FIXATIVES AND SUBSEQUENT TREATMENT OF SECTIONS. 61 To Fix Paraffin Sections on a Slide. There are several "fixatives" for serial sections, but the following will be found the most useful. (1.) Collodion and Clove-Oil.-Mix one part of collodion with three parts of clove-oil. By means of a brush paint a thin layer on a slide, and on it place the sections. Heat gently over the flame of a lamp, to fix them firmly and drive off the clove-oil. (2.) Albumen and Glycerine (P. Mayer).-Mix filtered fresh white of egg with an equal volume of glycerine, add a little carbolic acid or morsel of thymol to prevent putrefaction. White of egg filters very slowly. A very thin layer is painted on the slide, and made smooth by means of a clean glass rod, which is thus prepared to receive the sections. The sections are flattened on the albuminised surface by means of a fine brush, care being taken that no air-bubbles remain under the sections. Warm the slide to a temperature just sufficient to coagulate the albumen (70° C.). This may also be done by holding the slide for a few seconds over a jet of steam. Such substances as acids and alkalies which dissolve the albumen must not be applied to the sections, nor must the sections be stained with such substances as picrocarmine, which also dissolve the albumen. (3.) Method of Gaule. This method depends on capillary attraction. The slide is moistened with water or weak spirit, and on this the paraffin sections are carefully spread out. The surplus spirit or water is removed by blotting-paper, and the slide placed in a thermostat at 50° C. for twenty-four hours. Sections so dried are heated for a moment above the melting-point of the paraffin, and are then firmly fixed on the slide. To Remove Paraffin from the Sections.-Sections of tissues soaked and embedded in paraffin and fixed on a slide are placed in turpentine, toluol, or xylol. The extraction of the paraffin requires some time, and takes place more rapidly when the temperature is raised. The slides may be fitted into a zinc framework and lowered into a bath of turpentine or toluol. Clove-oil must not be used if collodion and clove-oil have been used as a fixative. that case clarify with creosote and turpentine. The turpentine dissolves out the paraffin. In After this, if the tissue has been previously stained in bulk, before it was embedded, drive away the turpentine with xylol or clove-oil, and mount the section in balsam. If, however, the sections are from an unstained tissue, after dissolving out the paraffin with turpentine, the latter must be displaced by absolute alcohol, and the slides are passed through alcohols of various strengths and then into water. i.e., provided the sections are to be stained in a watery solution of a dye. The sections are then coloured in situ on the slide. If the sections are to be mounted in balsam, they must go through the same process in the reverse order, viz., increasing strengths of alcohol-a clarifying agent, clove-oil, or xylol-and finally they are mounted in balsam. Staining. This process depends on the fact that different tissues, or different parts of the same tissue, have an affinity for certain dyes, and not for others. Thus some dyes stain only the nuclei, others however may cause a uniform stain, all the tissues being of the same colour. By using some decolorising reagent, it is possible to remove the stain from certain parts of the preparation, leaving other parts stained. A thin section of a tissue or an organ, as a rule, when examined shows but little differentiation of its several parts. Only in cases where pigment is naturally present is this difference very marked. Some substances when applied to the section stain one part and leave other parts unaffected, thus enabling one to differentiate more easily the several parts of a section. Those substances which stain the nuclei chiefly have been called nuclear stains. The section is placed in a weak solution of the dye, e.g., hæmatoxylin; and after it seems to be sufficiently stained, the surplus dye is removed by thoroughly washing the section in water or alcohol, a part of the dye remaining united with the chromatin of the nucleus and colouring the latter. Such stains may also colour to a less degree some other parts of the section. Amongst nuclear stains are carmine, hæmatoxylin, and some of the aniline colours. When a section is stained, it is called Section Staining, but the tissue may be stained in bulk before the sections are made (p. 44) -staining in bulk. A. Carmine and its Compounds. Carmine. In order to obtain a strong solution of this dye, certain solvents require to be employed. It is readily soluble in ammonia, yielding an ammoniacal solution, which may be made strong or weak. The ammoniacal solution may be diluted to any extent required with water, and practically the best results are obtained by allowing sections to remain for a long time (24-48 hours) in a weak solution. 