MOUNTING the force required grows greater, yet care must be taken not to use too much, or the vessels will burst, and render all the labour fruitless. The movement of the piston must be occasionally so slow as to be almost imperceptible, and for this reason the piston-rod is sometimes marked with lines about one-eighth of an inch apart. 19. Of course, during the whole process the injecting fluid and subject must be kept at a temperature high enough to allow the liquid to flow freely ; and the escape of a little of it need cause no fears to the operator, as it is almost im- possible to fill a subject without some loss. When the injected object has received sufficient fluid, it should have a plump appearance, owing to all the vessels being well filled. The vessel must then be tied up where the pipe was inserted, and the whole left in cold water two or three hours, after which time it may be mounted ; but it may be well to notice a few things which the beginner ought to know before enter- ing into that part of the process; and he may be here informed that it is not necessary to mount the objects immediately, otherwise it would be impossible for one person to make ase of half of any large subject, as it would be in a state of decay long before each part could have been examined and separated. Large pieces should be therefore immersed in equal parts of spirits of wine and water, or glycerine, which some think better still, and thus preserved in bottles until time can be given to a closer examination. 20. In operating upon large subjects, entire animals, &c., the constant pressure required by the piston of the syringe srows wearisome, besides occupying both hands, which is sometimes inconvenient when working without assistance. To obviate this, another way of driving the syringe was published in the Micrographic Dictionary which I will quote here :—‘ We have therefore contrived a very simple piece of apparatus, which any one can prepare for himself, and which effects the object by mechanical means. It con- sists of a rectangular piece of board, two feet long and ten inches wide, to one end of which is fastened an inclined OF MICROSCOPIC OBJECTS. 183 piece of wood (equal in width to the long board, and one foot high). The inclined portion is pierced with three holes, one above the other, into either of which the syringe may be placed—the uppermost being used for the larger, the lowermost for the smaller syringe; and these holes are of such size as freely to admit the syringe covered with flannel, but not to allow the rings to pass through them. The lower part of the syringe is supported upon a semiannular piece of wood, fastened to the upper end of an upright rod, which slides in a hollow cylinder fixed at its base to a small rectangular piece of wood; and by means of a horizontal wooden screw, the rod may be made to support the syringe at any height required. The handle of the syringe is let into 4 groove in a stout wooden rod connected by means of two catgut strings with a smaller rod, to the middle of which is fastened a string playing over a pulley, and at the end of which is a hook for supporting weights, the catgut strings passing through a longitudinal slit in the inclined piece of wood.” When in use the syringe is filled with injecting fluid, and passed through one of the three holes which is most suitable. The object being placed so that the pipe and syringe can be best joined, the rod and strings are ’ set in order, and a weight placed on the hook. The stop- cock must then be opened gradually, when the operator will be able to judge whether the weight is a proper one or not: if the piston is driven with any speed, there is danger of injuring the subject, and less weight may be used; if, however, the piston do not move, more must be added.* 21. Such is the method recommended in the Micro- graphic Dictionary, and perhaps it is as good as any mechanical plan could be; but where the operator is willing to undergo the labour of performing all this with the hand, he has a much better chance of succeeding, because the pressure can be regulated so accurately, and changed so * There is in the Monthly Microscopic Journal, vol. ii., page 48, another ingenious apparatus for injecting purposes. 184 PREPARATION AND MOUNTING quickly when requisite, that no mere machine can compete with it, however well contrived. 22. When the beginner attempts to inject a subject, one of his difficulties is finding the vessel from which to com- mence. Another consists in distinguishing the arteries from the veins; but this is partly removed by making a longitudinal incision in the vessel, and with a blunt thick needle probing a little distance into the tube. The artery will be found thicker in the coating than the vein, and the difference is easily perceived by this mode of testing: the vein is also of a bluer colour than the artery. I say above, a “longitudinal incision ” must be made: the reason for this is, that an artery when cut across contracts considerably, and is lost in the adjoining substance; but where the opening is made longitudinully all danger of this contraction is obviated. 