Nitrate of silver darkens by exposure; it is used in a half per cent. watery solution. Specimens to be acted upon should be washed in distilled water, to remove every trace of sodium chloride, and then steeped in the silver solution for some two or three minutes, after which they should be again washed until they cease to turn milky; then place them in glycerine and expose them to the action of light until they assume a dark brown colour, when they should be mounted in glycerine or glycerine jelly.
By means of this stain the endothelial cells of the lymphatics, blood vessels, &c., and the nodes of Ranvier, constrictions in medullary nerves, are rendered visible. Sections of any of these may subsequently be stained by logwood or carmine.
Several methods have been adopted for staining with gold chloride. Dr. Klein’s and Professor Schäfer’s are among the best.
Dr. Klein’s method of showing the nerves of the cornea is as follows:—Remove the cornea within fifteen minutes of death; place it in a half per cent. chloride of gold solution for half an hour, or an hour; wash in distilled water, and expose to the light for a few days; in the meantime occasionally change the water. Then immerse it in glycerine and distilled water, in the proportion of one to two; lastly, place it in water, and brush gently with a sable pencil to remove any precipitate, when it will be fit for mounting in glycerine. The colour of the cornea should be grey-violet.
Schäfer adopts another method—a double chloride of gold and potassium solution.
Osmic acid, first used by Schultze, is useful for the demonstration of fatty matters, all of which it colours black; it is also valuable for certain nerve preparations. Specimens should be allowed to remain in a one or two per cent. aqueous solution of the acid from a quarter to twenty-four hours, when the staining will be completed; but if it is desired to harden specimens at the same time, they should remain in it for some few days. Osmic acid does not penetrate very deeply, therefore small portions should be selected for immersion. This is a useful stain for infusorial animals.
Chloride of palladium, another of Schultze’s staining fluids, is used to stain and harden the retina, crystalline lens, and other tissues of the eye, the cornified fat and connective tissues remaining uncoloured. The solution should be used very weak:—Chloride of palladium, one part; distilled water, 1,000 parts. Specimens should be mounted in glycerine at once, or further stained with carmine.
Dr. Schäfer employs a silver nitrate and gelatine solution for demonstrating lung epithelium; this is made as follows:—Take of gelatine ten grammes, soak in cold water, dissolve, and add warm water to 100 cc. Dissolve a decigramme of nitrate of silver in a little distilled water, and add to the gelatine solution. Inject this with a glass syringe into the lung until distension is pretty complete. Leave it to rest in a cool place until the gelatine has set; then cut sections as thin as possible, place them on a slide with glycerine, and expose to light till ready for mounting.
Of the double stains Mr. Groves prefers only those where the double colour is produced by a single process—or stains in which one colour is first employed, and then another. Single stains are picro-carmine, carmine and indigo carmine, aniline blue and aniline red.
Picro-carmine is specially useful for staining sections hardened in picric acid. It is prepared in several ways:—