Golgi Method

GOLGI METHOD of staining histological preparations consists in that a small piece of tissue is first placed in either a pure solution of potassium bichromate or in one of the mixtures containing this salt, and then in a solution of silver nitrate or mercuric chloride. A precipitate of silver bichromate or mercury falls out on the structural elements, and their complete impregnation is obtained (silhouettes of cells and fibers). G. m. is used for revealing nerve cells and processes, glial cells, axial processes with collaterals and terminal endings, further for reproducing the boundaries between epithelial cells, for revealing secretory canals, bile capillaries, etc. The theory of the method has been thoroughly developed and concerns various questions. The precipitate obtained does not simply consist of Ag₂CrO₄, but represents a complex protein-chrome-silver compound. The dynamics of the process have been clarified by Hofmann and Liesegang (F. B. Hofmann, Liesegang). The latter's theory is based on the peculiarities of circulation and redistribution of K₂Cr₂O₇ and AgNO₃ particles, as well as Ag₂CrO₄ in the colloidal medium, and on the influence which the acids and ammonium salts of the tissue structural elements exert on the formation of the precipitate. Why only certain nerve cells are impregnated from among a whole group of identical ones is explained by the fact that precipitates fall out more easily on those structural elements which began to be impregnated earlier. An opinion has also been expressed that the deposition of precipitates goes along the surfaces of cells and fibers in the lymphatic spaces, and not in the elements themselves, but this does not correspond to the facts. Technique of the method. G. proposed several modifications, which are given below in order of frequency of use. -A. With which silver method of G. 1) A very small and thin piece of fresh material, preferably from an embryo or newborn, is placed in a mixture: 3% potassium bichromate-8 parts, 1% osmic acid-2 parts. Keep in the dark for 2-8 days. Previously, dry the piece with blotting paper and place it in the liquid also on filter paper. Duration of fixation: for neuroglia-2-3 days, for nerve cells-3-5 days, for nerve fibers-6-9 days. It is better to make trial sections after 2-3 days. 2) Quickly wash in distilled water or in a used solution of silver (¹/₁% silver nitrate). 3) 0.6-1% silver nitrate-2-6 days. Change the liquid when it turns yellow. 4) Cut, clamping the piece in liver tissue, or on a freezing microtome or after rapid embedding in celloidin (30 minutes), thick sections-50-100 μ. 5) Absolute alcohol, bergamot oil, Canadian balsam or dammar resin in xylene without a coverslip or else ascending alcohols up to 96%, creosote (briefly), balsam or resin. Allow to harden in the air.-B. Long-term silver method of G. 1) Fixation in Müller's fluid on cold for 3 months, then in warmth-1 month (in a dark bottle) or for acceleration-in a solution of potassium bichromate, from 2% gradually reach 5%. 2) Distilled water (briefly). 3) 0.5-1% silver nitrate and then, as in the first modification. Both modifications can be combined: first keep in Müller's fluid, then in a mixture of potassium bichromate with osmic acid.-C. Mercuric chloride method of G. 1) Müller's fluid-3-4 weeks. 2) ¹/₄-1% aqueous solution of mercuric chloride, from one week to 1-2 months. Change the liquid on the first day and then again when it begins to turn yellow. 3) Alcohols of ascending strength, celloidin, sections. 4) Distilled water. 5) Sections for 1-2 minutes are placed in liquid: a) Sodium hyposulfite 175 g, alum 20 g, Ammonium thiocyanate 10 g, table salt 40 g, distilled water-1,000 cubic cm. Let stand for 8 days, then filter. b) Chrome gold 1 g, distilled water-100 cubic cm. Take 60 parts of the first liquid and 7 of the second. Sections are made black. 6) Thorough washing. 7) Counterstaining with carmine. 8) Water. 9) Absolute alcohol, xylene, mounting in balsam. Of the modifications of G. m., the best is that of Cox (Cox).-Method of Cox. 1) Fixation and impregnation in fluid: a) 5% potassium bichromate 20 g, 5% mercuric chloride-20 g, distilled water 40 cubic cm; b) 5% potassium chromate-16 g. To avoid precipitation, first prepare fluid a (can be with heating) and add potassium chromate (b). Pieces (can also be large) are placed for 2 or more months. Keep shorter in summer. 2) Sections on a freezing microtome. 3) Sections are dropped into 5% sodium carbonate for 1-2 hours or into saturated sodium hyposulfite, where reduction occurs. 4) Water; quickly on the slide-absolute alcohol, oil; carefully filter paper. 5) Mounting in varnish: sandarac 75 g, camphor 15 g, turpentine 30 g, lavender oil 22.5 g, absolute alcohol 75 g, castor oil 5-10 drops. On the hardening layer of varnish, drop castor oil, cover with a coverslip and squeeze out the excess amount.-Indications for the method.* G. m. is not selective, gives precipitates, and yet in certain respects-for clarifying the connections of neurons, for detecting secretory canals-it is almost indispensable. Of the modifications given, the long-term one is older, while the rapid one is later and more perfected. The latter has advantages over the first. Similarly, mercury modifications tend to displace silver ones. But in practical activity, however, various modifications of Golgi's method should be used depending on the properties of the object being studied.