23. The different systems of vessels are often injected with various colours, so that their relative positions, may be shown more clearly. In some specimens, the veins are injected with white, and the arteries with red; in the kidney, the urinary tubes are often filled with white, and the arteries witn red. Then, again, the liver affords tubes. for three or four colours. But no written instructions on this point can benefit the young student, and he must be content for a while to employ himself with single colours until he has gained the mechanical skill and the primary knowledge which are necessary before he can make any advance. 24. We will now consider the best methods of mounting injected objects. They must always be well washed in water after they have been kept in any preservative liquid, using a camel-hair pencil to clean the surface if necessary. Many parts when injected are in masses, such as the lungs, liver, &c., of animals, and consequently sections of these must be cut. For this purpose Valentin’s knife is very convenient, as the thickness can be regulated so easily; but where the injections are opaque, there is no need to have the OF MICROSCOPIC OBJECTS. 185 sections very thin. Some few of this kind undergo compara- tively little change in drying, so that the section may be well washed and floated upon the glass slide in the place desired, where it will dry perfectly and adhere to it. It must then be moistened with turpentine and mounted in Canada balsam like other objects. No great heat should be used with these preparations, as it is very liable to injure them; and some of the colours seem to suffer a slight con- traction when any great degree of warmth is applied. There are many objects, however, which must be seen in the mass to be understood, and, indeed, lose all their form and beauty in drying, such as certain parts of the intestines, &c. These must be mounted in fluid, with the precautions noticed at length in Chapter V., and for this purpose either Goadby’s fluid, the chloride of zinc solution, or spirit diluted with ten parts of distilled water, may be employed. It is a good thing, when practicable, to mount similar objects on two separate slides, using different preservative liquids, and taking the precaution of marking each with the kind of liquid employed. This not only serves as a guide to what is best for certain subjects, but if one is injured, there will probably be a good specimen in the other. 25. It may be here mentioned that many are now mount- ing sections of injected substances with the balsam and chloroform before mentioned, instead of using balsam alone, and consider that the labour is much lessened thereby. 26. A description of that mode of injection which is most generally employed has now been given, but this is not the only method of effecting our object. A most ingenious process was invented by M. Doyers, requiring no artificial warmth, by which many beautiful objects have been pre- pared. Make a solution of bichromate of potash, 524 grains to a pint of water, and throw this into the vessels to be injected; then take 1,000 grains of acetate of lead dissolved in half a pint of water, and force this into the same vessels. A decomposition now takes place in the vessels, and the yellow chromate of lead is formed. In this decomposition, 186 PREPARATION AND MOUNTING however, the acetate of potash also is formed and as this salt has an injurious action upon the cells, Dr. Goadby recommends nitrate of lead to be used, which preserves rather than destroys them. THe also advises the addition -of two ounces of gelatine dissolved in eight ounces of water, to eight ounces of the saturated solution of each salt; but with this addition the hot-water bath would be required to keep the injecting fluid liquid. ; 27. Many of these are best mounted in balsam, in the same manner as those made in the ordinary way; whilst others are best shown when preserved in liquids, for which purpose Goadby’s fluid may be employed. 28. This mode of making injections with chromate of lead is deemed by many the best, especially where one colour only is employed. But it must be allowed that there is a little more danger of failure where two separate fluids are used for the same vessels. 