The preparation and mounting of microscopic objects

Front matter

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Med

THE

Preface To The Second Edition

rJ^HE reception accorded to this work has been so favourable as to induce the Publisher to issue a second edition, in which such new matter should be embodied as the progress of Microscopic science might require. He therefore applied to the Author, but he found to his regret that the state of Mr. Davies’s health was such as to forbid his undertaking the labour. He had, however, collected many valuable notes and memoranda, which he was willing to place at the disposal of any gentleman 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

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PREFACE TO THE SECOND EDITION.

his disposal, but also to make such additions as his experience might suggest in extension of the usefulness of the book to a new class of readers. — the Medical Student, and the Junior Medical Practitioner. 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 paragraph, to which the reader is now courteously referred.

Preface To The First Edition

J~N bringing this Handbook before the public, the Author believes that he is supplying a want which has been long felt. Much information concerning the “ Preparation and Mounting of Microscopic Objects ” has been already published ; but mostly as supplementary chapters only, in books written professedly upon the Microscope. From this it is evident that it was necessary to consult a number of works in order to obtain anything like a complete 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 oi many of his friends, in every department of

VLU 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 acknowledged 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 oversight, and his regret that such should have been the case.

W ARRINGTON

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MICROSCOPIC OBJECTS.

Introduction

This work having been written chiefly to help students, tne 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 of 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 preserving 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 which he should be familiar, so as to be able to speak with some degree of certainty of the nature of the tissues demonstrated 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 in 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 examination while fresh, viz. : —

1st. Fluid of the aqueous humour.

2nd. The serum of the blood.

3rd. Amniotic fluid, very fresh, in which a little iodine has been dissolved, making it of a faint yellow tint.

4th. Very dilute solutions of neutral salts, such as pnosphate 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 hereinafter 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 student, 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 is in the judicious selection of each one or more of them that the tact and discretion of the student will best

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be shown. He should bear in mind, too, that the same structure may well be submitted to various modes of inquiry, and that possibly new modes may occur to him which, though they may not serve to prove anything directly, may /3t 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, Oi 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 otherwise 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 preliminary to other modes of investigation.

10th. Electricity and heat.

11th. The moist chamber.

In dealing with structures by means of the agents comprised 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

B 2 i 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 beyond 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 contend with, viz., that surface markings only, or mostly, can be seen clearly (not to speak of the loss of light). The use of the draw-tube is the true remedy for this.

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1st Division.

Under onr first head may be ranged the following ; — Acetic acid.

Liquor potasses and sod©.

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 destroying 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 rendered opaque by it. Acetic acid renders some tissues transparent 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 potassaa and liquor sodse 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.

Concentrated sulphuric and hydrochloric acids, used cold, cause epidermic structures to swell up, so that their cells may be easily separated.

Tannin, dissolved in water or rectified spirit of wine, hardens gelatinous and albuminoid tissues • it also makes them shrink. Its solution in water has been used, as mentioned in another part of this treatise, as an injection preliminary to one of coloured gelatine, to prevent extravasation through the walls of the blood-vessels. It also colours the tissues a fawn-colour, or a very faint brown.

Lime water and baryta water, especially the latter, will, according to Rollet, dissolve the animal cement by which the fibres of connective tissue are held together. After a few days’ soaking such tissue, as well as tendon, may easily be teased out by needles.

Oxalic acid, in a cold saturated solution (1 acid, 15 water), according to Schultze, “ causes connective tissues to swell up and become transparent, while those formed of albuminous substances become hardened and isolated. Extremely 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 (Kiihne).

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 substances 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 transparent by means of a fluid which permeates them unequally , so that

OP MICROSCOPIC OBJECTS.

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.

2nd 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, heematoxyline, &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 maceration for 24 hours in the light.

For double staining by carmine and picric acid he recommends 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 carmine, 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 them. Id about half an hour they will become transparent, and may be mounted in Canada balsam.

“ A peculiar effect is thus obtained. Epithelial and glandular cells, muscles, and the walls of vessels show a yellowish colour, with reddened nuclei, while the connective tissue is not coloured by the picric acid, and only presents the carmine colour.”

Another mode of effecting the above is by adding to a saturated and filtered solution of picric acid in water, a strong ammoniacal solution of carmine, drop by drop, until neutralization takes place. Sections may be soaked in this solution, more or less dilute, for a sufficient time, and treated as in the previous method.

The other staining agents are : — nitrate of silver, osmic acid, chloride of gold, chloride of gold and potassium, proto-chloride of palladium. These are to be made into weak solutions in distilled water, in which the tissues, in section or otherwise, are to be placed, and then exposed to light for a sufficient time.

