STAINS, ETC. 



cess is recommended by Butschli for staining sections 

 of protozoa I ,u thick. Another method is as follows : 

 Treat sections for from )/ 2 an hour to 2 or 3 hours with 

 from a 1.5 to a 4 per cent, solution of ferric-ammonium 

 sulphate ; wash in water, and stain for from I to 12 hours 

 in an aqueous solution of hematoxylin, about o. 5 per cent. 

 Rinse with water and treat again with the iron solution. 

 soon as differentiation is complete, wash for 1 5 minutes 

 in running water, and mount. The results vary accord- 

 ing to the duration of the treatment with the iron and 

 hematoxylin solutions ; short baths give a blue prepara- 

 tion, in which the nuclear structures are highly differ- 

 entiated ; prolonged baths give black preparations, show- 

 ing connective-tissue fibers and red blood-corpuscles 

 black, central and polar bodies intensely black, cytoplasm 

 sometimes colorless, sometimes gray, in which case cell- 

 plates and achromatic spindle-fibers are stained. Micro- 

 organisms are sharply stained. Kleinenberg's Hema- 

 toxylin. Adapted to staining in the mass. Make a sat- 

 urated solution of calcium chloridin 70 per cent, alcohol. 

 Shake it, and let it stand ; decant, and add alum in ex- 

 cess ; shake again, and, after a day or two, filter. To I 

 volume of the filtrate add 8 volumes of a saturated solu- 



!tion of alum in 70 per cent, alcohol ; to this mixture 

 add, drop by drop, a saturated solution of hematoxylin 

 in absolute alcohol, until a purple color appears. It 

 becomes darker in time and on exposure to light. It 

 should be prepared some months before it is wanted. 

 Mallory's Phosphomolybdic-acid Hematoxylin. 

 Ten per cent, solution of phospho-molylxlic acid 1 part, 

 hematoxylin I part, water 100 parts, chloral from 6 to 10 

 parts. Expose to sunlight for a week. Filter before 

 using, and save the used portions. Stain sections for from 

 lominutes to an hour, wash in 401050 percent, alcohol, 

 changing it 2 or 3 times. Dehydrate and mount. If 

 the solution does not stain readily, add a little hema- 

 toxylin. The stain is blue, and in its general effect 

 similar to nigrosin. It is recommended for preparations 

 of the central nervous system. Mayer's Ammonium- 

 nitrate Hematei'n. Hemalum 10 c.c, 70 per cent, 

 alcohol 10 c.c, ammonium nitrate 5 gm. Dissolve, let 

 the solution stand until the excess of alum crystallizes out, 

 12 to 24 hours, and filter. Useful for staining small 

 objects in Mo. Mayer's Hemalum. An excellent 

 stain for large objects. It consists of two solutions ; one 

 of hemateln, or ammonium-hematein, I gm. , dissolved 

 by the aid of heat in 50 c.c. of 90 per cent, alcohol ; 

 the other of alum 50 grams and distilled water I liter. 

 The solutions are mixed, left to cool, and then filtered. 

 A crystal of thymol may be added to prevent the for- 

 mation of mold. For most purposes it is advisable to 

 dilute this stain with water or alum-solution. Hemalum 

 plus 2 per cent, glacial acetic acid gives a more precise 

 nuclear stain. Mayer's Hemacalcium. Rub together 

 in a mortar, very thoroughly, I gm. each of hemateln 

 or ammonium-hematein and aluminum chlorid, and dis- 

 solve in 600 c.c. of 70 per cent, alcohol, to which 10 

 c.c. of glacial acetic acid have been added ; then add 50 

 grams of calcium chlorid. The color of the fluid is red- 

 dish-violet. Objects overstained in it are treated with a 2 

 per cent, alcoholic solution of aluminum chlorid or from a 

 ji to a I per cent, solution of sodium or potassium acetate 

 in absolute alcohol. Clearing with bergamot-oil or clove- 

 oil causes early fading. Reeves' {/.£.) Hematoxylin. 