29. We will now consider the best manner of making transparent injections, which, for many purposes, possess an undoubted advantage over opaque ones. But it must be remembered that there are certain subjects to which no transparent injection could be applied, as they are too thick when in their natural state, and cutting would destroy all ‘that beauty which is shown by the different parts in their relative adaptation. For those objects, however, which must be cut into sections to display their wonders, or are naturally thin—such as some of the finer tissues, livers, kidneys, &c..—transparency is a great acquisition, and enables us to understand the arrangement of the vessels more perfectly. Again, another advantage is the simplicity of the process; no hot water is needed with some pre- parations, either for the subject or the injecting fluid, which runs into the minute vessels thoroughly and easily, whilst the cost is small. 30. For this kind of injection no colour is so commonly used as Prussian blue. It is not a good one, as was before stated, for any opaque object, the light reflected from it OF MICROSCOPIC OBJECTS. 187 appearing almost black; yet by transmitted light no colour is more useful, because its distinctness is equally great by artificial light and ordinary daylight. The method of pre- paring this, as given by Dr. Beale, is as follows :— GEEVCOTUIGS .. ven enon scessrsiseaecapacanes baaas?* LrOmnee. Wood naphtha, or pyroacetic acid ...... 1$ drachm. SPM OL WIHE! 2) cc cisdecacsacccnedorsaccas 1 ounce. Ferrocyanide (yellow prnssiate) of potas- POP san dieRavddavscmansies ndsee \iadaes dock . 12 grains. Tincture of sesquichloride of iron......... 1 drachm. DN i Mec clec a cunaiarnnanndascensh choc smasss, v4 OUNeaR Dissolve the ferrocyanide of potassium in one ounce of the water; add the tincture of sesquichloride of iron to another ounce, Mix these solutions gradually together, shaking the bottle well which contains them—it is best to add the iron to the potash solution. When thoroughly mixed, these solutions should produce a dark-blue mixture, perfectly free from any perceptible masses or flocculi. Next mix the naphtha and spirits of wine, and add the glycerine and the remaining two ounces of water. This must now be slowly mixed with the blue liquid, shaking the whole well in a large bottle whilst the two come together. The tincture of sesquichloride of iron is recommended, because it can always be obtained of a uniform strength. 31. Dr. Turnbull used a mixture slightly different from the above, which is made with the sulphate of iron :— Purified sulphate of iron ............08. 10 grains. Ferrocyanide of potassium ...........0.6 32 grains. J EE es 1 ounce. DPE I a. aca cic icasveccecnes 13 drachm. PN eae eo a ciate. ne ncinin de - 1 ounce. IES RE SORE, OY 5 TS ee Oe ee 4 ounces. Dissolve the sulphate of iron in one ounce of the water, 188 PREPARATION AND MOUNTING gradually add the ferrocyanide of potassium dissolved in another ounce, and proceed as before. 32. Dr. Beale also gives us the following carmine injection to be employed in the same way as the blue.* Take— CDATIIIE Ws cows.» diac we se mniaitonin GERRI 5 grains. Glycerine, with 8 or 10 drops of liydro- Ghlorie ead . ce, cv dad ape sntee eae eae > ounce, Glycerine (pure) ss.side icici dence 1 ounce. PCOS) gsssvin aon snoaandacishOanataen uae aoe 2 drachms. WRIT. es n'seeey ncdanans dene be canes ae 6 drachms. Mix the carmine with a few drops of water, and when well incorporated add about five drops of liquor ammoniz. To this dark-red solution about half an ounce of the glycerine is to be added, and the whole well shaken in a bottle. Next, very gradually pour in the acid glycerine, frequently shaking the bottle during admixture. Test the mixture with blue litmus-paper, and if not of a very decidedly acid reaction, a few more drops of acid may be added to the remainder of the glycerine and mixed as before. Lastly, mix the alcohol and water very gradually, shaking the bottle thoroughly after adding each successive portion till the whole is mixed. This fluid may be kept ready prepared, and injections made very rapidly with it. 33. The method of making injections with these colours is the same as with the gelatine mixtures before described, except that no heat is required, and consequently most of the trouble removed. The bottle of the fuid must be well shaken immediately before use; and when the object has been injected, we must allow it to remain in a cool place for a few hours before cutting it. Thin sections of the subject may be cut with Valentin’s knife, as before described, and * When, however, it is desirable to cut very thin sections of the injected subject, the carmine is sometimes added to a solution of fine gelatine—gelatine one part to water eight parts. But the warm water and mode of proceeding which are used with the size solutions before described will be necessary in this case also. OF MICROSCOPIC OBJECTS. 