tftf PREPAEATION AND MOUNTING >F MICEOSCOPIO OBJECTS. THE PREPARATION AND. MOUNTING OF MICROSCOPIC OBJECTS BY THOMAS DAVIES. \v EDITED BY JOHN MATTHEWS, M.D., F.E.M.S. Vice-P resident Quekett Microscopical Club. FOURTEENTH THOUSAND. LONDON: W. H. ALLEN AND CO., 13 WATERLOO PLACE, PALL MALL. S.W. LONDON: PRINTED BY W. H. ALLEN AND CO., 13, WATERLOO PLACE, S.W.- PKEFACE TO THE SECOND EDITION. rPHE 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 Vl PREFACE TO THE SECOND EDITION. his disposal, but also to make such additions as hia 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'etre in fact, may be found in the words of its concluding para- graph, to which the reader is now courteously referred. 4, MYLNE STREET, MYDDELTON SQUARE, B.C. October, 1873. PEEFACE TO THE FIEST EDITION. TN 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 viii 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 beer* the case. WAKRINGTON TUB PREPARATION AND MOUNTING or MICROSCOPIC OBJECTS. CHAPTER I. INTRODUCTION. THIS 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 rj&EPARATION 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. Strieker observes, " that it is to be borne iu mind that it is impossible to say of any fluid that it constitutes an indifferent, i.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, i.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 hj,s 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 Strieker, Frey, Klein, Schultze, Kiihne, Deiters, Leber, and others, many of whose processes he has personally verified, and of whose manuals, especially those of Beale, Strieker, 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 rs 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 ibe 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. 1st. 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. 3rd. 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 nther disturbing or confusing their structural relations. 4th. 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. llth. The moist chamber. In dealing Tvith 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 4 PREPARATION AND MOUNTING shall have been macerated in water, or some other agent, 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 is 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, i.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 cautioned 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 (i.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 usa of the draw-tube is the true remedy for this. OF MICHOSCOPIC OBJECTS. 5 IST DIVISION. Under our first head may be ranged the following : Acetic acid. Liquor potassae and sodas. 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. Iodine. 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 material, 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 potassae and liquor sodas act in much 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 recti6ed spirit of wine, hardens gelatinous and albuminoid ^issues : 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 teamed out by needles. Oxalic acid, in a cold saturated solution (1 acid, 15 water), according to Schultze, " causes connective tissues tc- swell up and become transparent, while those formed of al- buminous substances become hardened and isolated. Ex- 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 (Kuhne). Sulphuric 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 membranes, and thus renders them more visible. Finally, it may be affirmed that there often exists a need of making objects which are too dark more transparently means of a fluid which permeates them un> qually, so that OP 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. SND 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, hsematoxyline, &c., formulae 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 strength 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 S 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, pro to- 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 600 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. OP MICROSCOPIC OBJECTS. 9 BED DIVISION. 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- eired 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 peifect 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. Strieker, 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, &c., aro 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 Kiihne, claims for these solutions an effect ot the most important kind, viz., that of "preserving the finest textnral 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 MICKOSCOPIC OBJECTS. 11 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 Strieker 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 FB^PARATION 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 pemit 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 paraffine 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 secti >ns adhering. The cutting machines are generally graduated {by a screw and index) on the upward movement, so as to enable one to judgfe 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. 1$ 4iH DIVISION. This includes glycerine, liquor potassae and sodse, 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; bub 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 swell up. His practice is first to immerse the specimen in weak glycerine solution, and then gradually to increase th& 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 in 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 oilier medium. 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 textures 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. As in 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 let:d, is a very efficient means of softening horny substances, whale- OE MICROSCOPIC OBJECTS. 15 bone, &c., and rendering them plastic. Very thin lamin of these substances may also be procured by the employment of a well-sharpened scraper, such as that used by cabinet- maters. 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 eulphuric acid or, still better, by liquor potassse, 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, i.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, i.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 PiUSJfAilATION AND MOUNTING Teeth may be treated in the same manner as bone?, except for the examination of the enamel, which is best effected by sections and grinding. For that purpose developing teetb should le chosen, as in them the enamel prisms are most easily isolated. STH 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 th^ action of these, that effective histological research must chiefly depend. Albumen, when pure, is nearly insoluble in water, wholly BO when coagulated by heat. In dilute caustic alkali it dissolves with facility. Solution of nitrate of potash, acetia and 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 fre alkali in very small quantities. It is partly soluble also in very dilute acida. 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 in- soluble in cold water, but easily dissolved by the use of heat. Alcohol, corrosive sublimate in excess, nitrate of mercurv, 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 potassse, 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 in 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. 13 PEEPA RATION AND MOUNTING CTH DIVISIOX. 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. Fluoride 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 hy^ro- chloric acids. The shells of mollusca, and tcstse of echino- dermata, consisting principally of carbonate of lime, are also soluble in the same acids, as well as those of nummulites, forum inifera, &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." TTH 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 to Off MICROSCOPIC OBJECTS. 19 engrave names, numbers, &c., neatly upon slide?, 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 vaptour 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 at once. 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. STH 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 oilsj and are wholly soluble in ether and alcohol. 20 PREPARATION AND MOUNTING Camphor dissolves in only a very small proportion in water, but freely in alcohol, ether, and strong acetic acid. 9iH DIVISION. It is by no means intended to speak here of the general properties and uses of polarized light. But in relation to : ts 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 tli is work, where the preparation of the antennae 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 potassse, 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. 10iH 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. Strieker says " that the tissues become altered by it as they would be were they subjected to the action of weak acids or alkalies," and he describes a rather complicated apparatus for this purpose, of which it is impossible to give 22 PEEl'AEATIOX AX1) 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-hath, which may he 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 crystallization 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 objeot-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. HTH DIVISION. 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 3IICEOSCOPIC OBJECTS. 23 tubes into that which embraces the glass ring and the end of the microscope tube one for the entrance, tbe other for the exit of the gas. This is a simpler and less costly plan than that devised by Strieker. Prey observes that it is most advantageous to use immersion lenses and the moist chamber with the hot stage. 24 PREPABATIOX AND MOUNTING CHAPTER II. 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 bv unpleasant experience that we fully learn what "cleanli- ness is. An object which is to be viewed under the microscope must, of course, be supported in some way this is now usually done bv 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 objei/t 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 visually made of " sheet" glass; but is seldom found in those of the quality know.i 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, he taken to sort them, so that the more pelicate ohjects 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 greasiiiess is very obstinate upon the glass. It is then necessary to use a little liquor potassse 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 camhric 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 to remain. 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, bea^ole, and liquor sodse 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 up " 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 "//y" 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 from a 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 npt 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 he 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 to near to the object-glass that they admit of the very shinnest 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. For 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 4< 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 slides, 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 : Two 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 al ways 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 slides 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 docs not really belong to it. When anv object which it is devsired to mount is of con- siderable thickness, or will not bear pressure, it is evident that 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 couise, 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 hack with black varnish, and placed the object upon the smooth untouched side ; but a solution of the best Egyptian asphalt in benzole of moderate thickness may be painted on v\ith this further advantage, that 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 follow r s : 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 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 surface 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 process. But, of course, the gum must be perfectly dried before the ring is used. For cardboard, gntta-percha has been substituted, but cannot be recommended, as it always becomes 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. 31 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 icory cell. This is a 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 not expensive. 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 nre 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 32 PREPARATION AND MOUNTING of the objects is inconsiderable, especially with some of the Diatomacese, 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 tho refraction or interference to be reduced to the lowest point. A novice would naturally think the appearance of some of 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 slides, 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. 33 the edge is varnished with black or coloured rings, the appearance of finish is perfect. The trouble is ranch 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 contrary 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 34 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, f inch wide, 2 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 i.e. D is moved exactly 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 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. 35 DK. MATTHEWS' TURNTABLE, 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. Tn 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 l,o 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 FKBPAKATION 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. Two 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 arc 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 f 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 ^-in. iron wire is then run through the springs, which forms an axis to work upon, and also keeps them in theii 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 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. 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 42 PREPARATION AND Me UNTING be steeped in any liquid ; and th.e 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 knowin 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 OP MICROSCOPIC OBJECTS. 43 underneath, and worked with a small handle 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 beer* 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, CANADA. BALSAM 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 - r 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 ma;t 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 01' MICROSCOPIC OBJECTS. 4-5 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 GLUE. 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 Y. ; 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. GOLD SIZE. 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 CEMENT. 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 verj readily, and is specially 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. LIQUID GLUE 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. Even 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 Y.) Yet it makes excellent cells. BLACK 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- WATER 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 extra adhesive {rum-water, which is made with the addition of isinglass, thus : Dissolve two drachms of isinglass in four ounces of distilled vinegar; add as much gum 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. 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 parti}- 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. SEALING-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 les& liable. BLACK 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 Tarnishes 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 thr following possesses these qualities in a great degree : India-rubber drachm. Asphaltum 4 oz. Mineral naphtha 10 Dissolve the india-rubber in the naphtha, then add the asphaltum if necessary, heat must be employed. Some scientific friends have complained that they have been unable to dissolve either the india-rubber or the asphaltura in mineral naphtha. The frequency with which I have seen this solution thoroughly accomplished convince* OF MICROSCOPIC OBJECTS. 49 me that one of these things has occurred either the india- rubber or the asphaltum has not been pure, or the naphtha has been wood instead of mineral. In the early photo- graphic days every artist made a form of this varnish to use with glass positives, and I never heard a complaint of difficulty. This is often used by photographers as a black varnish for glass, and never cracks, whilst it is very adhesive. Dr. Carpenter, however, states that his experience has not been favourable to it ; but I have used it in great quantities and have never found it to leave the glass in a single instance when used in the above proportions. The objections to it are, however, I think easily explained, -when it is known that there are many kinds of pitch, &c., from coal, sold by the name of asphaltum, some of which are worthless in making a microscopic cement. When used for this purpose, the asphaltum must be genuine and of the best quality that can be bought. The above mixture serves a double purpose to unite the cell to the' slide, and also as a "finish- ing " varnish. But it is perhaps more convenient to have two bottles of this cement, one of which is thicker than common varnish, to use for uniting the cell, &c. ; the other liquid enough to flow readily, which may be employed as a surface varnish in finishing the slides. The brushes or camel-hair pencils should always be cleaned after use; but with the asphalt varnish above mentioned it is sufficient to wipe off as carefully as possible the superfluous quantity which adheres to the pencil, as, when again used, the varnish will readily soften it ; but, of course, it will be necessary to keep separate brushes for certain purposes. Here it may be observed that every object should be labelled with name and any other descriptive item as soon as mounted. There are many little differences in the methods of doing this. Some write with a diamond upon the slide itself; but this has the disadvantage of being not so easily seen. For this reason a small piece of paper is I 50 PREPARATION AND MOUNTING usually affixed to one end of the slide, on which, is written what is required. These labels may be bought of different colours and designs ; but the most simple are quite as good, and very readily procured. Take a sheet of thin writing paper and brush over one side a strong solution of gum, with the addition of a few drops of glycerine, or grains of moist sugar, as above recommended ; allow this to dry, and then with a common gun-punch stamp out the circles, which may be affixed to the slides by simply damping the gummed surface, taking care to write the required name, &c., upon it before damping it, or else allowing it to become perfectly dry first. There is one difficulty which a beginner often experiences in sorting and mounting certain specimens under the micro- scope, viz., the inversion of the objects ; and it is often stated to be almost impossible to work without an erector. But this difficulty soon vanishes, the young student becoming used to working what at first seems in contradiction to his sight. Let it be understood, that in giving the description of those articles which are usually esteemed necessary in the various parts of microscopic manipulation, I do not mean to say that without many of these no work of any value can be done. There are, as all will allow, certain forms of apparatus which aid the operator considerably ; but the cost may be too great for him. A little thought, however, will frequently overcome this difficulty, by enabling him to make, or get made, for himself, at a comparatively light expense, something which will accomplish all he desires. As an example of this, a friend of mine made what he terms his " universal stand," to carry various condensers, &c. &c., in the following way : Take a steel or brass wire, three- sixteenths or one-quarter inch thick and six or eight inches long ; " tap " into a solid, or make rough and fasten with melted lead into a hollow, ball. (The foot of a cabinet or work-box answers the purpose very well.) In the centre of a round piece of tough board, three inches in diameter, OF MICROSCOPIC OBJECTS. 