Pal’s Hæmatoxylin Stain.—Dissolve 0·75 Gm. of hæmatoxylin in 90 C.c. of distilled water and 10 C.c. of absolute alcohol. Just before use add saturated solution of lithium carbonate in the proportion of 3 drops to each 10 C.c. of hæmatoxylin solution. (See Weigert.)
Pal’s Hæmatoxylin Method.—Proceed at first as in Weigert’s process for nerve fibre, omitting the copper bath, and stain in Pal’s hæmatoxylin solution (see above) for 5 or 6 hours. Then wash the sections in distilled water (containing a trace of lithium carbonate if the sections are not deep blue), next treat for 15 to 30 seconds with a 0·25 per cent. potassium permanganate solution, rinse in water, and decolourise in Pal’s bleaching solution. (If black spots appear replace in the permanganate solution, again bleach, and wash for 15 minutes in water.) The grey substance of the sections is decolourised in a few sections; the sections should then be well washed out, and may be double-stained with picro-carmine or acetic acid carmine (see Schneider), Magdala red, or eosine. The nuclei may be stained with alum carmine. Finally dehydrate, clear, and mount.
Pal-Exner’s Osmic Acid Method.—Spinal cord or brain in 0·25 inch cubes is immersed in 0·5 per cent. osmic acid solution for 2 days, the solution being changed each day; then wash in water, transfer to absolute alcohol, and imbed in celloidin or paraffin. Place sections as cut in glycerine, then wash in water, treat with potassium permanganate and Pal’s solution, as in Pal’s hæmatoxylin method, counter-stain with carmine, dehydrate, clear, and mount in balsam.
Plant’s Method of Staining Actinomycosis.—Sections are placed for 10 minutes in Gibbes’ magenta solution or carbolic fuchsine, at 45° C.; next they are rinsed in water and placed in saturated aqueous solution of picric acid, mixed with an equal volume of absolute alcohol, for 5 or 10 minutes; they are then washed once more, passed through 50 per cent. alcohol into absolute alcohol, cleared in cedar oil, and mounted in balsam.
Ranvier’s Lemon Juice Method.—Soak pieces of fresh tissue in fresh lemon juice until transparent (5 to 10 minutes), then rapidly wash in distilled water, treat for 10 to 60 minutes with 1 per cent. gold chloride solution, again wash and expose to light in a bottle containing 50 C.c. of distilled water and 2 drops of acetic acid. Reduction is complete in 24 to 48 hours. If it is not desired to retain the superficial epithelium, reduction may be more completely effected in the dark, by treatment with formic acid (sp. gr. 1·2), diluted with 3 times its volume of water. The lemon juice in the above process may be replaced by an aqueous solution of citric acid (40 grains in each ounce).
Ranvier’s Picro-Carmine.—Carmine, 1 part; distilled water, 10 parts; solution of ammonia, 3 parts; mix and add of a cold saturated solution of picric acid 200 parts.
Renaut’s Hæmatoxylic Eosine.—Mix 30 C.c. of concentrated aqueous solution of eosine, 40 C.c. of saturated alcoholic solution of hæmatoxylin (which has been kept for some time and precipitated), and 130 C.c. of saturated solution of potash alum in glycerine (sp. gr. 1·26). Stand for 5 or 6 weeks in a partially covered vessel, protected from dust, until the alcohol is evaporated, and then filter. The filtrate can be diluted with glycerine if desired. Mount objects in this fluid diluted with 1 or 2 volumes of glycerine, or, stain separately for some days or weeks and mount in balsam, after washing in alcohol charged with a sufficient quantity of eosine.
Ranvier and Vignal’s Osmium Mixture.—Fix tissues in a freshly-prepared mixture of equal volumes of 1 per cent. osmic acid and 90 per cent. alcohol, then wash out in 80 per cent. alcohol, next with water, and stain for 48 hours with picro-carmine or hæmatoxylin. This method has been applied to the histology of insects.
Renaut’s Glycerine Hæmatoxylin.—To a saturated solution of potash alum in glycerine, add a saturated solution of homatoxylin in 90 per cent. alcohol drop by drop, so as to form a deeply coloured solution. Expose to daylight for a week, and then filter. This solution, like Renaut’s hæmatoxylic cosine, may be used for mounting unstained sections, which after some time absorb the colour from the liquid and become stained.
Safranine.—Safranine, 0·5 Gm.; rectified spirit, 20 C.c.; distilled water, 80 C.c.