189 are very beautiful transparent objects. Some of the finer tissues, also, are shown much better by this mode of in- jection than by the opaque, and are easily mounted by washing in clean water when first separated, and floating upon a slide, where they must be allowed to dry thoroughly. They may then be immediately mounted in balsam, or kept in the dry state until it is convenient to finish them; but in many cases this keeping, if too much prolonged, will injure the object. If it is desired to transfer the section to another slide, it will be necessary to wet it thoroughly with water by the aid of a camel-hair pencil, and then gently strip it off with the forceps. When it is wished to preserve injected subjects in masses, it must be done by immersion in spirit, and the sections may be cut at leisure. Most of these: transparent objects may be mounted in Canada balsam; but some recommend glycerine or glycerine jelly, as allowing the use of a higher power in their examination, and preserving them in a more natural form. 34. A few subjects may be noticed which are very beauti- ful when injected, and amongst these are the eyes of many animals. They must be injected by the artery in the back part, and when the blue transparent liquid is employed, nothing can exceed the delicate beauty displayed by some of the membrane. It must be dissected with care, but well repays us for the trouble. Water-newts and frogs are not difficult subjects, and in their skin and other parts are many interesting objects. Amongst the commoner animals—rats, rabbits, cats, &c.—almost endless employment may be found, making use either of portions or the whole animal at once. The intestines of many of these are very beautiful. We must divide them with a pair of scissors along the tube, and cleanse them from the contents; the specimen may then be laid upon a slide, and any remaining impurity removed by a camel-hair pencil and water. When dried it may be mounted in balsam, and having been injected with the transparent blue, its minute beauty is shown most per- fectly. In injecting a sheep’s foot, which is a good object, 190 PREPARATION AND MOUNTING the liquid should be forced into it until a slight paring of the hoof shows the colour in the fine channels, there. Perhaps one of the most beautiful and interesting objects is the skin or section of a cat’s tongue. On examination we shall readily learn the reason why we feel such a roughness when we allow the cat to apply her tongue to our hands. In appearance we shall be almost ready to say that there is no little resemblance in the arrangement betwixt this tongue and those of the molluscs already described. The liver of a rabbit or any other animal is an easy and beautiful object for injection. Sections made with Valentin’s knife, and mounted in balsam, are not at all difficult, and worth double the time they occupy. | 35. When the lungs of small animals are injected, the finest fluid must be used, as some of the capillaries are so small that it is not an easy matter to fill them properly. And before entering upon these subjects, a certain pro- ficiency in the mode of using the syringe, &c., should be obtained by practising upon simpler parts. 36. No subjects are more difficult to inject than fish, owing to the extreme softness of their tissues. Mr. Hogg recommends the tail of the fish to be cut off, and the pipe to be put into the divided vessel which lies just beneath the spinal column; by which method beautiful injections may be made. The gills, however, are the most interesting part as microscopic objects. 37. These instructions may seem very imperfect to those who have had much experience in this branch; but they will remember that their own knowledge was not gained from any written descriptions, but was forced upon them by frequent failures, some of which probably were very dis- heartening. As I before stated, it is very difficult (if not impossible) to accomplish much without some knowledge of anatomy. 38. I may here mention that the transparent injections sent over from the Continent are beautifully executed by Hyrtl of Vienna (who states that the injected fluid is com- OF MICROSCOPIC OBJECTS. 191 posed of gelatine and carmine), Dr. Oschatz of Berlin, the Microscopic Institute of Wabern, Schaffer and Co. of Magdeburg, and others. Some of these will bear examining with a high power. A friend informs me that he measured a vessel in a rat’s tongue by Hyrtl, which was 1-7200th of an inch in diameter, and had a clear outline with quarter- inch objective. He has also made many experiments with the same materials, but has as yet failed in producing perfectly distinct outlines, there being a tendency of the colouring matter (magenta, carmine, &c.) to diffuse itself through the coats of the vessels into the surrounding tissues, although he has varied the pressure from half a pound to sixty pounds. He believes the vessels are first washed out ' (injected with warm water and pressure applied), then some fluid introduced, probably solution of tannin, which renders. the arteries impervious to the coloured fluid afterwards in- jected. 