INTRODUCTION.

THE science of PATHOLOGICAL HISTOLOGY at the present day is not alone concerned with investigation of the minute structure of the tissues, but it has also in many instances to decide particularly as to the presence or absence of bacteria or other micro-organisms in them and their juices, as well as to determine the nature of such micro-organisms should they occur. As, however, widely different species of research are required for these two purposes, the methods of investigation employed in Pathological Histology are naturally divided into Histological and Bacteriological.

CHAPTER I.

HISTOLOGICAL METHODS OF INVESTIGATION.

1. The Examination of Fluids.-A medium-sized drop of the fluid to be examined should be placed on a slide by means of a loop of platinum wire or a glass rod. If the fluid is very poor in solid elements, the detection of the latter is facilitated by laying a hair or fine thread in the drop; if, on the other hand, it is very rich in solids, it must be diluted by the addition of an indifferent liquid. A 0.5 to 0.7 per cent. solution of common salt is most frequently used for the latter purpose, but this cannot be kept long in stock, as it readily becomes mouldy. Distilled or tap water may, however, also be used, if not dealing with structures which are very easily damaged.

When the examination is more protracted it is necessary, in order to prevent the object from drying up, from time to time to place at the margin of the cover-glass a drop of the fluid used for diluting, or to smear its edges with vaseline, melted paraffin or wax, glycerin, or the like; or, best of all, to carry out the examination in the hanging drop (p. 25). If it is desired to stain any cells which may be present in the fluid, a drop of picro-carmine, alum, cochineal, or of aqueous solutions of the anilin dyes (p. 26), can be placed at the margin of the cover-glass, whilst a piece of blotting-paper should be torn off and applied to the opposite margin, so as to increase the speed with which the staining fluid flows in. The superfluous stain can afterwards be removed by irrigation with water or a per cent. solution of acetic acid; and if it is wished to render such a preparation permanent, a 50 per cent. aqueous solution of potassium acetate, or glycerin, may then be similarly applied, and the preparation finally cemented (p. 22). For the mode of applying reagents, see p. 7.

Fluids are also in many cases examined in the dried condition, in what are called cover-glass preparations (p. 26).

2. The Examination of Fresh Tissues.-Tissues can be examined either in the fresh state, or after preliminary fication and hardening. The former of these methods must not be neglected; indeed, it is absolutely indispensable for the study of certain changes. The procedure consists in scraping the juice from the cut surface of the fresh preparation, or cutting out a very minute particle with a pair of scissors curved on the flat and then tearing it up as finely as possible by means of preparation needles (sewing needles firmly fixed by a clamp in wooden handles) in a drop of an indifferent fluid; or, lastly, by endeavouring to make thin sections, which may be done with a razor or double knife, or, which is far preferable, by means of the freezing microtome (p. 15).

Preparations made in this way are either simply examined in salt solution, or may be treated with stains and reagents, and, if desirable, preserved, in a manner analogous to preparations of fluids. They can also be kept in salt solution for a short period by depositing the slides in a moist chamber, such as is used for culture plates (Fig. 7).

The needling of fresh tissue can be very much facilitated by previous immersion in rather small pieces in macerating fluids, for which purpose the following may be used:—Müller's fluid (p. 8), or 0·01 to 0.05 per cent. solutions of chromic acid (especially for portions of the brain and spinal cord, which are left several days in the fluid); 01 per cent. osmic acid (particularly for tissue containing fat and for nerves-action twelve to twenty-four hours); 20 per cent. solution of nitric acid (isolates smooth muscle fibres in a few days); concentrated hydrochloric acid (for the canaliculi of glands—twelve to twenty-four hours' action); baryta water and lime water (for nerves, muscles, and connective tissue, which remain six hours in the former, several days in the latter); 33 per cent. solution of caustic potash or soda (for smooth and striated muscle, the cementing substance of which dissolves even within an hour); and, lastly, 33 per cent. alcohol prepared by mixing one volume of 90 per cent. alcohol with two of water (for epithelial structures, which are immersed for one to two days, the fluid being frequently shaken).

Macerated and needled preparations can be examined either in a drop of the macerating fluid itself—which is, indeed, unavoidable when using the 33 per cent. solution of caustic potash or sodaor else in salt solution or water, the latter being indicated after maceration in strong acids. For tearing up objects macerated in osmic acid glass needles must be used, which may easily be obtained by drawing out a glass rod in a gas-flame.

In preparing various objects for microscopic examination, it is advantageous to employ black and white backgrounds for unstained

and stained preparations respectively, for which purpose glass plates painted black and white, or the like, may be used.

3. Reagents. The ordinary mode of using reagents is either by applying them directly to the uncovered object, or by allowing them to flow in under the cover-glass from the side, a scrap of blottingpaper being laid at the opposite edge in order to secure more rapid penetration. Sections may also be placed for some time in a watchglass containing the reagent. The following are the most useful reagents :

(1) Acetic acid is usually employed only in very dilute form, as a to 2 per cent. solution, for the purpose of rendering cell nuclei and elastic fibres more distinctly visible, the albuminoid substances of the body of the cell and the connective-tissue fibres being caused by it to swell and thus become more transparent. Mucin is precipitated by acetic acid without re-dissolving in excess of it.

(2) Hydrochloric acid is used as a decalcifying reagent in a 3 to 5 per cent. solution. It dissolves calcium carbonate with, calcium phosphate without, liberation of bubbles of gas.

(3) Sulphuric acid (in 25 per cent. solution) in like manner dissolves out the calcium salts, with formation of crystals of gypsum—prisms grouped in bunches. It is also used as a reagent for amyloid substance and cholestearin.

(4) Osmic acid into 1 per cent. solution serves as a test for the recognition of fat and myelin, which are coloured by it of a tint varying from brown to black. A similar colour is, however, assumed by the granules in the so-called Mastzellen and occasionally even by those in cells which have undergone parenchymatous degeneration. The acid must be kept in brown glass bottles, and the vessels and implements used in its application must be made of glass, and be free from organic impurities.

(5) Caustic potash or soda, in 1 to 5 per cent. solution, causes all albuminoid bodies, those in cell-nuclei included, as well as gelatinous substances, to swell up and become transparent, so that all the remaining elements come out more distinctly, and thus these reagents can be used for the recognition of fat, lime, elastic fibres, pigment, etc.

(6) Iodine and potassium iodide are used in Luyol's solution (iodine, 1; potassium iodide, 2; distilled water, 300) in testing for glycogen, amyloid substance, and cholestearin.

(7) Ether and chloroform for removing fat (p. 53).

4. Fixation and Hardening.-Owing to the incompleteness of the methods for examining fresh tissue, fixation and hardening of the latter must in most cases be carried out, the object being, on the one hand, to ensure the least possible amount of change in the structure