51 make a hemispherical cavity to fit half of the ball, and bore a hole through from the middle of this cavity, to allow the wire to pass. Take another piece of board, about four inches in diameter, either round or square, and one and a half or two inches thick, make a similar cavity in its centre to receive the other half of the ball, but only so deep as to allow the ball to fit tightly when the two pieces of board are screwed together, which last operation must be done with three or four screws. Let the hole for the wire in the upper part be made conical (base upwards), and so large as only to prevent the ball from escaping from its socket, in order that the shaft may move about as freely as possible. Turn a cavity, or make holes, in the bottom of the under piece, and fill with lead to give weight and steadiness. This, painted green bronze and varnished, looks neat; and by having pieces of gutta-percha tubing to fit the shaft, a great variety of apparatus may be attached to it. Mr. Loy employs the following arrangement for dissecting insects or picking out Foraminifera, &c. : he fits an upright brass rod into a heavy leaden foot, this rod carries a hori- zontal arm bearing at its end a ring for holding a watch- maker's eye-glass; in focussing it to his work, he presses the eye-glass down with his head, the weight of the leaden foot keeping it in its place, and allowing it to follow his every movement. Again, a " condenser " is often required for the illumina- tion of opaque objects. My ingenious friend uses an "engraver's bottle" (price 6d.), fills it with water, and suspends it betwixt the light and the object. Where the light is very yellow, he tints the water with indigo, and so removes the objectionable colour. I merely mention these as examples of what may be done by a little thoughtful contrivance, and to remove the idea that nothing is of much value save that which is the work of professional workmen, and consequently expen -ive. 52 PREPARATION AND MOUNTING CHAPTER III. THE term " dry" is used when the object to be mounted Is not immersed in any liquid or medium, but preserved in its natural state, unless it requires cleaning and drying. I have before stated that thorough cleanliness is necessary in the mounting of all microscopic objects. I may here add that almost every kind of substance used by the microscopist suffers from careless handling. Many leaves with fine hairs are robbed of half their beauty, or the hairs, perhaps, forced into totally different shapes and groups ; many insects lose their scales, which constitute their chief value to the micro- scopist ; even glass itself distinctly shows the marks of the fingers if left uncleaned. Every object must also be thoroughly dry, otherwise dampness will arise and become condensed in small drops upon the inner surface of the thin glass cover. This defect is frequently met with in slide* which have been mounted quickly; the objects not being thoroughly dry when enclosed in the cell. Many cheap slides are thus rendered worthless. Even with every care it is not possible to get rid of this annoyance occasionally. A good plan is to fix the covers on to the cells temporarily by dropping on two sides of them a composition of equal parts of wax and resin : this allows of the easy removal of the cover at any time, while the object thoroughly dries and is protected from dust and damage. For the purpose of mounting opaque objects " dry '* discs were at one time very commonly used. These are circular pieces of cork, leather, or other soft substance, from one-quarter to half an inch in diameter, blackened with varnish OF MICROSCOPIC OBJECTS. 53 or covered with black paper, on which the object is fixed by gum or some other adhesive substance. They are usually pierced longitudinally by a strong pin, which serves for the forceps to lay hold of when being placed under the micro- scope for examination. Sometimes objects are affixed to both sides of the disc, which is readily turned when under the object-glass. The advantage of this method of mount- ing is the ease with which the disc may be moved, and so present every part of the object to the eye, save that by which it is fastened to the disc. On this account it is often used when some particular subject is undergoing investiga- tion, as a number of specimens may be placed upon the discs with very little labour, displaying all their parts. But where exposure to the atmosphere or small particles of dust will injure an object, no advantage which discs may possess should be considered, and an ordinary covered cell should be employed. Small pill-boxes have been used, to the bot- tom of which a piece of cork has been glued to afford a ground for the pin or other mode of attachment ; but this is liable to some of the same faults as the disc, and it would be unwise to use these for permanent objects. Messrs. Smith and Beck have lately invented, and are now making a beautiful small apparatus, by means of which the disc supporting the object can be worked with little or no trouble into any position that may prove most convenient, whilst a perforated cylinder serves for the recep- tion of the discs when out of use, and fits into a case to protect them from dust. A pair of forceps is made far the express purpose of removing them from the case and placing them in the holder. All dry objects, however, which are to be preserved should be mounted on glass slides in one of the cells (described in Chapter II,) best suited to them. Where the object is to be free from pressure, care must be taken that the cell is deep enough to ensure this. When the depth required is but email, it is often sufficient to omit the card, leather, or other -circles, and with the " turntable " before described by 54 PREPARATION AND MOUNTING means of a thick varnish and camel-hair pencil, to form a ring of the desired depth ; but should the varnish not be of sufficient substance to give such " walls " at once, the first application may be allowed to dry, and a second made upon it. A number of these may be prepared at the same time, and laid by for use. When liquids are used (see Chapter V.)> Dr. Carpenter recommends gold-size as a good varnish for the purpose, and this may be used in dry mountings also. I have used the asphaltum and india-rubber (men- tioned in Chapter II.), and found it to be everything I could wish. The cells, however, must be thoroughly dry, and when they will bear the heat they should be baked for an hour at least in a tolerably cool oven, by which treatment the latter becomes an excellent medium. All dry objects which will not bear pressure must be firmly fastened to the slide, otherwise the necessary movements often injure them, by destroying the fine hairs, &c. For this purpose thin varnishes are often used, and will serve well enough for large objects, but many smaller ones are lost by adopting this plan, as for a time, which may be deemed long enough to harden the varnish, they exhibit no defect, but in a while a " wall " of the plastic gum gathers around them, which refracts the light, and thus leads the student to false conclusions. In all finer work, where it is necessary to use any method of fixing them to the slide, a solution of common gum, with the addition of a few drops of glycerine (Chapter II.), will be found to serve the purpose perfectly. It must, however, be carefully filtered through blotting- paper, otherwise the minute particles in the solution interfere with the object, giving the slide a dusty appearance when, tinder the microscope. When mounting an object in any of these cells, the glass must be thoroughly cleaned, which may be done with a cambric handkerchief, after the washing mentioned in Chap- ter II. If the object be large, the point of a fine camel-hair pencil should be dipped into the gum solution, and a minute quantity of the liquid deposited in the cell where the object OP MICROSCOPIC OBJECTS. 54 is to be placed, but not to cover a greater surface than the object will totally hide from sight. This drop of gum musl be allowed to dry, which will take a few minutes. Breathe then upon it two or three times, holding the slide not far from the mouth, which will render the surface adhesive. Then draw a camel-hair pencil through the lips, so as to moisten it slightly (when anything small will adhere to it quite firmly enough), touch the object and place it upon the gum in the desired position. This must be done immediately to ensure perfect stability, otherwise the gum will become at least partially dry and only retain the object imperfectly. When, however, the objects are so minute that it would be impossible to deposit atoms of gum small enough for each one to cover, a different method of proceeding must be adopted. In this case a small portion of the same gum solution should be placed upon the slide, and by means of any small instrument a long needle will serve the purpose very well spread over the surface which will be required. The quantity thus extended will be very small, but by breathing upon it may be prevented drying whilst being dispersed. This, like the forementioned, should be then allowed to dry ; and whilst the objects are being placed on the prepared surface, breathing upon it as before will restore the power of adherence. A small patch of gold-size or gum dammar solution which has been allowed to become "tackey" is very useful in many cases. When gum or other liquid cement has been used to fix the objects to the glass, the thin covers must not be applied until the slide has been thoroughly dried, and all fear of dampness arising from the use of the solution done away with. Warmth may be safely applied for the purpose, as objects fastened by this method are seldom, if ever found to be loosened by it. As objects are met with of every thick- ness, the cells will be required of different depths. There is no difficulty in accommodating ourselves in this the deeper cells may be readily cut out of thick leather, card, or 56 PBEPARATION AND MOUNTING other substance preferred (as mentioned in Chapter II.). Cardboard is easily procured of almost any thickness; but sometimes it is convenient to find a thinner substance even, than this. When thin glass is laid upon a drop of any liquid upon a slide, every one must have observed how readily the liquid spreads betwixt the two : just so when any thin varnish is used to surround an object of little sub- stance, excessive care is needed lest the varnish should extend betwixt the cover and slide, and so render it worth- less. The slightest wall, however, prevents this from taking place, so that a ring of common paper may be used, and serve a double purpose where the objects require no deeper cell than this forms. Many objects, however, are of such tenuity as the leaves of many mosses, some of the Diatomacese, scales of insects, &c. that no cell is requisite excepting that which is neces- sarily formed by the medium used to attach the thin glass cover to the slide; and where the slide is covered by the ornamental papers mentioned in Chapter II., and pressure does not injure the object, even this is omitted, the thin glass being kept in position by the cover ; but slides mounted in this manner are frequently injured by dampness, which soon condenses upon the inner surfaces and interferes both with the object and the clearness of its appearance. The thin glass, then, is to be united to the slide, so as to form a perfect protection from dust, dampness, or other injurious matter, and yet allow a thoroughly distinct view of the object. This is to be done by applying to the glass slide round the object some adhesive substance, and with the forceps placing the thin glass cover (quite dry and clean) upon it. A gentle pressure round the edge will then ensure a perfect adhesion, and with ordinary care there will be little or no danger of breakage. For this purpose gold- size is frequently used. The asphalt and india-rubber varnish also will be found both durable and serviceable. Whatever cement may be used, it is well to allow it to become in some measure fixed and dried ; but where no OF MICROSCOPIC OBJECTS. 57 cell or wall is upon the slide, this is quite necessary, otherwise the varnish will be most certain to extend, as before mentioned, and ruin the object. It may be stated here that gold-size differs greatly in its drying powers, according to its age, mode of preparation, &c. (Chapter V.) : here gum dammar solution laid on in a very thin coating will be found most useful, as it dries so rapidly that it cannot run in unless laid on with an unsparing hand. Should any object be enclosed which requires to be kept flat during the drying of the cement, it will be necessary to use some of the contrivances mentioned in Chapter II. When the slide is thus far advanced, there remains the finishing only. Should the student, however, have no time to complete his work at once, he may safely leave it at this stage until he have a number of slides which he may finish at the same time. There are different methods of doing this, some of which may be here described. If ornamental papers are preferred, a small circle must be cut out from the centre a little less than the thin glass which covers the object. Another piece of coloured paper is made of the same size, and a similar circle taken from its centre also, or both may be cut at the same time. The slide is then covered round the edges with paper of any piain colour, so that it may extend about one-eighth of an inch over the glass on every side. The ornamental paper is then pasted on the " object " surface of the glass, so that the circle shows the object as nearly in the centre as possible, and covers the edges of the thin glass. The other coloured paper is then affixed underneath with the circle coinciding with that above. And here I may observe, that when this method is used there is no necessity for the edges of the slide to be ground, as all danger of scratching, &c., is obviated by the paper cover. Many now use paper covers, about one and a half inch long, on the upper side of the slide only, with the centre cut out as before, with no other purpose than that of hiding the edge of the thin glass where it is united to the slide. <30 PREPARATION AND MOUNTING very useful, and one is now made with a covering upon the broader portion of it to protect the enclosed matter from being so readily carried off whilst bringing it to the surface again. Where there is any depth of water, and the spoon will not reach the surface of the mud, the bottle must bo united to a long rod, and being then carried through the upper portion with the mouth downwards, no water will be received into it ; but on reaching the spot required, the bottle-mouth may be turned up, and thus become tilled with what is nearest. From the stomachs of common fish as the cod, sole, had- dock, &c. many specimens of Diatomaceae may be obtained, but especially from the crab, oyster, mussel, and other shell- fish. Professor Smith states that from these curious recep- tacles he has taken some with which he has not elsewhere met. To remove them from any of the small shell-fish, it is necessary to take the fish or stomach from the shell, and immerse it in strong hot acid (nitric is the best) until the animal matter is dissolved, when the residue must be washed and treated as the ordinary Diatomacese hereinafter described. Many diatoms are seen best when mounted in a dry state, the minute markings becoming much more indistinct if immersed in liquid or balsam ; and for this reason those which are used as test objects are usually mounted dry. Many kinds are also now prepared in this way as opaque objects, to be examined with the lieberkuhn, and are ex- quisitely beautiful. Others, however, are almost invariably mounted in balsam ; but as these will be again referred to in Chapter IV., and require the same treatment to fit them for the slide, it will not be out of place to describe the cleaning and preparation of them here. As before stated, there is much matter surrounding them which must be got rid of before the siliceous covering can be shown perfectly. As, however, we may first wish to become acquainted in some degree with what we have to do, it is well to take a small piece of talc, and place a few of the OF MICEOSCOPIC OBJECTS. 