STAINS, ETC. 



1397 



STAINING REAGENTS 



The sections are stained rather deeply in Delafield's 

 hematoxylin , washed in water, and then placed for a few 

 minutes in a solution of picric acid and acid fuchsin, made 

 by adding to a saturated aqueous solution of picric acid 

 a saturated aqueous solution of Griibler s acid fuchsin, 

 drop 1 >y drop, until a garnet color appears ; they are 

 again washed in water, then dehydrated in alcohol, 

 cleared in oil of origanum, and mounted in balsam. 

 The ganglion-cells, neuroglia, blood-vessels, and scle- 

 rosed areas are stained garnet, the axis-cylinders red, 

 ■xad the myelin yellow. This stain is well adapted to 

 ill tissues in which picrocarmin is used. Weigert's 

 Method. This method depends on the production in 

 he tissues of a chromium or copper lake, in consequence 

 1 >f which hematoxylin acquires the property of staining 

 ' he myelin of nerves in a specific way. The steps of 

 ■ he process are as follows : The tissue is hardened in 

 diiller's or Erlicki's fluid, and, when it has acquired a 

 irown coloration, is embedded by infiltration with cel- 

 oidin, and placed for I or 2 days in a saturated solution 

 if neutral copper acetate diluted with I volume of 

 ?ater and kept at the temperature of an incubating oven. 

 | n this the tissue becomes green and the celloidin 

 luish-green, and the change of color indicates that the 

 lordantage is complete. Preserve in 8o per cent, alco- 

 ol. Stain sections in Weigert's hematoxylin (see Stain- 

 ! ng Reagents). The time required varies according to 

 he tissue : for the spinal cord and the medullary layers 

 : f the brain 2 hours, for the cortical layers of the brain 24 

 ours ; rinse in water, and differentiate in a solution of 

 , orax 2 parts, potassium ferricyanid 2.5 parts, water 200 

 - to several hours being necessary. Wash in 

 vater, dehydrate, and mount in balsam. The nuclei 

 nay be demonstrated by previous staining in alum-car- 

 nin. Weigert's New Method. See Weigert's 

 \Wethod ■without Decolorizing. Weigert's Method 

 vithout Decolorizing. Tissues hardened in Miiller's 

 ■luid and alcohol are embedded in celloidin, and then 

 >ut into a mixture of equal parts of a 10 per cent, solu- 

 ion of sodium-potassium tartrate and a cold saturated 

 olution of copper acetate, which is kept at from 38 to 

 o° C. They are next placed in a half-saturated solution 

 f copper acetate at the same temperature for 48 hours, 

 "he blocks, rinsed in water, may be kept in 80 per cent, 

 lcohol and cut at any time. The staining fluid is com- 

 posed of I part of an alcoholic hematoxylin solution ( I 

 !) 10), and 9 parts of a saturated solution of lithium car- 

 onate ; this fluid is to be freshly made. Stain for from 4 

 ) 12 hours ; wash, dehydrate in 90 per cent, alcohol, 

 ■ nd clear in anilin- xylol (2 to l),then in pure xylol, and 

 lount in xylol- balsam. The advantage of the method 

 i the clearness with which the fine medullated fibers are 

 , istinguished from the cells and other parts, and it is less 

 i dious than the old method. Wolters' Method. 

 ■ larden either peripheral or central nervous tissue in 

 Nultschitzky's fluid, and follow by alcohol. Embed in 

 'illoidin or paraffin. Mordant sections 24 hours in the 

 ; inadium-chlorid and aluminum-acetate mixture used in 

 goiters' modification of Weigert's method ; wash for 10 

 ! linutes in water and stain for 24 hours in the hematoxy- 

 j n solution used in Wolters' modification of Weigert's 

 lethod. Wash out in acid alcohol until the sections 

 ; :quire a light blue-red color. Remove the acid in 

 are alcohol, dehydrate, clear in oil of origanum, and 

 j iount. Besides the axis-cylinders, which are sharply 

 ained, all the tissue-elements are colored. Ziehen'6 

 lethod. Pieces of nerve-tissue are put for 5 weeks 

 1 1 a mixture of equal parts of I per cent, solutions of 

 i ^ld chlorid and corrosive sublimate. The sections are 

 • at in o. 25 per cent, solution of iodin. The nerve-fibers, 

 Jiedullated and non-medullated, the nerve- cells, and 

 I ie neurolgia-cells are colored blue. 