 To one part of 5 or 10 c.c. of 5 per cent, cirbol- 

 lzed water add enough sodium sulphindigotate to 

 produce a deep bluish-green color, and 7 parts of 

 Delafield's hematoxylin. The mixing should be done 

 at the time of using. After staining, which requires from 

 % to I hour or more, immerse the section in water acid- 

 ulated with a few drops of nitric acid, and allow it to re- 

 main until it shows a clear, deep, sky-blue color ; wash 



1401 STAINING REAGENTS 



in water and dehydrate for 20 minutes in alcohol. This 

 stain differentiates the inclusions in carcinoma-cells — 

 chromatin, parasites, etc. Renaut's Glycerin-hema- 

 toxylin. To a saturated solution of alum in glycerin add, 

 drop by drop, a saturated alcoholic solution of hematoxy- 

 lin until the mixture has a deep color. Expose to light 

 and air for several weeks, and then filter. Sections may 

 be mounted in the stain. Sanfelice's Iodin-hema- 

 toxylin. Useful for staining in the mass. Make a solu- 

 tion of hematoxylin, 0.7 gm. in absolute alcohol 20 c.c, 

 and pour it, drop by drop, into a solution of alum 0.2 gm. 

 and distilled water 60 c c Let the mixture stand ex- 

 posed to the light for 3 or 4 days ; then add 10 to 15 

 drops of tincture of iodin, shake, and let it stand again 

 for 3 or 4 days. Tissues are immersed in this fluid for 

 from 12 to 24 hours, and then transferred for 24 hours 

 to 90 per cent, alcohol acidulated with acetic acid. 

 Weigert's Hematoxylin. See Staining of Nerve-tis- 

 sue, IVeigerfs Method. After using, this stain may be 

 regenerated as follows : Add about 5 per cent, of baryta- 

 water, shake it well, and let it stand for 24 hours ; then 

 pass carbon dioxid through it, let it stand another 24 

 hours, and filter {Fanny Berlinerblan). IV. Metallic 

 Stains. These are chiefly used in the study of epithelial, 

 connective, and nervous tissues, for which they exhibit 

 a remarkable selectivity. The results obtained vary ac- 

 cording to the method of impregnation, a negative or 

 primary impregnation coloring the intercellular sub- 

 stance, leaving the cells colorless; a positive or second- 

 ary impregnation staining the cells and not the inter- 

 cellular spaces. Ferric Chlorid. After impregnation 

 in a solution of ferric chlorid, reduction is effected in 

 tannic, gallic, or pyrogallic acid (Polaillon). Another 

 method is to fix the preparation in the iron-solution and 

 then treat for 24 hours with alcohol containing a trace 

 of gallic acid {Fol). Gold Chlorid. Recommended 

 for tracing nerve-endings in fresh tissues, and for stain- 

 ing connective-tissue and cartilage-cells. Place small 

 pieces of tissue, J{ inch square, in from a o. 5 to a I per 

 cent, solution of commercial gold chlorid in distilled 

 water. Keep in the dark, and when the tissue has be- 

 come yellow, wash in distilled water. Then expose to 

 the light in 50 c.c. of water containing 2 drops of 

 acetic acid for 48 hours, or until the tissue acquires a 

 purple tint. Mount in glycerin. Boiled Gold Chlorid. 

 Used in studying the terminations of nerves on sensory 

 surfaces. To 4 parts of a I per cent, solution of gold 

 chlorid add I part of formic acid, boil, and cool. In this 

 place small pieces of fresh tissue for from 10 minutes tc 

 I hour ; wash in water, and transfer to formic acid, I : 4, 

 keeping in the dark, where reduction occurs. Cohn- 

 hei/n's Method. Place fresh tissue in a 0.5 per cent, 

 solution of gold chlorid until it is yellow; then ex- 

 pose it to the light in water acidulated with acetic acid 

 until reduction occurs, and mount in acidulated gly- 

 cerin. Chrchtschonovitscfi 1 s Method. Place the fresh 

 tissue in a 0.5 per cent, solution of auric chlorid for 

 from 30 to 45 minutes ; then in distilled water for 24 

 hours ; then in a saturated solution of tartaric acid at a 

 temperature of 50 C. , until the gold is reduced. Wash 

 in water and harden in alcohol. Freud's Method. Har- 

 den the tissue in Miiller's fluid ; stain in equal parts of a 

 I per cent, gold chlorid solution and 95 percent, alcohol 

 for from 3 to 5 hours ; wash in water, and place in a so- 

 lution of caustic soda I c.c, and distilled water 6 c.c, for 

 from 2 to 3 minutes. Wash in water and carry to a IO 

 per cent, solution of potassium iodid for from 5 to 15 

 minutes; wash, dehydrate in alcohol, and mount. Glass 

 instruments should be used. This method, when 

 successful, gives islolated staining of axis-cylinders. 

 Gold Chlorid and Chromic Acid (A'o/osson). To 

 100 parts of a I per cent, solution of gold chlorid add 