61 diatoms upon it. This may be held over the flame of the spirit-lamp until all the surrounding matter is burnt away, and a tolerable idea may thus be obtained as to the quality of our treasure. In some cases it is well to use this burning operation alone in mounting specimens of diatoms, when they may be placed in their natural state upon thin glass, burnt for awhile upon the platinum plate, hereinafter described, and mounted dry or in balsam. In the preparation and cleaning of Diatomaceae, there is little satisfaction unless these operations have been success- fully performed, as a very small portion of foreign matter seriously interferes with the object. The mode of preparing them varies even amongst the most experienced. It will be found, therefore, most satisfactory to examine the principal of these separately, although it may be at the risk of some little repetition. The method which is most frequently employed is the following: Place the gathering containing the Diato- maces3 in a small glass or porcelain vessel, add strong nitric acid, and, by the aid of a Bunsen's burner or spirit- lamp, boil for some minutes. From time to time a drop of the mixture may be put upon a slide, and examined under the microscope to see if all foreign matter be got rid of. When the valves are clean, the vessel containing them must be filled with water, and the whole left for an hour or two, so that all the diatoms may settle perfectly. The liquid must then be poured off carefully, or drained away by the aid of a syphon, so that none of the diatoms are removed with it. Indeed, it is well to examine the liquid drained off each time with the microscope, as the finer forms are frequently lost in the washings. The vessel must then be refilled with pure water, allowed to settle, and drained as before. This washing must be repeated until a drop being placed upon a slide and evaporated leaves no crystals. "When it is desirable to preserve the diatoms in this state before mounting (which process will be described in another <52 PREPARATION AND MOUNTING place), they may be placed in a small phial with a little Distilled water. There are many cases in which the above method will not effect a perfect cleansing, as certain substances with which diatoms are frequently mixed are not soluble in nitric acid. For this reason the following method is resorted to : Take a, quantity of the matter containing the DiatomaceaB and wash first with pure water, to get rid of all the impurities possible. Allow this to settle perfectly and decant the water. Add hydrochloric acid gradually, and when all effervescence has subsided, boil for some minutes by aid of the lamp. When cool and the particles have subsided, decant the hydrochloric and add nitric acid. The boiling must then be repeated until a drop of the liquid when placed under the microscope shows the valves or " frustules " clean. After allowing the diatoms to settle, the acid must be decanted, and pure water substituted. The washing must be repeated as in the former process . until all the remains of crystals or acid are removed, when the specimens may be preserved in small phials. Such are the usual modes of treating the Diatomaceae, but there are certain cases in which particular methods are required to give anything like perfect results. Persons of great experience combine a variety of treatments, and thus obtain better and more uniform specimens. Perhaps it will be advantageous to give the young student the process adopted by one of the most successful preparers of these objects; but I will first state the different methods of mounting the cleaned diatoms dry : how to employ Canada balsam and fluid in their preservation will be elsewhere described. It was before stated that diatoms when cleansed might be preserved in small phials of distilled water. When re- quired for mounting, shake the phial, and with a thin glass tube or rod take up a drop of the fluid and spread it upon the surface of the slide in the desired position. This must be allowed to dry gradually, or by the aid of the lamp OF MICKOSCOPIC OBJECTS. 63 if necessary, without being shaken or interfered with, other- wise uniformity of dispersion will be prevented. When thoroughly dry, a thin ring of one of the adhesive varnishes gold-size will be found as good as any may be drawn round the diatoms, and allowed to dry in a slight degree. The slide and thin glass cover should then be warmed and the latter gently pressed upon the ring of varnish until the adhesion all round is complete. As some of the diatoms require object-glasses of extremely high power, and consequently short focus, to show them, they must be as close to the outer surface of the cover as possible. For this reason they are sometimes placed upon the under side of the thin glass, as follows. Clean the surfaces of the slide and cover, and with the rod or pipe place the liquid containing the diatoms upon the thin glass, and dry as before. Trace the ring to receive the cover upon the slide, and when almost dry, warm both and proceed as above. Whichever of these methods is employed, the outer ring of coloured varnish may be applied as elsewhere described and the slide finished. Diatoms are also sometimes mounted betwixt two thin glasses, as described in Chapter II., so that the light by which they are examined may receive as little interference as possible, and that an achromatic condenser may be brought into focus under the slide. Of the various modes of cleaning and mounting Diato- maceae, I believe that the following may be safely recom- mended, as affording results of the best quality. My friend, Mr. T. G. Eylands, gave it to me as that which he prefers, and I can safely say that his numerous slides are at least equal to any I have ever seen. I will give it just as I received it from him, though there may be some little repetition of what has been said elsewhere, as he does not appropriate any part of it as his own. He says : In this branch of mounting, general rules alone can be laid down, because the gatherings may contain iron, lime, fine silt, or vegetable matter under conditions for special treatment, and b4 PREPARATION AND MOUNTING consequently the first step should be to experiment on various kinds. In gathering diatoms much labour is saved by judgment and care ; hence it is desirable to get acquainted with them in their growing condition, so that when recognised upon the sand or other spots, they may be carefully removed by the aid of the spoon or small tin scoop before described. When growing upon algae or other plants, the plants and diatoms together may be carried home, in which case they must be simply drained and not washed or pressed, in order that the diatoms be not lost. As it is always desirable to examine the gathering on the ground, a " Gairdner's hand microscope " with powers from 80 to 200 diameters will be found very useful. The best gatherings are those which represent one species abundantly. Those which are mixed may be rejected, unless they are seen to contain something valuable or important, as the object should be not so much to supply microscopical curiosities as to collect material which is available for the study of nature. The gathering when carried home should always be care- fully examined before anything is done with it; not only or account of the additional information thus acquired, but also because it often happens that a specimen should be mounted in fluid (see Chapter V.) in the condition in which it is gathered, as well as cleaned and mounted in balsam (Chapter IY.) and dry. Where the gathering is taken from sand, the whole may be shaken up in water as a preliminary operation, when much of the sand will be separated by its own weight. The lime test, however, should be applied ; viz. a small portion of hydrochloric acid, and if there be effervescence, it must be dissolved out by this means. From Alga3 and other weeds diatoms may be detached by agitating the whole together in a weak solution of nitrie acid about one of pure acid to twenty or thirty of water, as it must be sufficiently weak to free the diatoms without destroying the matter to which they adhere. The diatoms may then be separated by OF MiCllOSCOPIC OBJECTS. 65 sifting through coarse muslin, which will retain the Algse, &c. The process of cleaning will vary according to circum- stances. Some gatherings require to be boiled only a few- minutes in nitric acid ; but the more general plan, where they are mixed with organic or other foreign matter, is to boil them in pure sulphuric acid until they cease to grow darker in colour (usually from a half to one minute), and then to add, drop by drop to avoid explosions, a cold saturated solution of chlorate of potash until the colour is discharged, or, in case the colour does not disappear, the quantity of the solution used is at least equal to that of the acid. This operation is best performed in a wide-mouthed ordinary beaker glass,* a test-tube being too narrow. The mixture whilst boiling should be poured into thirty times its bulk of cold water, and the whole allowed to subside. The fluid must then be carefully decanted and the vessel re-supplied once or twice with pure water, so as to get rid of all the acid. The gathering may then be transferred to a small boiling-glass or test-tube, and the water being carefully decanted boiled in the smallest available quantity of nitric acid, and washed as before. This last process has been found necessary from the frequent appearance of minute crystals, which cannot otherwise be readily dis- posed of without the loss of a considerable proportion of diatoms. I may here mention that the washing-glasses used by Mr. Eylands are stoppered conical bottles varying in capacity from two ounces to one quart; the conical form being employed to prevent the adherence of anything to the side : they are stoppered, to render them available in the shaking process about to be described. The gathering, freed from acid, is now put into two inches depth of water, shaken vigorously for a minute or two, and allowed to subside for half an hour, after which the turbid * These glasses are round, about six inches high, and usually contain about eight ounces. They are rather wider at the bottom, tapering gradually to the top, and may bo generally procured at the chemists, &c. t>4 PREPARATION AND MOUNTING consequently the first step should be to experiment or* various kinds. In gathering diatoms much labour is saved by judgment and care ; hence it is desirable to get acquainted with them in their growing condition, so that when recognised upon the sand or other spots, they may be carefully removed by the aid of the spoon or small tin scoop before described. When growing upon algae or other plants, the plants and diatoms together may be carried home, in which case they must be simply drained and not washed or pressed, in order that the diatoms be not lost. As it is always desirable to examine the gathering on the ground, a " Gairdner's hand microscope " with powers from 80 to 200 diameters will be found very useful. The best gatherings are those which represent one species abundantly. Those which are mixed may be rejected, unless they are seen to contain something valuable or important, as the object should be not so much to supply microscopical curiosities as to collect material which is available for the study of nature. The gathering when carried home should always be care- fully examined before anything is done with it ; not only or account of the additional information thus acquired, but also because it often happens that a specimen should be mounted in fluid (see Chapter Y.) in the condition in which it is gathered, as well as cleaned and mounted in balsam (Chapter IY.) and dry. Where the gathering is taken from sand, the whole may be shaken up in water as a preliminary operation, when- much of the sand will be separated by its own weight. The lime test, however, should be applied ; viz. a small portion of hydrochloric acid, and if there be effervescence, it must be dissolved out by this means. From Alga3 and other weeds diatoms may be detached by agitating the whole together in a weak solution of nitric acid about one of pure acid to twenty or thirty of water, as it must be sufficiently weak to free the diatoms without destroying the matter to which they adhere. The diatoms may then be separated by OF MICROSCOPIC OBJECTS. 65 sifting through coarse muslin, which will retain the Algse, &c. The process of cleaning will vary according to circum- stances. Some gatherings require to be boiled only a few minutes in nitric acid; but the more general plan, where they are mixed with organic or other foreign matter, is to boil them in pure sulphuric acid until they cease to grow darker in colour (usually from a half to one minute), and then to add, drop by drop to avoid explosions, a cold saturated solution of chlorate of potash until the colour is discharged, or, in case the colour does not disappear, the quantity of the solution used is at least equal to that of the acid. This operation is best performed in a wide-mouthed ordinary beaker glass,* a test-tube being too narrow. The mixture whilst boiling should be poured into thirty times its bulk of cold water, and the whole allowed to subside. The fluid must then be carefully decanted and the vessel re-supplied once or twice with pure water, so as to get rid of all the acid. The gathering may then be transferred to a small boiling-glass or test-tube, and the water being carefully decanted boiled in the smallest available quantity of nitric acid, and washed as before. This last process has been found necessary from the frequent appearance of minute crystals, which cannot otherwise be readily dis- posed of without the loss of a considerable proportion of diatoms. I may here mention that the washing-glasses used by Mr. Rylands are stoppered conical bottles varying in capacity from two ounces to one quart; the conical form being employed to prevent the adherence of anything to the side : they are stoppered, to render them available in the shaking process about to be described. The gathering, freed from acid, is now put into two inches depth of water, shaken vigorously for a minute or two, and allowed to subside for half an hour, after which the turbid * These glasses are round, about six inches high, and usually contain about eight ounces. They are rather wider at the bottom, tapering gradually to the top, and may bo generally procured at the chemists, &c. DO PREPARATION AND MOUNTING fluid must be carefully decanted. This operation must be repeated until all the matter is removed which will not settle in half an hour. The fluid removed should be ex- amined by a drop being put upon a slide, as in some cases very light diatoms have been found to come off almost pure in one or more of these earlier washings. The quan- tity of water and time of subsidence given may be taken generally, but may require to be modified according to circumstances and the judgment of the operator. By the repetition and variation of this process the shaking being the most important part the gathering, if a pure one, will be sufficiently clean. If, however, it contain a variety of species and forms, it may require to be divided into different densities. In some cases, however, it is best to divide the gathering as a preliminary operation, which may be done by agitating it in a quantity of water and decanting what does not readily subside. The heavier and the lighter portions are then to be treated as two separate boilings. But when the cleansing has been carried to the above stage and this division is required, the plan must be somewhat as follows : The gathering must be shaken in a test-tube with six inches of water, and then allowed to subside until one inch at the top remains pure. About three inches are then to be care- fully withdrawn by a pipette, when the tube may be filled up and the operation repeated. The three lower inches also may then be decanted and examined. The gathering is thus divided into three portions; viz. that which was withdrawn by the pipette, that which remained floating in the lower three inches of water in the tube, and that which had settled at the bottom. An examination of these will inform the operator how to obtain that particular density of gathering which he desires, and how far it is worth while to refine this process of elutriation; for in cases of necessity any one, or all three, of these densities may be operated upon in the same way to separate a particular diatom. OF MICROSCOPIC OBJECTS. 67 As an occasional aid, it may be remarked, that in some oases liquor ammonice may be used in one of the later washings in place of water, as it often separates fine dirt, which is not otherwise easily removed. Ammonia also dis- solves a flocculent matter which sometimes remains; and this method does not injure diatoms like some strong alkalies. Some fossil deposits require to be treated with a boiling solution of carbonate of soda to disintegrate them; but this operation requires great care, lest the alkali should destroy the diatoms. Vegetable silicates also sometimes require to be removed by a solution of carbonate of soda ; but as the frustules of the diatoms themselves are but vegetable silica, even more care is required in this case. It may be well to mention, that some diatoms are so imperfectly siliceous that they will not bear boiling in acid at all. Some f)f these may be allowed to stand in cold nitric acid some time, whilst others of a smaller and more delicate character should, when possible, be treated with distilled water alone. We will now consider the mode of mounting the prepared diatoms, which, if used dry (as described in this chapter), should be carefully washed two or three times with the purest distilled water. In this branch, as in every other, each collector gives preference to that method in which he is an adept. Thus the diatoms may be placed on the under side of the cover, to be as near to the object-glass as possible, or upon the slide itself; and each plan has its advocates. Whichsoever of these is used, nothing seems more simple to the novice than a tolerably equal dispersion of the objects upon the slide or cover; but this is by no means so readily accomplished ; consequently I give Mr. Rylands' method, as his elides are perfect in this respect also. He always places the diatoms upon the thin glass cover. It is not sufficient, as is frequently thought, to take a drop of liquid containing the cleansed material and spread it upon the cover or slide, as without some additional precaution that uniform and regular distribution of the F 2 68 PREPARATION ANI> MOUNTING specimeua, which is desirable, is not obtained. In order to effect this, let a drop of the cleansed gathering be diluted sufficiently for the purpose how much must be determined in each case by experiment and let the covers to be mounted be cleaned and laid upon the brass plate. (See Chapter II.) By means of a glass tube, about one-twelfth of an inch in diameter, stopped by the wetted finger at the upper end, take up as much of the diluted material as will form a moderately convex drop extending over the whole cover. When all the covers required are thus prepared, apply a lamp below the brass plate, and raise the tem- perature to a point just short of boiling. By this means the covers will be dried in a few minutes, and the specimens equally distributed over the whole area. The spread cf the fluid upon the covers is facilitated by breathing upon them ; and, to insure uniformity, care must be taken to avoid shaking them whilst drying. The best plan is to mount at least half a dozen at once. Before mounting, Mr. Eylands always burns the diatoms upon the glass at a dull red beat, whether they are used with balsam or dry. This burning, he says, is not only an additional cleaning process, but it effectually fixes the diatoms, and prevents them floating out if mounted with balsam. The thinnest covers may be burnt without damage if they are placed upon a small piece of platinum foil ot the size required, which should be about one-hundredth of an inch thick, perfectly flat, and having three of its edges slightly bent over, so as to prevent its warping with the heat. The small flame of a spirit-lamp, or, where there is gas, a Bunsen's burner, may be employed. The cover should be shaded from direct daylight, that the action of the flame may be observed more perfectly. Care must then be taken to raise the temperature only to the dull red heat before mentioned. The cover will then be in a fit state for mounting as required. It has been stated in another place that it is assumed that the operator is not mounting diatoms simply as micros- OE MICROSCOPIC OBJECTS. 