8. Penciling or Shaking. For the demonstration of the stroma or reticulum either fresh or fixed sections may be placed upon a slide in an abundance of fluid and gently penciled with a fine, blunt camel’s hair brush until the fluid becomes cloudy. The cloudy fluid is washed away and replaced by fresh as long as cells are given off. The same results may be obtained by shaking the sections in a test-tube until the cells are shaken out of the stroma. The removal of the cells from the section is shown by its greater transparency. One of the practical applications of these methods in diagnostic work is the differentiation between alveolar round-cell sarcomata and carcinomata.
9. Digestion. For the demonstration of stroma, parasites, etc., the tissues may be digested with gastric or pancreatic ferments until the required elements are freed. A freshly prepared pepsin in 0.2 per cent HCl in the incubator for 3-5 hours will digest fibrin in fresh clots. Sections of fixed tissues may be imbedded in paraffin, cut, and digested on the slide with Grübler’s pancreatin according to the method of Flint.
10. Intravital and Supravital Staining. Various methods have been advised for the intravital staining of cell-granules. Intravenous or intraperitoneal injections of methylene blue, alum carmine, neutral red and other stains will produce intracellular granule-staining in various organs of experimental animals. In the study of low forms of animal life staining solutions may be injected, or the animal or its parts may be examined in staining fluids. Human material can be examined by this method immediately after being removed from the body by operation or within 1-2 hours after death. (For details of these methods see article on “Färbungen, intravitale,” Encyklopädie der mikroskopischen Technik.)
11. Injection. Injections for the demonstration of blood-vessels, lymphatics, ducts of glands, etc., are rarely used in pathologic work. The organs to be injected must be fresh, warm from the body, if possible. The vessels should be washed out by a freshly filtered 8 per cent sodium nitrate or sodium sulphate solution, followed by physiologic salt-solution. A cannula is introduced into a main vessel, tightly secured, and then connected with a syringe or a gravity injection-apparatus giving a constant pressure. The injection-mass is then injected under a low pressure. In the case of injections into lymphatic vessels the cannula should be introduced into the largest lymphatics at the periphery of the blood-vessels where larger lymph-vessels are more easily found. Injections are made with either cold or warm solutions; the latter are preferable but require that the organ to be injected be warmed by immersing it in water at a temperature of 40°C. The injection fluid must be of the same temperature. After the warm injection is given the organ is put into ice-cold 10 per cent formol solution until fixed and then after-hardened, imbedded and stained as desired. After the use of cold injections the tissues are fixed in 10 per cent formol, alcohol, or any other desired solution, and treated according to the end sought. Nuclear and diffuse stains contrasting with the color of the injection mass should be used. The process of injection requires great care; the pressure must be carefully regulated to prevent extravasations, and the injection-fluid must be free from air-bubbles. The injection is continued until the organ appears diffusely stained. Blood-vessels are fixed in their natural blood-injection by such agents as formol and chromic acid, so that stains acting upon the red blood cells cause the veins and capillaries to appear as if they had been injected.
The following injection masses are advised:—
1. Cold Injection Mass (Beale’s Glycerin Carmine):—
Dissolve 0.3 grm. of carmine in a small quantity of water containing 5 drops of ammonia; add 15 cc. of glycerin and shake; add drop by drop 15 cc. of glycerin containing 8-10 drops of glacial acetic acid. Then add further glycerin 15 cc., alcohol 8 cc., and water 24 cc.
2. Warm Injection Mass (Thiersch’s Berlin-blue Gelatin):—
(a) Dissolve 1 part of gelatin in 2 parts of water by allowing it to soak 24 hours, and then warming. Filter through flannel.
(b) Saturated water solution of ferrous sulphate.