1. Strong Ammoniacal Carmine Solution.—Rub up in a mortar 2 grams of pure carmine with a few drops of water, add 5 cc. of strong liquor ammoniæ, mix thoroughly, and add 100 cc. of water. Place the whole in a bottle, and after a day or so any undissolved carmine is filtered off and the clear fluid kept as a stock solution. This solution may be diluted to any required extent. If it smell strongly of ammonia, the excess of ammonia must be allowed to evaporate. When the solution becomes neutral it is very liable to undergo putrefaction, but this may be avoided by placing a small piece of thymol in it to preserve it. 2. Frey's Carmine.-Ordinary carmine has two drawbacks: it is apt to undergo putrefaction, and as the ammonia escapes the carmine is precipitated. Carmine Distilled water 0.3 gram. 30 cc. Dissolve the carmine in the water, adding ammonia drop by drop until solution is complete. Then add— Glycerine 30 cc. 4 " and shake the mixture. Keep it in a stoppered bottle. It has no advantage, as far as coloration is concerned, over ordinary carmine, but it can be kept for a long time unchanged. 3. Alcoholic Borax Carmine (Grenacher). Dissolve the borax in the water and add the carmine, which is quickly dissolved, especially with the aid of gentle heat. Add 100 cc. of 75 per cent. alcohol, and filter. 4. Watery Borax-Carmine.-Rub up in a mortar 8 grams borax with 2 grams carmine, and add 150 cc. water. After twenty-four hours decant and filter. Tissues to be stained in bulk-e.g., after hardening with corrosive sublimate-are placed for twenty-four hours or longer in this fluid. They are then transferred to acid alcohol (1 per cent. HCl in 70 per cent. spirit for twenty-four hours), and then into alcohol. If it be desired to stain in bulk without bringing the tissue into contact with water, then use : 5. Carmine-Solution (P. Mayer).-For staining in bulk, and also for sections, Mayer recommends the following:-Suspend 4 grams carmine in 15 cc. water, and then add 30 drops hydrochloric acid, gently heating the mixture. Add 95 cc. alcohol (85 per cent.) and boil. Neutralise with ammonia and on cooling filter. 6. Borax Carmine (Grenacher). The borax dissolves the carmine. The whole is placed in a porcelain capsule and heated to boiling, when the fluid becomes of a dark-purplish or bluish-red. Add a few drops of 5 per cent. acetic acid, until the colour becomes more like that of carmine dissolved in ammonia. Let it stand for twenty-four hours, and filter. Add a drop or two of carbolic acid to preserve it. This gives a diffuse stain, so that the sections have to be treated with acid alcohol (p. 65).1 The original receipt is Borax-carmine is chiefly used for staining tissues "in bulk." Small pieces of tissue, to -inch cubes or larger, may be left in it for days, and they do not become over-stained. It gives by itself a diffuse stain; hence to get its effect concentrated upon the nuclei, for which it has a special affinity, the pieces of tissue must be placed for twenty-four hours or thereby in 70 per cent. alcohol containing I per cent. of hydrochloric acid. Acid Alcohol. This is called acid alcohol, and is prepared thus Hydrochloric acid Alcohol I cc. 70 " 30, When tissues are placed in the acid alcohol, they change in colour to a bright scarlet. A certain amount of the surplus carmine is extracted, but the nuclei become intensely stained. This method is particularly valuable for a large number of organs, and especially where nuclear staining is desired. 7. Álum Carmine.-Dissolve 5 grams of potash-alum in 100 cc. water. Add 1 gram carmine, and boil for a quarter of an hour. Make up the bulk with water and filter. Add a drop of carbolic acid to preserve it, as fungi rapidly form in it. It has the advan tage of not over-staining tissues left in it for a long time. 8. Lithium Carmine (Orth). Carmine Saturated solution of lithium carbonate 2.5 grams. Dissolve the carmine in the cold saturated solution of lithium carbonate; solution occurs very quickly. It gives a diffuse stain, to nearly all tissues very rapidly, and the sections must, therefore, be transferred, without previous washing in water, to acid alcohol (p. 65). They can then be mounted in glycerine or balsam as desired. The nuclei are stained a brilliant red. It cannot be used for sections fixed on a slide by means of white of egg. Application. (1.) Stain (2-3 minutes). (2.) Wash out surplus dye in acid alcohol (-1 minute). i.e., in 100 cc. of 70 per cent. spirit + 1 cc. HCl. (3.) Remove all acid by prolonged washing in water. (4.) Alcohol, oil, balsam. 1 Archiv f. Mik. Anat., vol. xvi. p. 363. E |