39. He finds that after washing out the vessels as above, the injecting fluid is much more easily introduced. He has used a strong solution of gallic acid previously to injecting with the colouring matter (in one experiment only), and the result was satisfactory. He puts the query,—Might not carbolic acid have a similar effect? He has often used it with injections to preserve the specimens, but not in suf- ficient quantity to act in the way indicated above. Since writing the above, Mr. J. G. Dale, F.C.S., and I have made numerous experiments with carmine injection, and have at length been favoured with what we deem success. Some of the vessels in a kitten lately injected do not exceed 1-2000th of an inch in diameter, and present a clear outline with one-fifth objective. There is no extra- vasation, neither does the colouring matter show any grain except when a very high power is employed. The following is our pracess :— 192 PREPARATION AND MOUNTING Take 180 grains best carmine. 3 fluid ounce of ammonia, commercial strength, viz., 0°92, or 15° ammonia meter. 3 or 4 ounces distilled water. Put these into a small flask, and allow them to digest with- out heat from twenty-four to thirty-six hours, or until the carmine is dissolved. Then take a Winchester quart bottle, and with a diamond mark the spot to which sixteen ounces of water extend. The coloured solution must be filtered into the bottle, and to this pure water should be added until the whole is equal to sixteen ounces. Dissolve 600 grains potash alum in ten fluid ounces of water, and add to this, under constant boiling, a solution of carbonate of soda until a slight permanent precipitate is produced, Filter and add water up to sixteen ounces. Boil and add the solution to the cold ammoniacal solution of carmine in the Winchester quart, and shake vigorously for a few minutes. e—_—— A. B, PAGE PAGE ACARIDA ‘ 110 | Bawtsam and Chloroform...... 44 B63 00 Wee to: ey oy ee eee D'| Balsam, Camada@ sis25-t.c0 5 fj Acide CgeOOliG ions casnarcanese 27 | Baryta water 22:.-. 24354 ces 6 ACiG GEN GR YS 555505 5550 caste shs 5 | Bath for injections ............ 177 ACIDS BIDBURIC Bees. 129 Cuticles, siliceous ............ 121 ME OUA, MOTRES. Lote niwcconcs vanes eae 31 GUISE igo eae ne 29 Cells, epithelial ............... 7 - D Cells, how to fillwith balsam 95 : Cells for test objects ......... 32 MR IVOEY \sscncclescantein>-cs OL” | DAMOWAR. «....:..idesedere nee 44 RGells -IOaGHEr .cvecseneese ess 30 | Dammar and benzole ......... 20 ACSUA, FIAPOM (.2355-m008 <6 xn ns ees 56 | Dammar cement ............... 45 Melis, pull-bOR oo...5.. ties sec nes 31 | Deane’s compound ............ 125 Relies, SPREE B.. fs pdedsiccec ser sanchdasear spat someanss if Vi SPUR ics ne narcnicceigne kee 175 - Syringe for balsam ............ 93 | VALENTIN’S knife......... 155, .F Synee, Plass. .....<.scscessnces 162."| Vaamish,, black...... c.cso.2-ee EEE or nin: sas ase gurigeienadae tee 14 | Varnish, caoutchouc ......... 48 Varnish, sealing-wax ......... 48 Vegetable tissues, dissection Aik Of. «.cxassabconan axes eae 162 "TPABE Ms DRAB G es ce «esis daeeeses 42 Tannic acid, or Tannin ...... 6 W. PP ERMBER Go cs daciasso- Seas 9; 11, 17 Teasing by needles ............ 3 | WATE€H-GLASSES ..............1 4] Teeth, sections of ......... 92, 146: | Water-bath, ..........:..s.coseer 22 Teeth, softened ............... 15 Wine-glasses, broken ......... 42 SE GBROR 234.5 ic ak eugene te 17 BISDOMA) ” ...cs..s.nccehteeee 14 RDREOS As os ogg ck aces Seecee ae toes 18 | Whalebone, sections of ...... 151 Thwaite's liquid ............... 1265'| Wiiod, forall ,.......<.s.0cteuee - 144 Tin tubes, collapsible ......... 44 | Wood, sections of............... 152 Tongues of molluscs ......... 169 Tongues or palates of mol- BUBGR: 5 nor02d siete didddaas waeennee 118 Z. Traches of insects ...... 109, 167 Trough, dissecting ............ 162: | Zunc, chloridevof ............... 126 Tubes; dippingy....:.......:.... at | Zoophyteasy. ...d:0:emnseennee 87 Turntable, Mr. Hislop’s...... 33 | Zoophytes, to mount ......... 108: Turntable, Dr. Matthews’... 34 | Zoophytes, to preserve, by Turntable, Mr..Shadbolt’s... 33 Dr. Bird’s method ......... 101 ROBERT HARDWICKE, PRINTER, 192,. PICCADILLY, i? ke i oe a, " ; wt ~~ " Line 5 ia ae vy wy LT wee SMITHSONIAN INSTITUTION LIBRARIES VALU TIM 3 9088 00572 6070 ii [ P é e3>" Te eit &