STAINING REAGENTS. 

 The stains employed in microscopic anatomy (histology) are 

 divided into two groups, according to their selective ac- 

 tion on the tissues : the histologic, or plasmatic stains, 

 and the cytologic, or nuclear stains. The substances 

 chiefly used are the coal-tar or anilin dyes, carmin, 

 hematoxylin, gold and silver. I. Anilin Stains. 

 These are classified by Ehrlich as acid, basic, and neu- 

 tral. The basic dyes are excellent nuclear stains, and 

 are most used. Some of them have special affinities for 

 certain tissues, and are of the utmost value in bacterio- 

 logic research. They are further classified as plas- 

 matic stains, and, according to the method of staining, 

 as direct and indirect nuclear stains. The indirect, 

 or " Flemming " Method, which is suitable only for 

 sections, consists in overstating all the tissue-elements 

 in a strong solution of the dye, and then decolorizing. 

 As the nuclei have the strongest affinity for these stains, 

 they resist the washing-out process longest, and still 

 retain the color when it has been yielded up by the 

 ground-substance. The washing-out is usually done in 

 alcohol, but in some instances may be effected by stain- 

 ing with another anilin, which displaces the first in 

 the ground-substance : this process is known as sub- 

 stitution. The initials attached to the names of anilin 

 dyes indicate a certain tint of a color and its depth or 

 intensity ; as, cyanin B, which means cyanin of a pecu- 

 liar shade of blue, cyanin BB meaning a deeper shade 

 of the same blue, and so on. The initials may also sig- 

 nify chemic change ; as, fuchsin S, which denotes sul- 

 phonation of the dye. Acid Fuchsin. A diffuse 

 stain, having a special affinity for axis-cylinders. A 

 solution of 2 gm. in 40 c.c. of 90 per cent alcohol and 

 160 c.c. of distilled water is employed. Wash out. in 90 

 per cent, alcohol. Weigert stains sections of tissue 

 hardened in Miiller's fluid in a saturated aqueous solution 

 of acid fuchsin for from I to 24 hours, then rinses them 

 quickly in water, immerses for a few minutes in a satur- 

 ated solution of potassium hydroxid I part, alcohol 10 

 parts. Wash thoroughly to remove the alkali, dehydrate, 

 clear, and mount. This process differentiates the finer 

 nerve-fibrils in the spinal cord. Acid Rubin. See 

 Acid Fuchsin. Anilin Blue-black. See Nigrosin. 

 Anilin Brown. Sections are stained in a saturated 

 solution of anilin brown in equal parts of water and 

 glycerin, and washed and preserved in glycerin. This 

 stain is used especially in microphotography. Anilin 

 Red. See Fuchsin. Artificial Indigo. See Nigro- 

 sin. Bengalin. See Xigrosin. Benzopurpurin. 

 A dark-red, plasmatic stain, affording a good contrast 

 to hematoxylin and other blue nuclear stains. Sections 

 are stained for from 2 to 5 minutes in a solution contain- 

 ing 0.25 gm. of purpurin to 20 c.c. of 90 per cent, 

 alcohol and 80 c. c. of distilled water. Bismarck 

 Brown.- A direct and indirect nuclear stain. A solu- 

 tion of 0.5 gm. in 20 c.c. of 90 per cent, alcohol and 

 80 c.c. of distilled water gives a good nuclear stain. 

 The addition of carbolic acid is advised. This dye has 

 also the property of staining certain cellular elements 

 during life. Blackley Blue. See A T igrosin. Chro- 

 motrop. See, under Cytologic Methods, IVatase's 

 M thod for Differentiating Sexual Cells. Congo 

 Red. An acid stain used in the study of the central 

 nervous system. Stain for 2 or 3 minutes in a 2 per cent, 

 aqueous solution. Corallin. See Fuchsin. Cyanin 

 (Quinolein Blue). A plasmatic dye. It stains fatty 

 matters a deep-blue, other tissues a pale-blue. Dissolve 

 1 gm. in 90 per cent, alcohol, and then dilute with 

 water. A weak solution should be used ; a very weak 

 solution, I : 500,000, in the medium that constitutes 

 the native habitat of the organism, stains Infusoria intra 

 vitam. Dahlia-violet. A nuclear stain, recom- 