69 usly ; that of the azalea came off without the least difficulty. The whole operation did not occupy more than five minutes. Undoubtedly many leaves, according to their texture, will require different strengths of acid, and longer or shorter periods of boiling." Closely connected with the leaves are the ANTHERS and POLLEN, of which a great number are beautiful and interesting subjects for the microscopist. The mallow tribe will furnish some exquisite objects, bearing the appearance of masses of costly jewels. These are usually ^dried with pressure, but the natural form may be more accurately preserved by allowing them to dry as they are taken from the flower, with no interference except thoroughly protecting them from all dust. Sometimes the anther is divided, so that the cell required to receive them may be of as little depth as possible. The common mallow is a beautiful object, but I think the lavatera is a better, as it shows the pollen-chambers well, when dried unpressed. The pollen is often set alone, and is well worth the trouble, as it then admits of more close examination. Often it is convenient to have the anther and pollen as seen in nature on one slide, and the pollen alone upon another. The former should be taken from the flowers before their fuil OF MICROSCOPIC OBJECTS. 81 development is attained, as, if overgrown, they lose much of their beauty. Some pollens are naturally so dark that it is necessary to mount them in Canada balsam or fluid, as described in other places ; but they are better mounted dry when they are not too opaque. Here we may also mention the SEEDS of many plants as most interesting, and some of them very beautiful, objects, requiring for the greater part but a low power to show them. Most of these are to be mounted dry, as opaque objects, in cells suited to them, but some are best seen in balsam, and will be mentioned in Chapter IV. The CORALLINES, many of which are found on almost every coast, afford some very valuable objects for the microscope. They must be well washed when first procured, to get rid of all the salts of the sea-water, dried and mounted in cells deep enough to protect them from all danger of pressure, as some of them are exceedingly fragile. The white ivory appearance which some of them present is given to them by an even covering of carbonate of lime; and should it be desired to examine the structure of these more closely, it may be accomplished by keeping them for some time in vinegar or dilute muriatic acid, which will remove the lime and allow of the substance being sliced in the same way as other Algae. (" Micrographic Dictionary," p. 183.) THE SCALES OF INSECTS. The fine dust upon the wings of moths and butterflies, which is so readily removed when they are handled carelessly, is what is called the scales. To these the wing owes the magnificent colours which so often are seen upon it ; every particle being what may be termed a distinct fiat feather. How these are placed (somewhat like tiles upon a roof) may be easily seen in the wing of any butterfly, a few being removed to aid the investigation. Their form is usually that of the battledore with which the common game is played, but the handle or base of the scale is often short, and the broad part varies in propor- tionate length and breadth in different specimens. The markings upon these also vary, some being mostly composed G 82 PREPARATION AND MOUNTING of lines running from the base to the apex, others reminding us of network bead-like spots only are seen in some indeed, almost endless changes are found amongst them. These scales are not confined to butterflies and moths, nor indeed to the wings of insects. The different gnats supply some most beautiful specimens, not only from the wings, but also from the proboscis, &c. ; whilst from still more minute insects, as the podura, scales are taken which are esteemed as a most delicate test. The gorgeous colours which the diamond beetles show when under the microscope are produced by light reflected from minute scales with which the insects are covered. In mounting these objects for the microscope it is well to have the part of the insect from which the scales are usually taken as a separate slide, so that the natural arrangement of them may be seen. This is easily accomplished with the wings of butterflies, gnats, &c. ; as they require no extra- ordinary care. In mounting the scales they may be placed upon slides, by passing the wings over the surface, or by gently scraping the wing upon the slide, when they must be covered with the thin glass. Of course, the extreme tenuity of these objects does away with the necessity of any cell excepting that formed by the gold-size or other cement used to attach the cover. The scales of the podura should be placed upon the slide in a somewhat different manner. This insect is without wings, and is no longer than the common flea. It is often found amongst the sawdust in wine-cellars, continually leaping about by the aid of its tail, which is bent underneath its body. Dr. Carpenter says : " Poduraa may be obtained by sprinkling a little oatmeal on a piece of black paper near their haunts ; and after leaving it there for a few hours, removing it carefully to a large glazed basin, so that, when they leap from the paper (as they will when brought to the light), they may fall into the basin, and may thus separate themselves from the meal. The best way of obtaining their scales, is to confine several of them together beneath a wine-glass inverted upon a piece of fine Mnooth OF MICROSCOPIC OBJECTS. 83 Paper; for the scales will become detached by their leaps Against the glass, .and will fall upon the paper." These oales are removed to the slide, and mounted as those from ^nats. &c. When the podura has been caught without the aid of meal, it may be placed upon the slide, under a test- tbbe, or by any other mode of confinement, and thus save the trouble of transfer from the paper before mentioned- Another method is to seize the insect by the leg with the forceps and drag it across the slide, when a sufficient quan- tity of scales will probably be left upon it. Mr. Mclntyre procures the scales in the following man- ner : He makes what he terms a breeding-cage, by taking a piece of plate-glass four inches long by two inches wide, and over this places a few sheets of blctting-paper. Upon these he lays a sheet of cork about a quarter of an inch thick, with a circle cut out of the centre one inch wide. This gives a kind of box, which he covers with glass, kept firm by two elastic bands. He says : " After capturing the insect by means of a tube and a camel-hair pencil, I let it remain for some days in one of the breeding-cages, into which I always transfer the newly-caught podura, until it has changed its skin ; then I stupefy it with chloroform, and drop it out on to a thin glass cover (previously cleaned) and with a very clean needle-point roll it backwards and forwards upon the cover till sufficient scales are removed. A very light pressure is indispensable, so as not to squeeze out any of the insect's fluids." These scales are usually mounted dry; but Hogg re- commends the use of Canada balsam (Chapter IV.) as ren- dering their structure more definite when illuminated with Wenham's parabolic reflector. Some advise other methods, which will be mentioned in Chapter Y. As most insects when undissected are mounted in Canada balsam, the dif- ferent modes of treatment which they require will be stated in another place. In mounting blood of any kind to show the corpuscles, or, as they are often called, globules, which are round or G2 84 PREPARATION AND MOUNTING oval discs, it is bat necessary to cover the slide on the spot required with a coating as thin as possible and allow it to dry before covering with thin glass. There is a slight con- traction in the globules when dried, but not enough to in- jure them for the microscope. The shape of these varies in different classes of animals, but the size varies much more, some being many times larger than others. Perhaps it will not be out of place to say a few words concerning the detec- tion of blood. Wherever the stains are, they must be care- fully scraped away and immersed for a few hours in a weak solution of bichloride of mercury. With a thin tube the more solid portion may then be removed to a glass slide and examined with a somewhat high power. A slight knowledge of the microscopic appearance of blood-discs will show us whether the suspicion of blood is correct. Some of the skins of larvcB are beautiful objects ; but, like many sections of animal and other fragile matter, are diffi- cult to extend upon the slide. This difficulty is easily over- come by floating the thin object in clear water, immersing the slide, and when the object is evenly spread gently lifting it. Allow it then to dry by slightly raising one end of the slide to aid the drainage, and cover with thin glass as other objects. The tails and fins of many small fish may be mounted in a similar manner, and are well worth the trouble. A few objects which are best shown by mounting dry may be here mentioned as a slight guide to the beginner, though some of them have been before noticed. Many of the Foraminifera, as elsewhere described. Some crystals are soluble in almost any fluid or balsam, and should be mounted dry; a few, however, deliquesce or effloresce, which render* them worthless as microscopic objects. The wings of butterflies, gnats, and moths will afford many specimens wherewith to supply the cabinet of the young student. A great variety of scales also may be found amongst the ferns; indeed, these alone will afford the student occupation for a long time. On the nnder-side of OP MICROSCOPIC OBJECTS. 85 the leaves are the reservoirs for the spores, which in many instances somewhat resemble green velvet, and are arranged in stripes, round masses, and other forms. The spores are usually covered with a thin skin, which is curiously marked in some specimens, often very like pollen-grains. The man- ner in which these spores with all their accompaniments are arranged, their changes and developments, afford almost endless subjects for study; different ferns presenting us with many variations in this respect totally invisible without the aid of the microscope. The hymenophyllnms (of which two only belong to England) are particularly interesting, and the structure of the leaves when dried makes them beautiful objects, often requiring no balsam to aid their transparency. Portions of the fronds of ferns should be mounted as opaque objects, after having been dried between blotting-paper, when they are not injured by pressure; but care must be taken to gather them at the right time, as they do not show their beauty before they are ripe, and if over-ripe the ar- rangement of the spores, &c., is altered. The spores may be mounted as separate objects in the same manner as pollen, before mentioned, and are exquisitely beautiful when viewed with a tolerably high power. The number of foreign ferns now cultivated in this country has greatly widened the field for research in this direction ; and it may also be mentioned that the under-sides of many are found to be covered with scales of very beautiful forms. A small piece of the frond of one of these may be mounted in its natural state, but the removal of the scales for es anima- tion by polarized light will be described in another place. The mosses also are quite a little world, requiring but a low power to show their beauties. The leaves are of various forms, some of which resemble beautiful net-work ; the " urns " or reservoirs for the spores, however, are perhaps the most interesting parts of these objects, as also of the liverworts which are closely allied to the mosses. These urns are generally covered by lids, which fail off when the fruit is ripe. At this period they are well fitted for th> 86 PIIEPARATION AND MOUNTING microscope. The common screw-moss may be found i great abundance, and shows this denudation of the spores- very perfectly. Many of these may be easily dried without much injury, but they should also be examined in their natural state. The student should not omit from his cabinet a leaf of- the nettle and the allied foreign species, the mystery ot which the microscope will make plain. The hairs or stings may also be removed, and viewed with a higher power than when on the leaf, being so transparent as to require no bal- sam or other preservative. There are few more interesting objects than the raphides or plant-crystals. These are far from being rare, but in some plants they are very minute, and consequently require care in the mounting, as well as a high magnifying power to render them visible; in others they are so large that about twenty-five of them placed point to point would reach, one inch. Some of these crystals are long and compara- tively very thin, which suggested the name (raphis, a needle) ; others are star-like, with long and slender rays - t while others again are of a somewhat similar form, each ray being solid and short. If the stem of rhubarb, or almost any of the hyacinth tribe, be bruised, so that the juice may flow upon the slide, in all probability some of these crys- tals will be found in the fluid. To obtain them clean,, they must be freed from all vegetable matter by maceration^ After this they must be thoroughly washed and mounted dry. They are also good polarizing objects, giving bril- liant colours; but when used for this purpose they must be mounted as described in Chapter IV. A few plants which contain them may be mentioned here. The Cac- tacese are very prolific ; the orchids, geraniums, tulips, and the outer coating of the onion, furnish the more unusual forms. The Fungi are generally looked upon as a very difficult class of objects to deal with, but amongst them some oi the most available may be found. The forms of many are or MICROSCOPIC OBJECTS. 87 very beautiful, but are so minute as to require a high mag- nifying power to show them. The mould which forms on many substances is a fungus, and in some cases may be dried and preserved in its natural state. A friend of mine brought me a rose-bush completely covered with a white blight. This was found to be a fungus, which required a high magnifying power to show it. Being a very interesting object, it was desirable to preserve it, and this was perfectly effected without injury to the form by simply drying the leaf in a room usually occupied. Amongst the fungi are many objects well worth looking for, one of which is the DiaclifRd elegans. This, the only species, says the Micro- graphic Dictionary, is found in England upon the living leaves of the lily-of-the-valley, &e. These little plants grow in masses, reminding one of mould, to a height of a quarter of an inch, and each " stem " is covered with a sheath, in shape somewhat like an elongated thimble. When ripe the sheath falls off and reveals the same shaped column, made up of beautifully fine network, with the spores lying here and there. This dries well, and is a good object for the middle powers. Amongst the fungi the blights of wheat and or" other articles of food may be included. Many of them may be mounted dry ; others, however, cannot be well pre- served except in liquids, and will be referred to in Chap- ter V. When rambling in a wood during the summer I sat down upon the fallen trunk of a tree, and here and there a few minute white spots caught my eye. I took my Cod- dington lens from my side-pocket and applied it to these. Judge of my surprise when I found each white speck a distinctly formed fungus resembling in size and form, to an amusing similarity, a disc of the arachnoidiscus. They were already dry, and I mounted them as ordinary dry objects ; and hitherto no change has taken place which I can detect. Amongst the zoophytes and sea-mats, commonly called sea-weeds, may be found very many interesting objects to be mounted dry. When this mode of preservation is used, it is necessary that all the sea-salt be thoroughly washed 88 PREPARATION AND MOUNTING from them. As they are, howerer, most frequently mounted in balsam or liquid, they will be more fully noticed in other places. The scales of fishes are generally mounted dry when used as ordinary objects ; but for polarized light, balsam or liquid must be used, as noticed in Chapter IV. To mount a fish-scale, however, in a satisfactory manner, care must be taken that it is perfectly clean. This can be accomplished only by careful washing, in which process soft camel-hair pencils will often be useful. When the slime or mucus has once dried, it is very difficult to remove. The variety and beauty of these are quite surprising to the novice. It is also very interesting to procure the skin of the fish when possible, and mount it on a separate slide to show how the scales are arranged. The sole is one of the most unusual forms, the projecting end of each scale being covered with spines, which radiate from a common centre, while those at the extremity are carried out somewhat resembling the rays of a star. One of the skates has a spine projecting from the centre of each scale, which is a very curious opaque object, especially when the skin is mounted in the manner described. The perch, roach, minnow, and others of the common fishes give the student good objects for his cabinet, and may be procured without difficulty. The scale of the turbot is a splendid object for the polariscope when mounted in balsam. Insects which are very transparent, or have the " metallic lustre" with which any medium would interfere, are mounted dry. The diamond -beetle, before mentioned, is a splendid example of this ; the back is generally used, but the logs, showing the curious feet, are very interesting objects. In- deed, amongst the legs and feet of insects there is a wide field of interest. When they are of a horny nature, it is best to dry them in any form preferred, but to use no pressure; when, however, they are wanted flat, so as to show the feet, &c., extended, they must be dried with a gentle pressure betwixt blotting-paper if possible. But this will be treated more fully in Chapter IY. OF MICROSCOPIC OBJECTS. 