Bacteria in Sections of Tissues.
Method of Hardening and Decalcifying Tissues.—To harden small organs, such as the viscera of a mouse, they should be placed on a piece of filter-paper at the bottom of a small wide-mouthed glass jar, and covered with about twenty times their volume of absolute alcohol. Larger organs are treated in the same way, but must be cut up into small pieces. Müller’s fluid, methylated spirit, or formalin may be used.
Teeth, or osseous structures, must first be placed in a decalcifying solution, as Kleinenberg’s. When sufficiently softened, soak in water, to wash out picric acid, and transfer to weak spirit. Ebner’s solution gives good results.
Methods of embedding, fixing, and cutting.—Crookshank finds that after hardening, the pieces of tissue are embedded in a mixture of ether and alcohol for an hour or more, then transferred to a solution of celloidin in equal parts of ether and alcohol, and left there for several hours.
The piece of tissue is then placed in a glass capsule, and some of the celloidin solution poured over it. The capsule can be placed bodily in 60 to 80 per cent. alcohol, and left there until the following morning. The celloidin should be of the consistency of wax. The piece of tissue is next cut out, and after trimming is put into water until it sinks, then transferred to gum, and cut with the freezing microtome.
Sections of fresh tissues are to be floated in ·8 per cent. salt solution, and then carefully transferred by a platinum lifter to a watch-glass containing absolute alcohol.
Staining Bacteria in Tissue Sections.—Weigert’s method is as follows:—Place sections for from six to eighteen hours in a one per cent. watery solution of any of the basic aniline dyes. To hasten, place the capsule containing solution in the incubator, or heat it to 45° C., or a stronger solution may be used. In the latter case the sections must be treated with a half-saturated solution of carbonate of potash, as they are easily over-stained. In either case the sections are next washed with distilled water, passed through sixty per cent. alcohol into absolute alcohol. When almost decolourised, spread out on a platinum lifter and transfer to clove oil, or stain with picro-carmine solution (Weigert’s) for half an hour, wash in water, alcohol, and treat with clove oil, and transfer to clean glass slide.
Gram’s Method.—Sections are stained for ten minutes in a capsule containing aniline-gentian-violet solution, then placed in the iodine and iodide solution until uniformly brown, then placed in absolute alcohol, and washed by carefully moving sections in the liquid with a glass rod. When completely decolourised, they are transferred to clove oil and then to a slide.
Double-staining is obtained by transferring the sections after decolourisation to eosin, Bismarck brown, or vesuvin (Crookshank).
Formalin is an excellent preservative fluid; one part to 20,000 is sufficient to prevent fermentation. For the preservation of vegetable sections, a one per cent. solution is required; even the fresh appearance of vegetable structures is preserved for some time when immersed in it. In the nutrient gelatine for biological specimens, if used early, will arrest the liquefaction of the gelatine by bacteria. For hardening it saves time, and is even better than alcohol, chromic acid, pot. bich., and many others. It does not cause shrinkage of the cells. Tissue ½ to ¾ inch thick hardens in twenty-four hours in pure formalin; five to ten per cent. is best for loose tissue. In another method, by which time can be saved, instead of placing the specimen in the formalin and afterwards in mucilage, prior to cutting sections, make the mucilage with two per cent. (or stronger) formalin water, and it will then answer both purposes at the same time.