EMBEDDING IN PARAFFIN 13 paraffin, then for five to ten minutes in chloroform, and from that, in case the pieces have already been previously stained en massethough this is but seldom possible in Pathological Histology-are transferred to absolute (or 96 per cent.) alcohol, ethereal oil, and balsam (pp. 21-22); otherwise, however, they are removed from the chloroform into 80 per cent. alcohol, then into water, and finally into aqueous staining solutions. When the sections are very fragile, or would fall to pieces on dissolving out the paraffin, they (and especially ribbon sections) must, before undergoing further treatment, be fixed upon well-cleaned microscopic slides, either by smearing the latter with a thin layer of a medium consisting of 1 vol. collodion and 2 vols. oil of cloves, and laying and arranging the sections upon this, after which the slides are further placed for ten to thirty minutes in a thermostat at 52° C.; or more simply, by spreading out the sections upon the slide in alcohol, pressing them with Swedish filter paper several times folded, and then transferring the slides for several hours. to a thermostat at 35° C. When cool, the slides are treated by the method already given for sections, in order to dissolve the paraffin, for which purpose vessels of such a shape (Fig. 1) as to admit of 1 Microscopic slides are cleansed first in absolute alcohol, and then in distilled water; or with still greater certainty by depositing them in concentrated nitric acid, which is then washed away with distilled water, this being further followed by cleansing with absolute alcohol and distilled water, and finally drying with a clean cloth. A very excellent mode of cleaning and disinfecting slides which have already been in use consists in boiling them for a quarter- to half-an-hour, with repeated stirring, in a 5 to 10 per cent. solution of lysol (in which they may also be kept for a longer time), and immediately afterwards, that is, before they have begun to cool, washing them in a strong jet of water until it flows away pure, and finally wiping them in a cloth free from grease. Cover-glasses which have been used can also be cleaned in like manner, but these are first removed from the slides by warming the latter over a flame, and are then boiled separately, in order, as far as possible, to avoid breaking them. several slides at once being arranged in them in rows may be used with advantage. The kind of paraffin embedding just described, i.e., saturation, allows of the preparation of very thin sections, and is especially suitable for obtaining serial sections; but it has, on the other hand, the disadvantage that, owing to the temperature required to melt the paraffin, shrinkages take place in the tissues, or during the subsequent solution of the medium small particles may drop out of sections if their elements are very loosely connected. 8. The Preparation of Sections. Microtomes. For rough examination it may be sufficient to make sections by hand only, with a razor ground hollow on both sides or at least on the upper, a smooth cut surface being first secured, and the razor made to work more by moistened, in cutting fresh specimens, with a fluid consisting of 2 SECTION CUTTING 15 parts of alcohol and 1 part of glycerin diluted with water to half its strength; in cutting hardened preparations alcohol alone is used. Nowadays, however, special microtomes, of which there are several types, are far more frequently used-that is, when fine and even sections are desired. Those made by Jung of Heidelberg, Reichert (Fig. 2) and Fromme of Vienna, Katsch of Munich, and Schanze of Leipzig, can be recommended: their modes of action may be seen from the catalogues of the respective makers. The objects to be cut are fixed in a suitable clamp, for which purpose preparations which are not embedded must be wedged into amyloid liver, or attached to blocks of cork or wood with glycerin jelly. This is prepared as follows:-Fine gelatin is cut up and allowed to swell for some hours in water; the water is then poured off, and the swollen gelatin boiled up with an equal volume of glycerin to which a little camphor or a trace of corrosive sublimate is added to guard against the growth of mould, filtered through linen, and then allowed to set. In use a small piece is liquefied over a flame and placed on the roughened surface of a cork or wooden block; the preparation, which must not be more than cm. in height, is pressed upon it, and the whole is deposited in strong alcohol to set. In cutting, the knife of the microtome is, as a rule, fixed at the most acute possible angle with the preparation,-indeed, in such a way that the whole of the edge can act; but occasionally a different position is more advantageous. The knife, as well as the preparation, must be kept continually moistened with a hair pencil dipped in alcohol, and the sections are removed by the same means. preparations embedded in paraffin are best cut dry (p. 12). Only To avoid tearing very large sections, particularly of the brain, it is advisable to carry out the cutting altogether under fluid, and for this purpose the instruments known as immersion microtomes are adapted. Another special variety is the freezing microtome (Fig. 3), in which the preparation is laid on a metal plate