89 The eyes of insects are sometimes allowed to dry in their natural shape, and mounted as opaque objects; but generally they are used as transparencies in balsam or liquid, so the description of the treatment which they require will be deferred to Chapter IV. Hairs when not too dark, are sometimes transparent enough when mounted dry, but are usually mounted in balsam. These will be more fully noticed in another place, but there are some without which no cabinet is deemed in anywise complete. Many different species of bats, English and foreign, present us with hairs the form of which we should never have dared to imagine without microscopic aid. Other curious objects are found in the antennae of crabs. You can also readily know whether you are being deceived when you buy what you deem a real sealskin or sable. From some of the common caterpillars I have ob- tained exquisitely beautiful slides, and a kangaroo is a true friend to an object-gatherer. The hair of the ornithorhynchus is a very curious object, having a thin place in the middle of its length, and so pre- senting somewhat the appearance of a flail. These are a few of the objects which are often mounted dry, but some of them should be shown in balsam or liquid also, and there is much difference of opinion as to the best way of preserving others. This, however, is explained by the fact, that the transparency which balsam gives, inter- feres with one property of the object, and yet develops another which would have remained invisible if preserved dry. The only method of overcoming this difficulty is to keep the object mounted in both ways, which is comparatively little trouble. I may here mention that many prefer the lieberknhn for the illumination of opaque objects; and a good back- ground is gained by putting upon the under-side of the slide, immediately beneath the object, a spot of black varnish, which does not interfere materially with the light. 90 PBEPABATION AND MOUNTING CHAPTER IV. MOUNTING IN CANADA BALSAM. THE nature and use of this substance has been before spoken of, so that the method of working with, it may be at once described. Perfect dryness of the objects is, if possible, more neces- sary in this mode of mounting than any other, as dampness remaining in the object will assuredly cause a cloudiness to make its appearance in a short time after it is fixed. Where pressure does not injure the specimens, they are most suc- cessfully treated when first dried betwixt the leaves of a book, or in any other way which may prove -most convenient, as noticed in Chapter III. Before describing the methods of proceeding with par- ticular objects, general rules may be given which should be observed in order to succeed in this branch of mounting. As the object is to be thoroughly immersed in the balsam, it is evident that when it has once been covered, so it must remain, unless we again free it by a process hereafter men- tioned, which is very troublesome ; and on this account there must be nothing whatever in the balsam except the object. The inexperienced may think this an unnecessary caution ; but the greatest difficulty he will meet with is to get rid of minute bubbles of air, perhaps invisible to the naked eye, which appear like small globules when under the microscope, and render the slide unsightly, or even woithless. Balsam dissolved in benzole will be found invaluable in mounting without air-bubbles ; if a few are left in the specimen, by the next morning they will have entirely disappeared. In making this solution the balsam should be first boiled gently till on dropping a small quantity into water it i* OF MICROSCOPIC OBJECTS. found to be as hard as resin, the softened and warm solution may be now poured into a bottle, and when cool the benzole added in sufficient quantity to make it of a desirable thick- ness. Ten objects out of eleven contain air, or at least aie full of minute holes which are necessarily filled with it ; so that if they should be immersed in any liquid of thick consistency, these cells of air would be imprisoned, and become bubbles. The air, then, must be removed, and this is usually accomplished by soaking for some time in turpen- tine, the period required differing according to the nature of the object. In some cases, the turpentine acts upon the colour, or even removes it altogether, so that it must be watched carefully. Often, however, this is an advantage, as where the structure alone is wanted, the removal of the colouring matter renders it more transparent. There are objects, however, which retain the air with such tenacity that soaking alone will not remove it. If these will bear heat without being injured, they may be boiled in turpentine, or even in balsam, when the air will be partly or totally expelled. But where heat is objectionable, they must be immersed in the turpentine, and so submitted to the action of the air-pump. Even with this aid, sometimes days are required to accomplish it perfectly, during which time the air should be exhausted at intervals of five or six hours, if convenient, and the objects turned over now and then. Many complaints are made concerning turpentine, both as to its cleanliness and penetrating power. Most of these spring from the fact that few substances in the market vary so much as turpentine in purity ; all sorts of rubbish are sold under this name, and now benzole is employed by many in all cases where turpentine alone was once used. Sometimes the objects are so minute that it is impossible to submit them to any soaking, and in this case they must be laid upon the slide at once, and the turpentine applied to them there. But it must not be forgotten that there are some few which are much better mounted in such a way that the balsam may thoroughly surround, and yet not 92 PREPARATION AND MOUNTING penetrate, the substance more than necessary. Sections of teeth are amongst these, but they will be noticed in another place, and some insects (see Dr. Carpenter) when required to show the ramifications of the tracheae. Having freed the object, then, from these two enemies dampness and air we now proceed to mount it. The slide must first be cleaned ; then on the centre a quantity of balsam must be placed with a bluntly-pointed glass rod, according to the size of the object about to be mounted. To this a slight heat must be applied, which will cause any bubbles to rise from the surface of the slide, so that they may be readily removed with a needle. The object having been freed from all air by steeping in turpen- tine, as before described, and then from superfluous liquid by a short drainage, or touch upon blotting-paper, is to be carefully laid upon, or where it is practicable thrust into, the balsam just prepared on the slide. In the former case, or where the balsam has not totally covered the object, a small quantity must be taken, warmed, and dropped upon it, and any bubbles removed by the needle as before. To cover this, the thin glass must be warmed, and beginning at one side, allowed to fall upon the balsam, driving a email " wave " before it, and thus expelling any bubbles which may remain. This is quite as safely performed (if not more so) by making a solution of balsam in turpentine of the consistency of thick varnish or by the use of chloroform and balsam, as mentioned in Chapter IT. The thin glass cover (may be slightly coated with this, and will then be much >^less liable to imprison any air, which frequently happens when the cover is dry. Bubbles, however, will sometimes make their appearance in spite of all care ; but when the object is comparatively strong, they may be removed by keeping the slide rather warm, and working the cover a little, so as to press them to one side, when they should be immediately removed with a needle point, otherwise they are again drawn under. Where the slide requires keeping warm for any length of OP MICROSCOPIC OBJECTS. 93 time, a hot-water bath is sometimes made use of, which is simply a flat tin, or other metal case, with a mouth at the side, that when the hot water is introduced it may be closed up, and so retain its warmth for a long time. An excellent bath may be made of an ordinary water-plate costing about Is. 9d. This may be filled either with hot water or sand, and if to it be added a flat tin cover such as is used in eating-houses, costing about 6d. a very effective oven for baking slides is the result. It may be placed on the hob, or over or near any source of heat. It is easy to add a thermometer if necessary. In working, the slide is laid upon it, and so admits of longer operations, when required, without growing cold. Sometimes a spirit-lamp is placed under it to keep up an equal heat through excessively long processes. Where the time required, however, is but short,. a thick brass plate is sometimes used (see Chapter II.), which may be heated to any degree that is required, the slide being previously placed upon it. Some objects, which are so thin that they are usually floated upon the slide, as before stated, require no steeping in turpentine or other liquid. These are best mounted by covering with a little diluted balsam, and after this has had time to penetrate the substance, ordinary balsam is laid upon it, and the slide finished in the usual manner. I have stated that balsam is usually applied to the slide and objects with a bluntly-pointed glass rod ; but for the purpose of drawing the balsam from the bottle, and convey- ing it to the desired place, Dr. Carpenter uses a glass syringe with a free opening. These are his instructions : " This (the syringe) is most readily filled with balsam, in the first instance, by drawing out the piston, and pouring in balsam previously rendered more liquid by gentle warmth^ and nothing else is required to enable the operator at any time to expel precisely the amount of balsam he may require,, than to warm the point of the syringe, if the balsam should have hardened in it, and to apply a very gentle heat to tho syringe generally, if the piston should not then be readily Di PREPARATION AND MOUNTING pressed down. When a number of balsam objects are being mounted at one time, the advantage of this plan in regard to facility and cleanliness (no superfluous balsam being deposited on the slide) will make itself sensibly felt," but the collapsible metal capsules are certainly the best and most easily managed. When the mounting has been thus far accomplished, the outer wall of balsam may be roughly removed after a few hours have elapsed; but great care is necessary lest the cover be moved or disturbed in any way. In this state it may be left for the final cleansing until the balsam becomes hard, which takes place sooner or later, according to the degree of warmth to which it has been subjected. Many advocate baking in a slow oven to accelerate this drying; but with some objects even this heat would be too great, and generally a mantel-piece, or other place about equal to it in temperature, is the best suited to this purpose; and when the requisite hardness is attained, the slide may be finished as follows: With a pointed knife the balsam must be scraped away, taking care that the thin glass be not cracked by the point getting under it. If used carefully, the knife will render the slide almost clean; but any minute portions which still adhere to the glass must be rubbed with linen dipped in turpentine or spirit. If the balsam is not very hard, these small fragments are readily removed by folding a piece of paper <.V,btly in a triangular form with many folds, and damping Ih? point with which the glass is rubbed. As the paper becomes worn with the friction, the balsam will be carried off with it. In some cases I have found this simple ex- pedient very useful. Sometimes the object to be mounted is of such a thick- r.ess as to require a cell. For this purpose glass rings are nsed (as described in Chapter V.), and filled with balsf m. The best mode of doing this is thus described by Mr. T. S. Halph in the Microscopic Journal : " The question was asked me when I was in England, if I knew how to fill a OF MICROSCOPIC OBJECTS. 95 cell with Canada balsam and leave behind no air-bubbles ? I replied in the negative ; but now I can state how to accomplish this. Fill the cell with clear spirit of turpentine, place the specimen in it, have ready some balsam just fluid enough to flow out of the bottle when warmed by the hand ; pour this on the object at one end, and gradually inclining the slide, allow the spirit of turpentine to flow out on the opposite side of the cell till it is full of balsam ; then take up the cover, and carefully place upon it a small streak of Canada balsam from one end to the other. This, if laid on the cell with one edge first, and then gradually lowered until it lies flat, will drive all the air before it, and prevent any bubbles from being included in the cell. It can be easily put on so neatly as to require no cleaning when dry. If the cover be pressed down too rapidly, the balsam will flow over it, and require to be cleaned off when hardened, for it cannot be done safely while fluid at the Sometimes with every care bubbles are enclosed in the balsam, injuring objects which are perhaps rare and valuable. If the object will not be injured by heat, carefully warming the slide over a lamp will often set loose and remove these pests ; but should heat be objectionable, or the bubbles too closely imprisoned, the whole slide must be immersed in turpentine until the cover is removed by the solution of the balsam ; and the object must be cleansed by a similar steep- ing. It may then be remounted as if new in the manner before described. The balsam and chloroform described in Chapter II. is thus used; and where the object is thin, the mounting is very easily accomplished. When the object is laid upon the slide with a piece of glass upon it, and the balsam and chloroform placed at the edge of the cover, the mixture will gradually flow into the space betwixt the glasses until the object is surrounded by it, and the unoccupied portion filled. The chloroform will evaporate so quickly that the outer edge will become hard in a very short time, when it may be 96 PREPARATION AND MOUNTING cleaned in the ordinary way. Sometimes the balsam isr dissolved in the chloroform without being first hardened; but this is only to render it more fluid, and so give the operator less chance of leaving bubbles in the finished slide, as, the thicker the medium is, the more difficult is it to get rid of these intruders. It has been before noticed that some have objected to chloroform and balsam, believing that it became clouded after a certain time. Perhaps this may be accounted for in part by the fact that almost all objects have a certain amount of dampness in them. Others are kept in some preservative liquid until the time of mounting, and these liquids generally contain certain salts (Chapter V.). If this dampness, as well as all traces of these salts, however small, are not totally removed the former by drying, the latter by repeated washings the addition of chloroform will render the balsam much more liable to cloudiness than when balsam alone was used. It may safely be affirmed that benzole will be found in all cases a more valuable solvent for Canada balsam than chloroform. This mode of employing the balsam, however, will not be always applicable, as chloroform acts upon some substances on which balsam alone does not. Some salts are even soluble in it, the crystals appearing after a few days or weeks, whereas in balsam alone they are quite permanent. Ex- perience is the only guide in some cases, whilst in others a little forethought will be all that is required. The particular methods used for certain objects may now be entered upon. Many of the Diatomacese and fossil Infusoria, as they are sometimes termed, are mounted dry, and cleaned in the way described in Chapter III. Others are almost always placed in balsam, except where they are intended to be used with the lieberkuhn and dark back- ground, by which means some of them are beautifully shown. The usual way of mounting them in balsam is as follows : Take a drop of the water containing them, place it upon tiie slide, and evaporate over the lamp, whilst with a needle OF MICKOSCOPIC OBJECTS. 97 they may be dispersed over any space desired. When they are thoroughly dry, drop a little balsam on one side, and exclude the bubbles. The slide may then be warmed to such a degree that the balsam, by lifting the glass at one end, will be carried over the specimens, which may then be covered with thin glass, made warm as before described. Where the objects are quite dry, and loose upon the glass, it requires great care in placing the cover upon them, other- wise they are forced to one edge, or altogether from under it, in the wave of the balsam. For this reason, Professor Williamson adds a few drops of gum-water to the last washing, which causes them to adhere sufficiently to the glass to prevent any such mishap. Mr. T. G. Eylands's method differs in some degree from the above, and is, to use his own words, as follows : Thick balsam is preferable, and the burnt covers (see Chapter III.) to be mounted are laid in a convenient position, with the diatoms upwards. The slides required having been care- fully cleaned and marked on the under side with a ring of ink about half an inch in diameter by the aid of a turntable to point out the centre, a drop of benzole is applied by a large pin to the diatoms on the cover, so as to exclude the air from the valves and frustules. The slide is then held over the lamp, and when warm, a sufficiently large drop of balsam is put upon it, and heated until it begins to steam. If small bubbles appear, a puff of breath removes them, The slide being held slightly inclined from the operator, and the drop of balsam becoming convex at its lower edge, the cover is brought in contact with it at that point, gradually laid down, pressed with the forceps, and brought to its central position. When cool the superfluous balsam (if any) is removed with a heated knife-blade, the slide cleaned with a little turpentine, and finished by washing in a hand- basin with soap and water. In this process there is no delay if the balsam be sufficiently thick, as the slide may be cleaned off almost before it is cold. It is now well known that from common chalk it is an PREPARATION AND MOUNTING easy matter to obtain interesting specimens of Foraminifera. Scrape a small quantity of chalk from the mass and shake it in water ; leave this a few minutes, pour the water away and add a fresh quantity, shake up as before, and repeat two or three times. Take a little of the residue, and spread it upon the slide, and when quite dry, add a little turpentine. When viewed with a power of two hundred and fifty dia- meters, this will generally show the organisms very well. If it is desired to preserve the slides, they may then be mounted in Canada balsam. Mr. Guyon, in " Recreative Science," observes that the accumulation of the powder, by the action of the rain or exposure to the atmospheric action, at the foot or any projection of the chalk cliffs, will afford us better specimens than that which is scraped, as the organisms are less broken in the former. Take a piece of chalk, and with a soft tooth or nail brush, brush it under water, and then wash the sediment well till the water is not coloured, when the residue will be nearly all Foraminifera. The above is the most simple method of obtaining Fora- minifera from chalk, but is not so satisfactory when any number of perfect slides is wanted for the cabinet. I shall, therefore, give additional particulars from the experience of good men. But some specimens of chalk seem to have undergone such powerful action that no perfect forms are found in them. This accounts for that disappointment which I have now and then heard expressed. One student says, Take about one ounce of chalk, place it in a quart bottle with about a pint of water, shake it, and after a few moments pour off the milky fluid to about one fourth. Add more fresh water, shake and pour off, waiting longer each time for it to settle. Continue ten or twelve times in one day, and repeat two or three times a day for a few days, and the result will be a sediment entirely composed of Foraminifera. If fragments of chalk remain, the bottle has not been sufficiently shaken. Mr. Kobertaon uses a somewhat different method. Break OF MICROSCOPIC OBJECTS. 