Leber recommends a mode of staining by Prussian blue, as follows : — Immerse the specimen in a weak solution of a protosalt of iron for five minutes, more or less, according to size or the thinness of the section. Then wash and move it to and fro for a few minutes in a 1 per cent, solution of ferro-cyanide of potassium until it assumes an intense and uniform blue colour. Then wash in water, soak in alcohol, and mount as usual. The effect is that of partial tingeing ; the colour penetrates very deeply, and the tissue may be subsequently stained with iodine, carmine, or fuchsine. This method has been used for the cornea of the frog.

Iodine 1 part, with 3 of iodide of potassium, dissolved in 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.

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3rd Division.

Under the third division of our list may be ranged the following agents : — alcohol, solutions of chromic acid, bichromate 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 yer 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 turpentine, and then easily and speedily put up for examination in balsam.

Dr. Beale recommends a mixture of alcohol and a solution of caustic soda for the preservation of delicate tissues. He observes, “ that alcohol alone tends to coagulate albuminous 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, 025 — 0*5 to 1 and 2 per cent, of distilled water is much used. On account of its deliquescence, it is most conveniently kept in a saturated solution, which may be diluted as desired; and very often the weaker this solution the better. When it has had the desired 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 solutions. There are some precautions needed for perfect success with this agent, for which the reader is referred to Frey’s “ Microscopic Technology.”

Bichromate of potash, in solutions of similar strength to those of chromic acid, may he 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 removing the preparation into alcohol for twenty-four hours.”

It is always advisable to divide the substances to be hardened into portions as small as convenient, since the larger often putrefy in the centre, though they harden at the surface. 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., are better prepared with this than by the preceding agent. One great element of success in these two processes is, that the volume of the solution should be very large in proportion to the size of the object; another, that the action should be commenced with a weak solution, and continued with a stronger.

It sometimes happens that objects may be hardened too much by these solutions, though there is less risk by the bichromate of potash. In such cases Frey recommends that they be soaked in glycerine for a few days, and even that it be added to the solutions at first. He, with Deiters, Arnold, Schultze, and Kiihne, claims for these solutions an effect of the most important kind, viz., that of “preserving the finest textural relations, while exerting a somewhat macerating action on them, so that very delicate organizations, especially in nerve tissues, may be made visible which were previously hidden, or not visible in examination of the fresh tissue.”

Hyperosmic acid and chloride of palladium are sometimes used for this purpose also. Their solutions may contain from one-fifth to one-tenth per cent, of distilled water.

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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 solutions of starch and gelatine, solid muscular fibre and skin, &c., which then acquire the power of resisting putrefaction. 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 firmness admirably adapted for section; but if they remain too long uncut they become of a consistence unfit for that purpose. 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.

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 assumes a consistency which will permit fine sections to be made with a cooled razor. The object is more convenient to handle if it is allowed to freeze on a piece of cork. Nerves and muscles have been treated in this manner with good results. Glands (salivary), livers, spleens, the lungs, skin, and the bodies of embryos (see Beale’s process for the same in this work), also ganglia, afford excellent appearances. Indifferent (or neutral) media, such as iodine serum, are to be used in examining such sections. Or the preparation may be held in paraffine wax (diluted or not with oil), or tallow, which have been melted, and the object suspended or plunged in them until they cool, and the cooling may be carried further, if needed, by freezing.”

In reference to this subject, Mr. Kesteven informs the author that he has found the 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 B-eports). If many sections are to be made from a brain, machinecutting saves much time. The razor should have some spirit of wine dropped on it, so as to prevent the sections adhering. The cutting machines are generally graduated (by a screw and index) on the upward movement, so as to enable one to judge of the thickness of the section ; but as the brain substance and paraffine are both yielding to a certain extent, the reading must be taken with allowance.

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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; but Dr. Beale speaks in the highest terms of its uses and advantages, and declares that the tissues gradually regain their original volume if left in it for a sufficient time. They then soften, and even swell up. His practice is first to immerse the specimen in weak glycerine solution, and then gradually to increase the density of the fluid. He recommends, also, “in order that tissues may be uniformly permeated with a fluid within a very short time after the death of an animal, that the fluid should come quickly in contact with every part of the texture.” This, he says, may be effected in two ways, by

A. Soaking very thin pieces in the fluid;

B. By injecting the fluid into the vessels of the animal.

He thinks that these properties more particularly appertain 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 io a degree of minuteness impossible in any other 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 increasing pressure and careful manipulation exceedingly thin layers can be obtained, without the relation of the anatomical 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 advantage which results from the use of glycerine for the preparation 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 investigated 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 lead, is a very efficient means of softening horny substances, whale-

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bone, <fec., and rendering them plastic. Very thin laminae of these substances may also be procured by the employment of a well-sharpened scraper, such as that used by cabinetmakers. 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.