and frozen by causing an ether spray to act on the lower surface of the latter. The method is applicable to both fresh and hardened objects; in the former, however, the freezing causes certain alterations of structure, and is therefore only to be recommended in cases where it is wished to obtain tolerably fine sections quickly. In order that the preparations may freeze well, they must be thoroughly saturated with water; so that objects hardened in alcohol are to be completely freed from the latter by protracted soaking in water, say for twelve to twenty-four hours. Preparations preserved in Müller's fluid, however, may be used at once, or after lying in water for a short time. If the red corpuscles are to be retained in fresh objects which it is intended to cut with the freezing microtome, the specimens are previously immersed for some hours in Müller's fluid, a proceeding which is, indeed, to be recommended in other cases also. FIG. 3.-FREEZING MICROTOME [known in this country as The pieces destined for freezing ought not to be more than about cm. in height, and, while the ether spray is acting, are lightly pressed down upon the plate until they become frozen to it. If there is difficulty in getting this to succeed, the under surface of the preparation may be smeared with liquid glue. The piece, although completely frozen, should not be too hard. Cutting is usually done dry, and the sections transferred to salt solution, or to Müller's fluid diluted with water. The sections are either examined unstained in one of the fluids already mentioned, or are carefully transferred to alcohol (first to weaker, then to stronger), and then stained and treated like sections of hardened. objects. Lastly, preparations which have already been embedded in celloidin may also be cut with the freezing microtome, but they must previously be soaked in water for at least twelve hours. 9. Preparation of Serial Sections. (a) Of Celloidin Preparations.Narrow strips, about twice the width of the sections, are cut from tough paper, such as sanitary paper, and with these the sections are removed from the knife of the microtome by bringing one of the strips, lightly strained, down upon the section as it lies floating in a moderate quantity of alcohol close to the edge of the knife, and then drawing it away to the left in the plane of the surface of the blade. In this way rows of sections are obtained upon the paper strip, in which each SERIAL SECTIONS. STAINING 17 The section must always come to the right of the preceding one. paper strips must, until their transference to the slides, be kept moist with alcohol by spreading them with the sections uppermost upon several layers of blotting paper lying in a flat dish and well saturated with alcohol. As soon as the cutting is finished, the strips are placed, stretched tight and with the sections downwards, upon wellcleaned slides, over which some time previously a thin layer of collodion has been poured. They are then quickly covered with a few layers of filter-paper, pressed with the latter against the slide, and lastly drawn away, when the sections remain adherent to the glass and should still be somewhat moist. The slides are now placed with the sections uppermost in a wide but low cylindrical specimen glass, upon a table-shaped plate made of sheet metal perforated like a sieve; ether is poured on the bottom of the glass, which is closed, and thus the sections are exposed for any desired length of time to the ether vapour. In this manner the sections become fixed to the slide, and may then be stained and subjected to further treatment. (b) Of Paraffin Preparations.—If preparations of not too large dimensions are saturated with a paraffin suitable for room temperature (p. 12), and the paraffin block cut to shape so that two of its sides are parallel with the edge of the knife fixed transversely, the margins of the successive sections adhere, one below the other, when the knife is rapidly worked, and thus ribbons of sections are formed, which can be fixed and further treated by the methods given on page 13. 10. Treatment of Sections by Pencilling or Shaking. This is done, for the purpose of demonstrating the stroma, either by treating the section with perpendicular dabs from a pencil of badger's hair, while it is held fast on a slide by means of a needle and covered with plenty of fluid (water or glycerin), which must be repeatedly changed; or by shaking it up vigorously with water in a test tube. 11. Staining. The object of staining processes is to cause certain constituents of the tissue or of the cells to stand out with special distinctness; but since such processes invariably bring about alterations in the tissues, preparations should always, at least for more thorough study, be examined in the unstained condition also, which, when they are made from hardened tissues, is done in glycerin, either plain or diluted to half strength with water. In the case of unstained sections the reagents detailed on page 7 can be employed, with the exception of caustic potash and soda, which can act only on fresh tissue. In all staining processes, the following general rules must be observed: (1) The staining solutions should be filtered before use. (2) The sections, on removal from the alcohol in which they are |