99 a lump of fresh chalk into pieces not larger than a walnut; then crush, but not grind, lest you destroy the forms, into a coarse powder that will pass a somewhat wide sieve. Tie this, as a pudding, in a stout piece of calico. Drop into water and allow the bundle to become saturated, and then knead with the hands. This will expel a quantity of milky water. From time to time, after allowing the fluid to drain off, the cloth should be untied, and retied more closely to the mass ; and when the contents are reduced to about one- third of their original bulk, all large pieces of chalk, portions of spines of echini, &c., should be removed, lest they injure the more delicate forms. Care must be taken in the kneading when the greater portion of the chalk has escaped, and at last the bag only shaken until the water flows from it almost clear. The whole may then be trans- ferred to a bottle of clear water and treated as before described. The results, Mr. Robertson says, will be satis- factory, and the chalk must be very poor in fossils if 2 Ib. would not satisfy any microscopic observer. When the Foraminifera are of a larger size, though trans- parent enough to be mounted in balsam, the air must be first expelled from the interior, otherwise the objects will be altogether unsatisfactory. To accomplish this they must be immersed in turpentine and submitted to the action of the air-pump. So difficult is it to get rid of this enemy, that it is often necessary to employ three or four exhaus- tions, leaving them for some time under each. When all air has given place to the turpentine, they must be mounted in the ordinary way. Of all objects which are commonly met with, few are such general favourites as the POLYCYSTIN.S; ; and deservedly so. Their forms are most beautiful, and often peculiar stars varying in design, others closely resembling crowns ; the Astromma Aristotelis like a cross, and many whose shapes no words could describe. The greater part, perhaps, of those which are usually sold, are from the rocky parts of Bermuda ; but they are also found in Sicily, some parts of n2 100 PREPARATION AND MOUNTING Africa and America. They are usually mounted in balsam, but are equally beautiful mounted dry, and used with the lieberkuhn. They require as much care in cleaning as the Diatomacese, but the process is a different one. Sometimes this is effected by simply washing until they are freed from all extraneous matter ; but this is seldom as effectual as it should be. In the Microscopic Journal Mr. Furlong gives the following method of treatment as the best he knows : Procure A large glass vessel with 3 or 4 quarts of water. New tin saucepan holding 1 pint. 2 thin precipitating glasses holding 10 oz. each. Take 3 oz. of dry Barbadoes earth (lumps are best), and break into rather small fragments. Put 3 or 4 oz. of com- mon washing soda into the tin and half fill it with water. Boil strongly, and having thrown in the earth, boil it for half an hour. Pour nine-tenths of this into the large glass vessel, and gently crush the remaining lumps with a soft bristle brush. Add soda and water as before, and boil again ; then pour off the liquid into the large vessel, and repeat until nothing of value remains. Stir the large vessel with an ivory spatula, let it stand for three minutes, and pour gently off nine-tenths of the contents, when the shells will be left, partially freed only, like sand. 2ND PROCESS. Put common washing soda and water into the tin as before, and having placed the shells therein, boil for an hour. Transfer to the large vessel as before, and after allowing it to stand for one minute, pour off. Each washing brings off a kind of " flock," which seems to be skins. 3RD PROCESS. Put the shells in a precipitating glass and drain off the water until not more than ^ oz. remains. Add half a teaspoonful of bicarbonate of soda, dissolve, and then pour in gently 1 oz. of strong sulphuric acid. This liberates the "flock," &c., and leaves the shells beautifully trans- parent. Wash well now with water to get rid of all salts and other soluble matter. OF MICROSCOPIC OBJECTS. 101 Some of the large shells are destroyed by this method, but none that are fit for microscopic use. An oblique light shows these objects best. These are sometimes treated in the manner described in Chapter III., where the diatoms are spoken of, but many forms are liable to be injured by this severe process. It has been before stated that some of the zoophytes may be mounted dry, and others examined as opaque or trans- parent objects according to their substance. They are very interesting when examined in the trough whilst living, but to preserve many of them for future examination they must be mounted in some preservative medium. Sometimes this may be one of the liquids mentioned in Chapter V., but if possible they should be kept in balsam, as there is less danger of injury by accident to this kind of slide. This method of mounting presents some difficulties, but I think that all agree as to the trustworthiness of Dr. Golding Bird's information on the subject, which appeared in the Microscopic Journal. Of this, space forbids me to give more than a condensed account, but I hope to omit nothing of moment to the reader for whom these pages are written. After stating that there are few who are not familiar with these exquisite forms, and have not regretted the great loss of beauty they sustain in drying, he informs us that from their so obstinately retaining air in the cells and tubes when dried, it is hardly practicable to get rid of it ; and they also shrivel up very seriously in the process of drying. The following plan, however, he has found almost faultless in their preparation. To preserve them with extended tentacles, they should be plunged in cold fresh water, which kills them so quickly that these are not often retracted.* The specimens should be preserved in spirit until there is leisure to prepare them ; * It has been stated that the best method of killing zoophytes is to drop alcohol, French brandy, or benzole into th^e salt water in which they are placed ; as this will cause no retraction of tentacles if it be done gradually. 102 PKEPARATION AND MOUNTING if, however, they have been dried, they should be soaked in cold water for a day or two before being submitted to the following processes : 1. After selecting perfect specimens of suitable size, im- merse them in water heated to about 120, and place them under the receiver of an air-pump. Slowly exhaust the air, when bubbles will rise and the water appear to be in a state of active ebullition. After a few minutes re-admit the air and again exhaust, repeating the process three or four times. This will displace the air from most, if not all, of the class. 2. Remove the specimens and allow them to drain upon blotting-paper for a few seconds ; then place them in an earthen vessel fitted with a cover, and previously heated to about 200. This heat may be easily got by placing the vessel for a short time in boiling water, wiping it imme- diately before using, with a thick cloth. The specimens are then dropped into this, covered with the lid, and imme- diately placed under the receiver of the air-pump, and the air rapidly exhausted. By this means they are dried completely, and so quickly that the cells have no time to wrinkle. 3. In an hour or two remove them from the air-pump and drop them into a vessel of perfectly transparent cam- phine. This may be quite cold when the horny, tubular polypidoms, as those of the Sertulariae, are used ; but should be previously heated to lOO^ 3 when the calcareous, cellular Polyzoa are the objects to be preserved. The vessel should be covered with a watch-glass and placed under the re- ceiver, the air being exhausted and re-admitted two or three times. 4. The slide which is to receive the specimen should be well cleaned and warmed so as to allow the balsam to flow freely over it. This must be applied in good quantity, and air-bubbles removed with the needle-point. Take the poly- pidom from the camphine, drain it a little, and with the forceps immerse it fully in the balsam. The glass to be OF MICROSCOPIC OBJECTS. 103 laid upon it should be warmed and its surface covered with a thin layer of balsam, and then lowered gradually upon it, when no bubbles should be imprisoned. A narrow piece of card-board at each end of the object, for the cover to rest upon, prevents any danger of crushing the specimen. This mode of mounting polypidoms, &c., seems to give almost the complete beauty of the fresh specimens. They are very beautiful objects when viewed with common light, but much more so when the polarizer is used (in the manner described a little farther on). To the above instructions there can be little to add ; but I may here mention that some young students may not be possessed of the air-pump, and on this account put aside all search for those specimens which need little looking for at the seaside. Many of these, however, though they lose some beauty by the ordinary mode of drying, will, by steeping for some time in turpentine, not only be freed from the air- bubbles, but suffer so little contraction that they are a worthy addition to the cabinet. Another class of objects is the spicula met with in sponges, &c. These are often glass-like in appearance and of various shapes ; many are found resembling needles (whence their name) ; some from the synapta are anchor- like, whilst others are star-like and of complex and almost indescribable combinations. As some of these are composed of silex and are consequently not injured by the use of nitric acid, the animal substance may be removed by boiling them in it. Those, however, which are calcareous must be treated with a strong solution of potash instead; but whichever way is used, of course they must afterwards oe freed from every trace of residue by careful washing. These spicules may be often found amongst the sand which generally accumulates at the bottom of the jars in which sponges are kept by those who deal in them, and must be picked out with a camel-hair pencil. The speci- mens obtained by this means will seldom if ever require 104 PREPARATION AND MOUNTING any cleaning process, as they are quite free from animal matter. In the former chapter were noticed those insects or parts of them whbh are usually mounted dry. When they are large and too opaque to admit of the dry treatment, they must be preserved in Canada balsam or fluid. The first of these may now be considered. It may be here mentioned, that with these objects much heat must not be employed, as it would in some instances give rise to a cloudiness, and almost invariably injure them. In killing the insect it is necessary not to rub or break any part of it. This may be performed by placing it in a small box half filled with fragments of fresh laurel-leaves, by immersion in turpentine or strong spirit, as also in solu- tions of various poisonous salts. After which it may be preserved for some time in turpentine or other preservative liquid (Chapter Y.) until required. As an assistance to the student, I believe that I can do no better than give him the plan pursued by my friend Mr. Hepworth, whose specimens are in every way satisfactory; but when his method is used, the insects must not have been placed in turpentine for preservation : " After destroying the insects in chloroform or sulphuric ether (methylated being cheaper), wash them thoroughly in a wide-necked bottle, half-filled, with two or three waters; the delicate ones requiring great care. Then immerse them in liquid potash (or Brandish's solution, which is stronger than the usual preparation), and let them remain a longer or shorter time according to their texture. When ready to remove, put one by one into a small saucer of clear water, and with a camel-hair pencil in each hand press them flat to the bottom, holding the head and thorax with the left- hand brush, and applying pressure with the other from above, downwards, giving the brush a rolling motion, which gene- rally expels the contents of the abdomen from the thorax. A minute roller of pith or cork might be used instead of the brush. In larger objects, use the end of the finger to flatten OF MICKOSCOPIC OBJECTS. 105 them. Large objects require more frequent washing, as it is desirable to remove the potash thoroughly, or crystals are apt to form after mounting. Having placed them on the slides with thin glass covers, tied down with thread,* dry and immerse them in rectified spirits of turpentine ; place the vessel under the receiver of an air-pump, and keep it exhausted until the turpentine has taken the place of the air-bubbles ; they are then ready for the application of the balsam. Larger objects may often with advantage be trans- ferred to a clean slide, as during the drying there is con- siderable contraction, and an outline showing this often remains beyond the margin. When closely corked, they may remain in the spirits two or three months. As you take them from the bottle, wipe as much turpentine off as possi- ble before removing the thread, and when untied carefully wipe again, placing the finger on one end of the cover whilst you wipe the other, and vice versa. By this means you remove as much turpentine from under the cover as is necessary ; then drop the balsam, thinned with chloroform (see Chapter II.), upon the slide, letting the fluid touch the cover, when it will be taken in between the surfaces by capillary attraction; and after pressing the cover down, it may be left to dry, or you may hold the slide over a spirit- lamp for a few seconds before pressing down the cover. If heat is not applied, they are much longer in drying, but are more transparent. If made too hot, the boiling disarranges the objects, and if carried too far, will leave only the resin of the balsam, rendering it so brittle that the cover is apt to fly off by a fall or any jar producing sufficient concussion Never lift the cover up, if possible, during the operation, as there is danger of admitting air. A few bubbles may appear immediately after mounting, but they generally subside after a few hours, being only the chloroform or turpentine in a state of vapour, which becomes condensed." * This applies to the more delicate, which will not bear transfei ring after being once spread out and dried. 106 PREPARATION AND MOUNTING This method of preparing and mounting insects I cac strongly recommend as giving first-rate results ; but where the specimens are small, they seldom need the soaking in caustic potash which larger ones must have. It is only necessary to leave them awhile in turpentine, especially when they have been first dried with gentle pressure between two glasses, and then mount with balsam in the ordinary way. With many, even of the larger insects, by soaking them in turpentine or oil of cloves for a longer time, they are made so much more transparent and even colourless, as to exhibit their internal organs (which are visible in layers, by the aid of the binocular microscope), the muscles of the legs, &c. They become also very beautiful objects for the polariscope. Amongst the insect tribes there is abundant employment, especially for the lower powers of the microscope. But if the deeper wonders and beauties of the animal economy are to be sought out aad studied, it is desirable that the various parts should be set separately, in order that they may receive a more undivided attention, as well as be ren- dered capable of being dealt with under the higher powers. We will therefore briefly consider the treatment which the different portions require. The eyes of butterflies, and indeed of almost all insects, afford materials for a study which is complete in itself. When examined with a tolerably high power, instead of finding each eye with an unbroken spherical surface, it is seen that many are composed of thousands of hexagonal divisions, each being the outer surface of a separate portion termed the ocellus. In others these divisions are square; but in all there is a layer of dark pigment surrounding their lower parts. The ocelli may be partly removed from tho eye, which will show how their tapering forms are arranged. But here we have to consider how to place them in balsam for preservation. The eye being removed from the insect, and the dark pigment removed by the use of a camel-hair pencil, must be allowed to remain in turpentine at least for OF MICKOSCOPIC OBJECTS. 