Nissl's Method

NISSL'S METHOD of staining (Nissl) is used for nerve cells. In proposing it, Nissl put forward the principle that the microscopic picture of the structure of a nerve cell in tissue taken from an animal killed in a specific way, with a specific histological processing, is repeated with a known regularity, and thus comparable "equivalent pictures" (Aquivalentbilder) are obtained. (For the significance of the method, see Nerve cells.) The basic "classical" Nissl method: fixation in 96° alcohol. The pieces should not be too large, but also not smaller than 1 cm³. The material must be as fresh as possible, taken no later than 18 hours after death. The amount of alcohol must be sufficiently large, and it must be changed several times. The duration of fixation is at least five days. Preparation of sections: from the piece, a plate no thicker than 6-8 mm is cut with a sharp razor; its area can be up to 2 cm² and even larger. One of the large surfaces is dried with filter paper, and the piece is glued with this side onto a wooden block using gum arabic, which is hardened by immersion in 96° alcohol. Sections are prepared with the microtome knife at the most oblique angle possible, moistened with 70° alcohol. Staining: the sections, well-flattened, are transferred with a spatula or glass hook into a watch glass with dye of the following composition: Methylene Blue B (Methylenblau B) - 3.75 g, scraped Venetian soap - 1.75 g, distilled water - 1,000 cm³. The dye with the sections is carefully heated until steam appears. After cooling, the sections are transferred, also flattened, into a mixture of aniline oil (colorless, from the Hoechst firm) - 10 cm³ and 96° alcohol - 90 cm³. The sections remain in it until clouds of dye come off them. Then the sections are transferred to a microscope slide, slightly dried with filter paper, and rinsed with cajuput oil, which is quickly drained; its excess is removed with filter paper. Further, the sections are washed on the slide with benzene and mounted in a saturated solution of rosin in xylene; when applying the coverslip, the preparation is slightly heated. At the present time, the following modifications are used with good results. For the preparation of sections, the specimen can be embedded in celloidin. For staining, a 0.1% solution of toluidine blue (Toluidinblau), thionine (Thionin), or cresyl violet (Kresylechtviolett) is used. When using toluidine blue, the preparation is heated until steam appears. It is also possible to work without heating. Differentiation can be done in 96° alcohol with subsequent passage through absolute alcohol and xylene. With strong overstaining, one can differentiate with alcohols of lower concentration (70, 80°). The rosin solution can be replaced with ordinary Canada balsam, also heating the preparation for its faster drying. With good staining, the intensely stained chromatin and chromatophilic substance should clearly stand out against a colorless background. Brain pieces fixed with formalin and chrome mixtures (Zenker, Helly, Orth, Zenker-formol, etc.) do not give correct pictures. Therefore, for pathological histology, it is not recommended to use such preparations. In some cases, however, it is necessary to use material fixed in this way. In this case, it is necessary: 1) prolonged (about 2 days) washing in running water; 2) prolonged (according to Spielmeyer, 2-3 months) processing of small pieces first with 70°, then with 96° alcohol (to dissolve lipoids fixed by formalin); 3) embedding in celloidin; before staining, the sections are left in 70° alcohol for a day in an incubator at a temperature of 37° (Spielmeyer). The disadvantage of staining preparations fixed with formalin is an unclean background and uneven differentiation.