THE

PREPARATION AND MOUNTING

OF

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.

Refer

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. 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. Stricker 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 phosphate 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 Stricker, Frey, Klein, Schultze, Kühne, Deiters, Leber, and others, many of whose processes he has personally verified, and of whose manuals, especially those of Beale, Stricker, and Frey, the student is advised to possess himself. He believes also, from his own early experiences, that some short rationale of the intentions of the processes and means of investigation used by well-known workers may be acceptable to the 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

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 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 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 either 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

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 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.

ÌST DIVISION.

Under our first head may be ranged the following:Acetic acid.

Liquor potassæ and sodæ.

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 (Kölliker),) 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 potassæ and liquor sodæ 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.