STAINS, ETC. 



1370 



CYTOLOGIC METHODS 



lute alcohol. Infiltrate with thick cedar-oil, embed in 

 paraffin which is overheated, and section. (Gustav 

 Platner, Arch. f. mikr. Anat., 1889, Bd. 33, Heft I.) 

 Achromatin and Plasma Stains. Foremost among 

 these is the Ehrlich-Biondi fluid, which is used by 

 Heidenhain as follows : Dilute 6 parts of the staining 

 fluid with 400 parts of water. Fill two beakers with 

 distilled water, and add to each a few drops of the 

 diluted staining solution. To one beaker add, with 

 continual agitation, drop by drop, a I : 500 solution of 

 acetic acid, until a crimson color appears. The con- 

 tents of the two beakers are to' serve as controls. The 

 dilute solution first prepared is now acidified with dilute 

 acetic acid, added drop by drop, with continual agita- 

 tion, and from time to time a few drops are added to a 

 beaker of distilled water, until a crimson tint is ob- 

 tained corresponding to that of the test-beaker, when 

 the staining bath is ready. Treat sections for two 

 hours with o. 1 per cent, acetic acid, then for 10 to 15 

 minutes with official tincture of iodin, rinse in alcohol, 

 and place in the stain for from 12 to 18 hours. 

 Altmann's Granules, Altmann's (R.) Method. The 

 tissue is fixed in osmic acid and stained with cyanin. 

 The granules then appear in chains of a violet color. 

 The substance between the granules stains readily with 

 hematoxylin or carmin, but not with cyanin, and vice 

 versa. (" Die Structur des Zellkernes," Arch. v. Dn 

 B. Reymond, Anat. Abth. , 1889. ) Chromatin Stains. 

 Of these the foremost in importance, for fresh tissues, 

 is methyl-green, which may be used alone or in the 

 Ehrlich-Biondi mixture (see Staining Reagents) ; also 

 Bismarck-brown in dilute glycerin, or in aqueous solu- 

 tion with acetic acid. For osmium objects, Mayer's hem- 

 alum. For sections of hardened tissues, Bohmer's hema- 

 toxylin, the finer hematein stains, safranin, gentian-vio- 

 let, Victoria-blue, and other anilins, used according to 

 the indirect method. Babes stains in safranin, as fol- 

 lows : A supersaturated solution of safranin in water 

 is warmed to 60° C. and filtered warm. On cooling it 

 becomes turbid through the formation of small crystals. 

 Place the sections in a watch-glassful of this turbid 

 solution, and warm for a few seconds (until the liquid 

 clears) ; after one minute wash in water and treat with 

 alcohol and turpentine in the usual way. Do not 

 clear in clove-oil. Ehrlich's Granules and Gran- 

 ular Cells (Mastzellen). See Staining of the Blood. 

 Fresh Cells. Tease out a piece of living tissue in a 

 drop of a solution of methyl-green containing 0.75 per 

 cent, of acetic acid; then expose for 15 minutes to 

 vapor of osmic acid, by inverting the slide over the 

 mouth of a bottle containing a small quantity of a one 

 per cent, solution ; remove when the cells are brown ; 

 add a drop of solution of Ripart and Petit, and cover. 

 Scrapings from the freshly-cut surface of a recently ex- 

 cised liver or lymphatic gland, having been treated with 

 I to 2 per cent, acetic acid, may be stained with fuch- 

 sin added in sufficient quantity to a 2 per cent, acetic 

 acid to saturate it. This renders the nuclei visible (v. 

 Kahlden) . Segmenting ova of Echinodermata may be 

 stained on the slide by placing a drop of safranin at the 

 edge of the cover-glass. When the ova are dark, the 

 excess of stain is removed by means of bibulous paper, 

 and one per cent, acetic acid is allowed to flow under 

 the cover. Karyokinesis. 1. Place small pieces of 

 tissue hardened in strong Flemming's solution in an 

 alcoholic solution of safranin (2gm. to 6oc.c ) for from 

 24 to 48 hours. Wash for a few minutes in water, and 

 carry to acidulated absolute alcohol (10 drops of acetic 

 acid to 100 c.c.) for from ^ to I minute. When thick 

 clouds of color are no longer given off, carry to abso- 

 lute alcohol. After I or 2 minutes, clear and mount. 