107 some days. The turpentine should then be renewed and the eye well washed in it just before it is to be mounted. It may then be set in balsam in the same way as any other object ; but here a difficulty is met with. The eye being spherical upon the surface required, must necessarily be " folded " or broken in attempting to flatten it. This diffi- culty may be often overcome by cutting a number of slits round the edges ; but some object to this mode of treatment, and, where it is practicable, it is much more satisfactory to- mount one in the natural rounded form and another flat. Instead, however, of mounting the organ whole, four or five slides may be procured from each of the larger ones, such aa those of the dragon-fly, &c. The antenncB also are often mounted on separate slides, as being better suited for higher powers and more minute examination than when connected with the insect. These two projecting organs, issuing from the head, are jointed, and moveable at will. They differ very much in form amongst the various species, and are well worth the atten- tion of the microscopist. They are usually mounted with the head attached, and perhaps they are more interesting when thus seen. Some few are very opaque ; to prepare these the following method has been advised, though it i& far better to view them as opaque objects : Bleach the antennae by soaking in the following solution- for a day or two : Hydrochloric acid, 10 drops. Chlorate of potash, ^ drachm. Water, 1 oz. This will render them transparent. "Wash well, dry, and mount in Canada balsam. Instead of the above, a weak solution of chloride of lime may be used, by which means the nerves will be well shown. Many, however, are rendered transparent enough bv simply soaking in turpentine for a longer or shorter time. Where the antennas, however, are " Plumose," or feather-like, extreme care is required ia 108 PREPARATION AND MOUNTING mounting, though the difficulty is not so great as some seem to think. If they are first dried with gentle pressure, and then subjected to the action of the air-pump in a small quantity of turpentine until the air is thoroughly expelled, they can be easily finished upon the slide, especially when balsam and chloroform are used. Insects supply us with another series of beautiful objects, viz., the feet* These are sometimes simply dried and mounted without any medium, as before mentioned; but most of them are rendered much more fit for examination by using balsam in their preservation, as it greatly increases their transparency. The smaller kinds may be dried with gentle pressure betwixt blotting-paper, and then immersed :or some days in turpentine, without requiring the treat- ment with liquor potassae. This immersion will render them beautifully transparent, when they may be mounted in balsam, in the usual manner. It is, however, sometimes found difficult to fix the feet when expanded, in which state the interest of the object is greatly increased. Mr. Ralph recommends the following mode : " First wash the feet, while the insect is yet alive, with spirits of wine ; then, holding it by a pair of forceps close to the edge of a clean piece of glass, the insect will lay hold of the upper surface by its foot ; suddenly drop another small piece of glass over it, so as to retain the foot expanded, and cut it off with a pair of scissors, tie up and soak to get rid of air." Mr. Hepworth says that he never found any difficulty in expanding the foot on a drop of water or well-wetted slide, and laying a thin glass oover over it, tying with thread, drying, and immersing in turpentine. The mouth, also, with its organs, is an interesting object in many insects. That of the common fly is often used, and is comparatively easy to prepare. By pressing the head, the tongue (as it is commonly termed) will be forced * See Mr. Hepworth's interesting articles on the fly's foot in the second and third volumes of the Microscopic Journal. OF MICROSCOPIC OBJECTS. 109 to protrude, when it must be secured by the same means a the foot, and may be subjected to the soaking in turpentine, and mounted as usual. The honey-bee is, however, very different in formation, and is well worth another slide j indeed, even in insects of the same class, the differences are many and interesting. There is another good friend to the Microscopic Cabinet, the large water-beetle, " Dyticuz marginalia" ; and he is by no means uncommon, as he may be met with in many old ponds. If his wings are taken, dried, and mounted in balsam, beautiful circles with crosses make their appearance when examined by the aid of polarized light. But what are commonly termed his suckers are perhaps, his most popular gifts. On his an- terior legs will be found small discs attached to central members (making the whole an exact resemblance of a boy's sucker), which may be readily cut off, placed on the slide, and mounted in balsam. The Dyticus also gives splendid examples of spiracles ; but this will be mentioned where dissection is treated of. Another worthy object of study is the respiration of insects, which is effected by tracheae or hollow tubes, which generally run through the body in one or more large trunks, branching out on every side. These terminate at the sur- face in openings, which are termed spiracles, or breathing organs. The trachece often present the appearance of tubes, constructed of a spiral thread, somewhat resembling the spiral fibres of some plants. These are very beautiful objects, and are generally mounted in balsam, for which reason they are mentioned here; but as they evidently belong to the dissecting portion, they will be fully treated of in another place. Amongst the parasitic insects a great variety of micro- scopic subjects will be found. As these are usually small, they may be killed by immersion in spirits of turpentine ; and, if at all opaque, may be allowed to remain in the liquid until transparent enough, and then mounted in Canada balsam. 110 PREPARATION AND MOUNTING The acarida, or mites and ticks, are well known; none, perhaps, better than those which are so often found upon cheese. Flour, sugar, figs, and other eatables are much infested by them ; whilst the diseases called the itch in man, and the mange in animals, are produced by creatures belonging to this tribe. These insects are sometimes mounted by simply steeping them in turpentine, and proceeding as with other insects. The Micrographic Dictionary gives the following directions as to mounting parts of these : ''The parts of the mouth and the legs, upon which the characters are usually founded, may be best made out by crushing the animals upon a slide with a thin glass cover, and washing away the exuding substance with water : some- times hot solution of potash is requisite, with the subsequent addition of acetic acid, and further washing. When after- wards dried and immersed in Canada balsam, the various parts become beautifully distinct, and may be permanently preserved." Feathers of different kinds of birds are usually mounted in balsam when required to show much of the structure. This is particularly interesting when the feathers are small, as they then show the inner substance, or pith, as it may be termed, with the cells, &c. The " pinnaB," or soft branches of the feathers, will be found of various constructions ; some possessing hooks along one side, whereby they fasten them- selves to their neighbours; others branching out, with straight points somewhat resembling the hairs from certain caterpillars. But, of course, when the metallic-looking gorgeous colours are all that is required to be shown, and reflected light used (as with the feathers of the humming- bird, peacock, &c.), it is much better that they should be mounted dry, as in Chapter III. The seeds and pollen of plants are most frequently mounted dry, as mentioned in Chapter III. ; but the more transparent of the former, and the darker kinds of the latter, are perhaps better seen in Canada balsam. There is nothing particular to be observed in the manipulation, except OF MICROSCOPIC OBJECTS. Ill that the glass cover must be applied lightly, otherwise the grains may be crushed. There are some objects which cannot be shown in a perfect manner when mounted dry, but when immersed in balsam become so very transparent that they are almost useless. To avoid this, it has been re- commended to stain the objects any colour that may be con- venient, and afterwards mount in balsam in the ordinary manner. Permanent dyes, however, for these minute objects are not so readily procurable. My friend Mr. Abbey showed me that what was permanent with vegetable matter of one kind was totally untrustworthy with another. The most useful that I have tried is Magenta, and the colour is a convenient one. Whatever is used for this purpose should be in solution, and the object steeped for awhile and after- wards thoroughly washed, in order that no superfluous salt may remain, There are many liquids now sold by every chemist which will help the student in this respect. Most objects intended for the polariscope may be mounted in Canada balsam ; but there are some exceptions to this. Many of the salts are soluble in this medium, or their forms so injured by it, that glycerine or oil has to be used (see Chapter V.) : others must be left in the dry form, as before mentioned; and some few it is impossible to preserve un- changed for any length of time. Crystals, however, are amongst the most beautiful and interesting subjects for polarization ; and it is very probable that, by the aid of the polariscope, new and valuable facts are yet to be made known. For one who finds pleasure in form and colour, there is a field here which will only open wider upon him as he advances ; and instead of being in anywise a merely mechanical occupation, it requires deep and careful study. The little here said on the subject will show this in some degree. With almost every salt the method of crystallization must be modified to obtain the best forms ; I may even go further than this, and say that it is possible to change these forms 112 PREPARATION AND MOUNTING to such a degree that the eye can perceive no relationship to exist between them. If a solution of sulphate of iron be made, a small quantity spread evenly upon a slide, and then- suffered to dry whilst in a flat position, the crystals often resemble the fronds of the common fern in shape. But if, whilst the liquid is evaporating, it is kept in motion by stirring with a thin glass rod, the crystals form separately, each rhombic prism having its angles well defined, and giving beautiful colours with the polarized light. Again, pyro-gallic acid, when allowed to flow evenly over the slide in a saturated solution, covers the surface in long needles, which are richly coloured by polarized light ; but if any small portion of dust or other matter should form a nucleus around which these needles may gather, the beauty is wonderfully increased. A form very closely resembling the " eye " of the peacock's tail, both in form and colour, is then produced, which to one uninitiated in crystallography bears very little resemblance to the original crystal. From these simple facts it will be clearly seen that in this, as in every other department, study and experience are needful to give the best results. By dropping a saturated solution of any salt into alcohol where it" is not soluble in the alcohol crystals are in- stantaneously produced, and the results are often very curious and beautiful. These crystals can easily be taken up by a pipette deposited upon a slide, and, after having been allowed to dry spontaneously mounted in balsam. To obtain anything like uniformity in the formation of crystals upon the glass slide, every trace of grease must be removed by cleaning with liquor potassaa or ammonias im- mediately before using, care also being taken that none of the agent is left upon the slide, otherwise it may in- terrupt and change their relative position, and even their form. Amongst those which are generally esteemed the most beautiful, are the crystals of oxalurate of ammonia. The preparation of this salt from uric acid and ammonia is a OF MICROSCOPIC OBJECTS. 113 rather difficult process, and will not, on that account, be de- scribed here ; but when possessed, a small quantity of a strong solution in water must be made, and a little placed on the slide, and evaporated slowly. Part of the salt will then be deposited in circles with the needle-like crystals extending from common centres. They should then be mounted in pure Canada balsam ; and, when the best colours are wanted, used with the selenite plate. Of this class of crystal, salicine is a universal favourite, and can be easily procured of most chemists. The crystals may be produced in two ways : A small portion of the salt must be placed upon the slide, and a strong heat applied underneath until fusion ensues ; the matter should then be evenly and thinly spread over the surface. In a short time the crystals will form, and are generally larger than those procured by the following process ; but the uncertainty is increased a little when fusion is used, which, however, is desirable with many salts. Secondly, make a saturated solution of salicine, which is effected by boiling one part of the salt in eighteen of water, and allowing it to cool. Place a little upon the slide, and let it evaporate spontaneously, or with the aid of gentle heat. The crystals are generally uniform, and with ordinary powers quite large enough to make a beautiful object. Their circular shape and gorgeous colours even without a selenite plate have made them such great favourites that there are few cabinets without them. There are also some salts which are crystallized in a some- what different manner from those before mentioned. San- tonine is one of the most beautiful, and will illustrate my meaning. Place a small portion upon a slide, and heat over a lamp until it is fused. With a hot needle spread the salt over the surface required. As the slide cools, the formation of crystals takes place, until it becomes one mass. This salt is slightly soluble in the ordinary balsam, and should be mounted in castor oil. If, however, the slide is well covered under the thin glass, the balsam soon becomes; saturated, and very little injury results. According to the i 114 PREPARATION AND MOUNTING temperature during crystallization the character of the crystals is affected. If the fused salt is very hot, the crystals run in straight lines from a common centre. If the heat is (what I may term) medium, the crystals show concentric waves of very decided form. If the slide is cool, the crystals, still concentric, are exceedingly minute. The most beautiful crystals for the microscopist are those formed at a tem- perature betwixt the second and third above mentioned, as the minute and wavy forms are then combined, and long feathery crystals are the result. As this method requires some little practice, many crystallize the s.alt in a simpler manner, which I will give ; but the variations obtainable by fusion give that mode the precedence. Dissolve a few grains of santonine in a drachm of chloroform, and drop the solution upon a glass slide. Allow the liquid to evaporate, and beautiful crystals will be the result. Mount as above. In fear of being somewhat uninteresting to part of my readers, I feel as though I should not be fulfilling my desire of giving every information, if I omitted to show another method of crystallization, which a novice would cast away as a failure before he had completed his experiment. Tartrata of soda, made by neutralizing a strong solution of tartaric acid by the addition of carbonate of soda, is spread in solu- tion over a glass slide, and must be then warmed, but net boiled. It must now be laid in a dry place, protected from all chance of dust. In time from one or two days to as many weeks some of the slides will prove beautiful objects, showing the cross form surrounded by rays. Some of these slides never crystallize, though I can find no reason for this, and even the application of heat to these calls out no decided form. Hippuric acid will be found most interesting to those who are fond of beautiful polariscopic effects, inasmuch as this salt is capable of giving an astounding variety in the forms of its crystals. Make a saturated solution in absolate alcohol, and use it warm ; by dropping a small quantity from a warm pipette on to a warm slide a film will spread OF MICROSCOPIC OBJECTS. 115 over the slide and crystals of a circular form will begin to appear, and may be modified by the atmosphere in which they are allowed to grow ; thus a moist atmosphere or the reverse, an atmosphere of vapour of ammonia, spirit, benzole, or sulphureous fumes, will each produce a different result, and the modifications thus produced will afford food for very serious reflection on the changes one salt may be made to assume in contact with other agents. These crystals are best mounted in castor oil balsam that is very liquid not balsam in benzole, as the benzole changes the character of the crystal. Many new forms may be procured by uniting two totally different salts in solution in certain proportions. This is a field affording new facts and beauties ; but requires some chemical knowledge and much perseverance to obtain very valuable results. One of the most beautiful I have met with has been composed of sulphate of copper and sulphate of magnesia. The flower-like forms and uniformity of crystallization when successful make it well worth a few failures at first ; and as I became acquainted with some new facts in my frequent trials, I will give the preparation of the double salt from the beginning. Make a saturated solution of the two sulphates, combined in the proportion of three parts copper to one part mag- nesia, and then add to the solution one-tenth of pure water. No dust or other impurities should have access to the slide, and it should be freed from all traces of grease by cleaning immediately before use with liquor potassse or ammonias. A drop of the solution should then be placed upon it, and by a thin glass rod spread evenly upon the surface. Heat this whilst in a horizontal position until the salt remains as a viscous transparent substance, which will not be effected until it is raised to a high degree. The slide may now be allowed to cool, and when this is accomplished, the flower- like crystals will be perceived forming here and there upon the plate. When these are at any stage in which it is wished to preserve them, a few seconds' exposure to the fire, T 2 116 PREPARATION AND MOUNTING AS warm as the hand can comfortably bear, will stop the expansion, when the portion which we wish to mount should be cut off from the mass of salt by simply scratching the film around, and pure Canada balsam with the thin glass used. Breathing upon the film, or allowing the slide to become cold and attract the moisture from the atmosphere, will cause the crystallization to extend, and sometimes greatly rob the effect ; so it is necessary to mount quickly when the desired forms are obtained. As the crystals are very uncertain as to the place of their formation, I may here mention that they may be got in any part of the slide by piercing the film with a needle-point ; but in some degree this necessarily interferes with the centre. Into the cause of this we have no need to enter here, and as it has been flsewhere discussed, I can only give the above directions, and say that there is a great field in this branch of study which the microscope alone has opened. It would be useless to enter into particulars respecting the various salts and treatment they require, as a great differ- ence is effected even by the strength of the solution. There are some crystals, also, which are called forth in insulated portions, showing no formation upon the ground ; but even when mounted in any preserving fluid, and unchanged for a year, a new action seems to arise, and a groundwork is pro- duced which bears little resemblance to the original crystal. Sometimes this new formation adds to the beauty of the slide ; in other cases the reverse is the result, the slide being rendered almost worthless. This action, I believe, frequently arises from some liquid being contained in the balsam or other mounting medium used ; and this is rendered the more probable by the crystallization being called forth in an hour after the balsam diluted with chloroform is employed, whereas no change would have taken place for months (if at all) had pure balsam been used. Sections of some of the salts are very interesting objects ; but the method of procuring these and their nature will be described in Chapter VI. OF MICROSCOPIC OBJECTS. 