Double and Treble Staining.
Dr. W. Stirling[46] furnishes a brief but useful account of the methods he has employed with much success.
Osmic Acid and Picro-carmine.—Mix on a glass slide a drop of the blood of newt or frog and a drop of a one per cent. aqueous solution of osmic acid, and allow the slide to stand by. This will fix the corpuscles without altering their shape. At the end of five minutes remove any excess of acid with blotting-paper, add a drop of a solution of picro-carmine, and a trace of glycerine to prevent evaporation, and set aside for three or four hours to see that no overstaining takes place. At the end of this time the nucleus will be found to be stained red, and the perinuclear part yellow.
Picric Acid and Picro-carmine.—Place a drop of the blood of a frog or newt on a glass slide, and add a drop of a saturated solution of picric acid: put the slide aside and allow it to remain for five minutes; at the end of that time, when the acid has fixed the corpuscles (that is, coagulated their contents), any excess of acid should be removed as before. A drop of solution of picro-carmine should now be added, and a trace of glycerine, and the preparation set aside for an hour. At the end of that time remove the picro-carmine solution by means of a narrow slip of blotting-paper, and add a drop of Farrant’s solution of glycerine and apply glass-cover. The perinuclear part of the corpuscles will be seen to be highly granular and of a deep orange colour, whilst the nucleus is stained red. Some of the corpuscles will appear of a delicate yellow colour, and threads are seen extending from the nucleus to the envelopes. The preparation should be preserved and mounted in glycerine.
Picro-carmine and Aniline Dye.—For glandular tissue, none of the aniline dyes answer so well as iodine green, used in the form of a one per cent. watery solution. Stain the tissue in picro-carmine, wash it in distilled water acidulated with acetic acid, and stain it in a solution of iodine green. As it acts rapidly, care must be taken not to overstain. Wash the section in water, and then transfer it to alcohol; finally clear with oil of cloves. The washing should be done rapidly, as the spirit dissolves out the green dye. All preparations stained with iodine green must be mounted in dammar.
Picro-carmine and Iodine Green.—Stain a section of the cancellated head of a very young bone (fœtal bone) in picro-carmine, wash it in distilled water, and stain it with iodine green, and mount in dammar. All newly-formed bone is stained red; that in the centre of the osseous trabeculæ, the residue of the calcified cartilage in which the bone is deposited, is stained green. Many of the bone corpuscles are also stained green.
Ossifying cartilage, the back part of the tongue, Peyer’s Patches, solitary-glands, trachea, and bronchus, may all be treated in the same way. In preparing the skin, take a vertical section from the sole of the foot of a fœtus. The cuticle and superficial layers of the epithelium are dyed yellow, the rete Malpighii green; and the continuation of these cells can be traced into the ducts of the sweat-glands, which are green, and form a marked contrast to the red stained connective tissue of the cutis vera, through which they have to ascend to reach the surface. The outer layer of the grey matter of the cerebellum with Purkinge’s cells is, when double stained, red, while the inner or granular layer is green. Logwood and iodine green stains the mucous glands of the tongue green, and the serous glands, lilac logwood stain.
Eosin and Iodine Green.—Eosin is used as the ground colour. Stain the tissue in an alcoholic solution of eosin, which will colour it very rapidly, usually in a few seconds. Wash the section thoroughly in water acidulated with acetic or hydrochloric acid, a one per cent. solution, and stain with iodine green. This will double stain bone and cerebellum; but if logwood is substituted for the latter, the cerebrum and general substance become stained by the eosin, while the logwood colours the nerve-cells a lilac.
Gold Chloride and Aniline Dyes.—The tissue must be impregnated with chloride of gold, and then stained with either aniline blue, iodine green, or rosin. The tail of a young rat, containing as it does so many different structures, is an excellent material for experimenting upon. Remove the skin from the tail, and place pieces half an inch long into the juice of a fresh lemon for five minutes, wash it to get rid of the acid. The fine tendons swell up under the action of the lemon acid, and permit of the more ready action of the chloride of gold solution. Place the piece for an hour or more in a one per cent. solution of gold, remove it and wash it thoroughly, and then place it in a twenty-five per cent. solution of formic acid for twenty-four hours. This reduces the gold. During the process of reduction the preparation must be kept in the dark. The osseous portion has then to be decalcified in the ordinary way, with a mixture of chromic and nitric acid. After decalcification preserve the whole in alcohol. Transverse sections of the decalcified tail are made, and may be stained with a red dye, as rosin, and afterwards with a watery solution of iodine green. Mount in dammar.