 2. Baumgarten's Method. This method may be em- 



ployed conjointly with a stain for bacteria. Harden the 

 tissue for several weeks in a dilute solution of chro- 

 mic acid ; stain for from 5 to IO minutes in a concen- 

 trated alcoholic solution of fuchsin ; rinse quickly in 

 absolute alcohol ; stain for from 5 to 10 minutes in 

 an aqueous solution of methylene-blue. In exam- 

 ining for bacteria also, stain first for 24 hours in anilin- 

 water methyl -violet (decolorize with dilute acid if 

 staining for tubercle-bacilli) ; then follow with fuchsin 

 and methylene-blue, as indicated. 3. Bendd [ s Method. 

 Fix in Flemming's fluid, imbed in paraffin, and stain 

 the sections as follows : Place for 24 hours in a concen- 

 trated solution of neutral copper acetate, kept at a 

 temperature of 40 C. Wash well with water, and 

 stain to a dark-gray tint in aqueous hematoxylin solu- 

 tion. Decolorize in 0.2 per cent, hydrochloric acid, 

 until of a light-yellow, and then neutralize the acid by 

 returning the sections to the copper solution, in which 

 they should remain until they acquire a grayish-blue 

 tint. Wash, dehydrate, and mount in balsam. 4. 

 Bizzozero- Vassale Method. Fix in absolute alcohol. 

 Stain IO minutes in Ehrlich's gentian- violet solution; 

 wash quickly in absolute alcohol ; transfer to dram's 

 solution for two minutes, then pass into absolute alco- 

 hol for 30 seconds ; o. I per cent, chromic acid, 30 to 40 

 seconds ; absolute alcohol, 20 to 30 seconds ; o. 1 per 

 cent, chromic acid, 30 seconds ; absolute alcohol, 30 

 seconds; oil of cloves ; renew the last until no more 

 color is given off. Treat with xylol, and mount in 

 xylol-balsam. 5- Gram' s Method. This is the same as 

 for bacteria. The resting nuclei are either wholly or par- 

 tially decolorized, while the dividing nuclei retain the 

 dye. 6. Mitosis in the Amnion. Kill the pregnant ani-i 

 mal, and place the uterus in a saturated watei 

 tion of picric acid, opening the organ and the mem j 

 branes under the fluid. Harden for 24 hours, was': 

 in alcohol, and harden in alcohols, beginning with 7c) 

 per cent. Tinge a small part of the membrane ir 

 Ehrlich's acid hematoxylin diluted one-half. 7 

 Mitosis in Lieberkiikn ' s Glands. Harden a section of 

 small intestine in mercuric chlorid, and stain with ack| 

 fuchsin and methyl-green. The resting nuclei will tx : 

 blue and those in active mitosis green. 8. M 

 the Vermiform Appendix. Fix in Flemming's solution 1 

 Fol's solution, or absolute alcohol ; stain 5 to 10 min 

 utes in the following mixture: gentian-violet, 1 gm. 

 absolute alcohol, 15 c.c. ; anilin-oil, 3 c.c. ; water, 8" 

 c.c. Wash in absolute alcohol; immerse 30 to 4, 

 seconds in I per cent, chromic acid, then for th; 

 same length of time in absolute alcohol ; repeat thi 

 chromic acid and absolute alcohol to remove all exce 

 of dye ; clear, and mount in balsam. Living Cell;, 

 Young larvae of Amphibia are the best objects for thlj 

 study of cells intra vitam. Place the larva' o\~ Soli 

 mandra in a watch-glassful of water containing 5 to I 1 

 drops of a solution of I part curare in 100 parts eai 

 of water and glycerin. Half to one hour's immersic 

 is required for curarization. It is not necessarv to WM 

 until the larva 1 are motionless ; they may be remove, 

 as soon as their movements have become slow, 

 gills and the caudal " fin " may then be studied. 11; 

 tail maybe excised from the living animal and studU 

 for some time in one percent, salt-solution or other i 

 different medium. The adult animal offers for stiK 

 the thin, transparent bladder. Larvae may 1»>- bred fro 

 adults, if well fed with aquatic worms, and suppli<j 

 with a vessel of water. The larvx will be deposit* 1 

 in the water. The cytoplasm of living cell- 

 stained with methylene-blue, dahlia, or gentian-violt 

 dissolved in water or in an indifferent liquid. Micr> 

 chemic Reactions. 1. Chromatin is distinguish' 

 from lecithins and albuminoids by treating in 