117 Few objects are more beautiful with polarized light than young oysters. Good colours and a decided cross are given by them when well prepared. The following is the method pursued by Mr. Henry Lee : Having found a 41 black-sick " oyster (to use the dredgemen's term), the spawn of which is quite mature and ready for extrusion, pour off from the shell the da,rk slate-coloured fluid into a long narrow two-ounce phial ; fill up the bottle with distilled water ; shake it gently ; allow the deposit to settle, and change the water two or three times, repeating the agitation to get rid of the salt. Then substitute for the water liquor potassse, diluted with equal quantity of distilled water. Allow the young oysters to remain in this for two days, agitating occasionally ; and as often as the liquid becomes discoloured pour it off, and renew the same until no colour is given off and the shells are seen to be thoroughly cleansed from all animal matter by their sinking freely and rapidly to the bottom. When this stage is arrived at, stop the process, that the two valves of the shells may not be sepa- rated by the destruction of the hinge. Wash repeatedly in distilled water, to remove all trace of alkali, and finally wash and preserve the shells in a little rectified spirits of wine (not methylated spirit). These objects are frequently mounted in balsam, to increase their transparency for the polariscope, but where they are sufficiently clear they may be mounted dry like the foraminifera. The scales of various fish have been before mentioned as mounted dry ; when, however, they are required for polarizing objects, they are generally mounted in balsam, and some few in liquid. The former method will be con- sidered here. The jifiLaffords a beautiful object for this purpose. The scales are covered by a thin skin, which may be slightly raised with a knife and then torn off, in the same manner as the covering of the geranium and other petals, described in Chapter III. The required portion may then be removed ; or if a piece of skin can be procured as stripped off in 118 PREPARATION AND MOUNTING cooking, the scales may be easily taken from the inner surface. They must then be washed and thoroughly cleaned. After drying, soak for a day in turpentine, and mount in the ordinary manner with balsam. This is a good polarizing object; but the interest, and I think the beauty, is increased by procuring a piece of eel's skin with the scales in situ, washing and drying under pressure, and mounting in balsam as before. The arrangement of the scales produces beautiful " waves " of colour, which aro quite soothing to the eye after examining some of the very gorgeous salts. There are many scales of fish which are good subjects for the polariscope when mounted in balsam ; but as they re- quire no particular treatment, they need no mention by name. Among hairs we find some which are beautiful when mounted in balsam and examined by polarized light. Some, when wanted as common objects, are always used dry, as before mentioned ; but if they are intended to be shown as polarizing objects, they must be placed in some medium. The Micrographic Dictionary mentions a mode of making an interesting object by plaiting two series of white horse- hairs at an angle, mounting in balsam, and using with the polariscope. All hairs, however, must be steeped in tur- pentine for a short time before mounting, as they will thus be rendered cleaner and more transparent. When this is done, there is no difficulty in mounting them. Many of the " tongues " of fresh-water and marine mollusca are deeply interesting and most beautiful objects when examined by polarized light. As these are usually mounted in balsam, I mention them in this place ; but as they must be removed from the animals by dissection, particulars respecting them will not be entered into until we come to the part in which that operation is described (Chapter VI.). The manner of preparing and mounting many of the Polyzoa and Zoophytes has been before described ; but any OP MICROSCOPIC OBJECTS. 119 notice of polarizing objects would be incomplete without some allusion to them. A small piece of the Flustra avicularis, well prepared, is beautiful when examined in this manner. No selenite is needed, and yet the colours are truly gorgeous. It is often met with upon shells and zoophytes of a large size, and will well repay the trouble of searching for. Many of the Sertularidae are very beau- tiful with polarized light, and, indeed, no ramble upon the seaside need be fruitless in this direction. The different starches are quite a study in themselves, and are peculiarly connected with polarized light. They are found in the cellular tissue of almost every plant in small white grains, which vary considerably in size ; that from the potato averages one-three-hundredth of an inch in diameter, and 1 that from arrow-root about one-six-hundredth. To procure starch from any plant, the texture must first be broken up or ground coarsely ; the mass of matter must be then well washed in gently-flowing water, and, as all starch is totally insoluble in cold water, the grains are carried off by the current and deposited where this is stayed. In pro- curing it from the potato, as well as many other vegetables, it is but necessary to reduce the substance to a coarse pulp by the aid of a culinary grater ; the pulp should then be well agitated in water, and allowed to rest a short time, when the starch will be found at the bottom, its lighter colour rendering it easily distinguishable from the pulp. It should, however, be washed through two or three waters to render it perfectly clean. These grains have no crystalline structure, but present a very peculiar appearance when examined with polarized light. Each grain shows a dark cross whose lines meet at the point where it was attached to the plant, called the hilum. Bound the grain also a series of lines is seen, as though it were put together in plates. This is more dis- tinctly visible in some kinds than others. As to the mounting of these starches there is little to be said. If the grains are laid upon the slide, and as small a 120 PREPARATION AND MOUNTING portion as possible of the balsam diluted with turpentine, as before mentioned, be applied, they will cling to the glass and allow the pure balsam to flow readily orer them with- out being so liable to imprison air-bubbles when the thin glass is put upon them. The raphides, which were fully described in Chapter III., when required for use with polarized light, must be mounted in balsam, and many are found which give beautiful colours. They require no peculiar treatment, but must be washed quite clean before putting up. But in order to understand anything of the natural arrangement of raphides, it is necessary to mount certain parts of plants with these objects in situ. The most common is the coating of the onion, which must be soaked some time in turpentine or benzole, in order to render it transparent, and must then be mounted in balsam, as before said We shall then be able to obtain such colours by the aid of polarized light, that the raphides are shown in wonderful distinctness, and somewhat of their nature will be perceived. There is one class of objects for the polariscope which differs in preparation from any we have yet considered, and affords very beautiful specimens. Some of the plants, in- cluding many of the grasses and the Equisetacese (i.e. horse- tails), contain so large a quantity of silica, that when the vegetable and other perishable parts are removed, a skeleton of wonderful perfection remains. This skeleton must be mounted in balsam, the method of performing which will now be considered. Sometimes the cuticle of the equisetum is removed from the plant, others dry the stem under pressure, whilst the grasses, of course, require no such preparation. They should then be immersed in strong nitric acid and boiled for a short time ; an effervescence will go on as the organic matter is decomposed, and when this has ceased, more acid should be added. At this point the modes of treatment differ; some remove the object from the acid and wash, and having OF MICROSCOPIC OBJECTS. 121 dried, burn it upon thin glass until all appears white, when it must be carefully mounted in balsam. I think, however, it is better to leave it in strong acid until all the substance, except the required portion, is removed ; but this will take a length of time, varying according to the mass of the plant. Of course, when this latter method is used, the skeleton must be washed from the acid, &c., before being mounted in balsam. These siliceous cuticles are readily found. The straws of most of the cereals, wheat, oat, &c. ; the JiusJes, also, of some of these ; many canes ; the equisetum, as before described ; and some of the grasses. Many of these are everywhere procurable, so that the student can never want material for a splendid object for the polariscope. In Chapter III. the scales (or hairs) which are often found upon the leaves of plants were mentioned as beautiful objects when mounted dry; but some of these when detached from the leaf which is easily done by gently scraping it, when dried, with a knife present brilliant starlike and other forms, if mounted in balsam and used with the polariscope. There is a little danger, when placing the thin glass upon the balsam, of forcing out the scales in the wave of matter which is always ejected ; this may be overcome by applying to the slide, previously to placing the objects upon it, an extremely thin covering of balsam diluted with turpentine as before mentioned, letting it dry more or less with the objects placed in it, and then, after the addition of a little more balsam, putting the cover on, and thus giving them every chance of adherence; or by using the balsam with chloroform, as before noticed. This method is peculiarly successful in cases where it is desired to arrange several objects symmetrically on a slide, and to obviate their sub- sequent disturbance by placing the cover on. Type slides with several parts of an insect displayed upon them, scales of fish, or of plants, &c., may thus be shown, so that the number of slides may by this plan be seriously diminished. 122 PREPARATION AND MOUNTING These scales are much more abundant than was formerly supposed, and new specimens are discovered daily ; so that the student should always be on the look-out for them in his researches in the vegetable world. Most classes of objects, and the treatment they require when mounting them in balsam, have now been considered. The next chapter will be devoted to preservative liquids, and the best method of using them. OF MICROSCOPIC OBJECTS. 12$ CHAPTER V. PRESERVATIVE LIQUIDS, ETC., PARTICULARLY WHERE CELL* ARE USED. THERE are many objects which would lose all their distinc- tive peculiarities if allowed to become dry, especially those belonging to the fresh-water Algse, many animal tissues, and most of the very delicate animal and vegetable sub- stances in which structure is to be shown. These must be preserved by immersion in some fluid ; but it is evident that the fluid must be suited to the kind of matter which it is intended to preserve. As it often requires much study and trouble to obtain microscopic objects of this class, it is well that their preservation should be rendered as perfect as possible; and for this reason the CELLS, or receptacles of the fluids, should be so closed that all possibility of escape should be prevented. The accomplishment of this is not so easy a matter as it might appear to the inexperienced. Before giving any directions as to the manipulation required in mounting the objects, we must consider the different liquids and cells which are requisite for their pre- servation. Of the former there are a great number, of which the principal may be mentioned. DISTILLED WATER is strongly recommended by many for Diatomacese and other Protophytes. It has been, however, stated that confervoid growths often disturb the clearness of the liquid, and on this account various additions are made to it. A lump of camphor is often left in the bottle, so that the water may dissolve as much as possible. One grain of bay salt and one of alum are added to each ounce of water ; or a drop or two of creosote shaken up with an ounce of water, which should be afterwards filtered. These additions 124 PREPARATION AND MOUNTING are often made; perhaps each of them good for certain objects. GLYCERINE. Some affirm this to be one of the best pre- servative liquids, especially for vegetable objects ; but others think that it is much better when diluted with two parts of camphor-water, prepared as above.* Mr. A. E. Verrill, of Yale College, U.S., says glycerine preserves the natural colours of marine animals ; and the only precaution to be taken is to use very heavy glycerine, and to keep up the strength by transferring the specimens to new as soon as they have given out water enough to weaken it much, re- peating the transfer till all the water is removed before finally mounting on the slide. GLYCERINE AND GUM. This is also believed to be a very good liquid for vegetable tissues, and is thus prepared : Pure gum-arabic... 1 oz. Glycerine 1 Water (distilled)... 1 Arsenious acid ... 1^ grain. Dissolve the arsenious acid in the cold water, then the gum, add the glycerine, and mix without bubbles. Dr. Carpenter states that the proportions used ultimately by the late Mr. Farrants are : Picked gum-arabic ... 4 parts by weight Distilled water (cold).. 4 Glycerine 2 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." or MICROSCOPIC OBJECTS. 125 per) a small piece of camphor. This requires no cell, as tht* adhesive power is sufficient. DEANE'S COMPOUND. This is usually deemed about the best medium for preserving Alga3, 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 cooled, but not enough to become stiff, add | 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 egg. 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 of 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. Eemove, 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. GOADBY'S FLUID. 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 qual 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. THWAITES'S LIQUID. This is recommended for the preser- vation of Algae, &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 OF ZINC SOLUTION. 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 MICKOSCOPIC OBJECTS. 127 plaints that this mixture becomes turbid with keeping ; but 1 think this must only be the case when some impurity has got into the bottle. CARBOLIC ACID.* 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 : Arsenious 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 Algae, 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 OIL. 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. Grace 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, &c., 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 CELLS. 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, e. Dr. Bastian says the best c&mtmt far lignfrj cells is one, much used in Germany, made by adding a considerable quantity of nitrntp of hifitnm|i to a solution of guni mastjcj rM nrnfn rm 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 RINGS 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 trustworthy 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 330 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. GLASS RINGS. AVhere anv 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 rou<*h, 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. LSI 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 RINGS. 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. Zinc and pure tin rings may also be procured, and are excellent, especially the latter. VULCANITE. 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. 132 PREPARATION AND MOUNTING We will now suppose the cell employed hns been made by placing a glass ring upon tiie siicle 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-fours 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 tie 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 under the receiver of an air-pump, and the air withdrawn. Almost immediately it will be perceived that the bottom and sides of tne cell are covered with minute bubbles, which are formed by the air that is held in sus- pension by the liquid. The slide may row 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. 133 the air-pump and exhausted. It may then be transferred to the cell, and will probably cause the liquid to overflow a little. The cover with the gold-size applied to the ed 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. L2 148 PREPARATION AND MOUNTING SECTIONS or BONE. With the aid of the microscope few fragmentary remains have proved so useful to the geologists 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 maybe 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 ground 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 down upon the 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. 0. 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 debris" 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 Tessels which penetrate the lacuna* and eanaliculi 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 lacunce, bone cells, and eanaliculi (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, &c , ia dissolved away and the cartilage is left, which may be cut into sections: ia 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 in 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 also 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 Micrographic 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 conies 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 a short 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 PllEPARATION 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 solidiby 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, aa 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 inicroscopist may find endless amusement or study in it. From a single section the das* 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 J. 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. 15S 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 sanoe 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, it is 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 rerolve, 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, nd the section cafe by a circular act'on. 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 Y. 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 number of specimens is required it iy 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. To cut 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. 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 subiects as anatomical prepara- 156 PREPARATION AND MOUNTING lions 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 shav- 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, Hie 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 or Mir.aosnopTO OBJECT*. t*iT rings vrith a cross, the long line of which passes through both, the short line dividing it in the miad^e. The process of cutting these sections is rather difis