STAINS 



488 



STAINS 



Anilin-blue, used in a 0.2J& alcoholic solution as a 

 counterstain with carmin or safranin as the nuclear 

 stain, it is recommended f6r the differentiation of nerve 

 tissue and of cartilage. See Carmin and Anilin-blue. 

 Bencke's Method, i. A modification of Weigert's 

 method for fibrin, which consists in diminishing the 

 bleaching power of the anilin-xylol by increasing the 

 proportion of xylol. By using a mixture of 2 volumes 

 of anilin and 3 volumes of xylol the following struc- 

 tural elements can be demonstrated : mitotic figures; 

 connective- tissue fibers; elastic fibers; Sharpey T s 

 fibers and the fibrils of osseous tissue ; striated muscle ; 

 neuroglia; nuclei of ganglion cells; the reticulum of 

 squamous epithelium. Benda's Copper-hematoxy- 

 lin. Treat paraffin sections of tissue fixed in Flem- 

 ming's mixture for 24 hours with concentrated solution 

 of copper acetate at 40 C, or for 48 hours at normal 

 temperature ; wash well in water and stain until dark 

 gray or black in 1 % aqueous solution of hematoxylin. 

 Decolor in 0.2% hydrochloric acid until the sections 

 are light yellow; neutralize in the solution of copper 

 acetate until bluish-gray. Benda's Iron-hematox- 

 ylin, mordant sections for 24 hours in a mixture of 

 iron sulfate 80 parts, sulfuric acid 15 parts, nitric acid 

 18 parts, and water 200 parts (or liquor ferri sulfurici 

 oxidati, P. G., diluted with one or two volumes of 

 water) ; wash in distilled water and stain until black 

 in I % aqueous solution of hematoxylin ; differentiate 

 in 30% or weaker acetic acid or in the iron-sulfate 

 solution diluted to a pale straw color. Benda's 

 Method. I. {a) For kinetic nuclei. Stain sections 

 for 24 hours in anilin-water safranin solution (see saf- 

 ranin formula b of Babes, Staining Reagents'), and 

 then for a half minute in a solution of light green or 

 acid violet, 0.5 gm. in 200 c.c. of alcohol. Chroma- 

 tin red; archoplasm green (or violet) ; centrosomes of 

 spermatozoa sometimes red, sometimes green. (6) 

 Stain with iron hematoxylin and afterstain with safra- 

 nin Chromosomes and centrosomes black ; linin fibrils 

 and nuclear spindle red. 2. For frozen sections of 

 organs of the central nervous system. Treat small 

 pieces of tissue for one or more hours with 2.5% for- 

 malin; wash and freeze in distilled water. The sec- 

 tions are not brittle and have a consistency like soap. 

 Benda's Method. For neuroglia: Fix in 10% for- 

 malin. Mordant in Weigert's chromium alum and 

 copper acetate mordant and then in 0.5^ chromic 

 acid. Wash in water. Embed in paraffin. Mordant 

 the sections for 24 hours in 4% iron alum, wash in 

 water and stain in a weak solution of sodium sulfaliza- 

 rinate and then in I % toluidin blue ; wash in 1 fa 

 acetic acid, dry, dehydrate, and differentiate in crea- 

 sote. Benda's Reaction, a macro-chemic and mi- 

 cro-chemic reaction of fatty tissue necrosis. Harden 

 the tissue in 10% formalin and treat with Weigert's 

 copper acetate mordant for neuroglia (see JFeigert's 

 Method); after 24 hours in the incubator the necrotic 

 areas are covered with green flakes. Microscopically 

 the necrotic tissue is blue-green, the fatty acid crystals 

 being most intensely colored. The normal fat-cells 

 show no trace of the blue or blue-green hue. Bens- 

 ley's Method for the study of the mammalian gastric 

 glands: Fix the gastric mucosa in Foa's mixture; 

 after from a half to 2 hours wash in 70% alcohol until 

 all the bichromate is removed; transfer to 95^ alco- 

 hol. Embed in paraffin and stain with nuclear anil 

 granule dyes. Benzoazurin, a plasma or nuclear 

 stain according to the progressive or regressive methods 

 respectively. It is recommended by Bonnet, in par- 

 ticular for the nuclear staining of preparations that are 

 difficult to stain. Berkley's Method, a modification 

 of Golgi's silver method. Harden in osmium-bichro- 



mate and impregnate in a freshly prepared solution of 



2 drops of 10% phosphomolybdic acid to 60 c.c. of 

 \ c /c silver nitrate, in winter to be kept at 25 C. 

 Bernard's Method for the demonstration of the cen- 

 trosome in plant cells: Fix in alcohol or Flemming's 

 reagent and stain in a mixture of 2 parts each of 1^ 

 aqueous solution of fuchsin and iodin green and 40 

 parts of water. Bethe's Anilin-black for staining 

 chitin : Fix the sections to the slide and treat them for 



3 or 4 minutes with freshly prepared 10% solution of 

 anilin hydrochlorid containing I drop of hydrochloric 

 acid to each 10 c.c. Rinse in water and treat with 

 10% solution of potassium bichromate. Rinse and 

 repeat the process until the stain has the desired inten- 

 sity. The stain is at first green, but becomes blue on 

 washing in tap-water or in alcohol containing ammo- 

 nia. Bethe's Fluid. 1. Dissolve I gm. of ammo- 

 nium molybdate (or sodium phosphomolybdate) in 20 

 c.c. of water. 2. Ammonium molybdate (or sodium 

 phosphomolybdate) I gm., water icc.c, and 0.5^ 

 osmic acid (or 2 ( / c chromic acid) 10 c.c. To each so- 

 lution add I drop of hydrochloric acid, and if desired 

 I gm. of hydrogen dioxid. 3. (a) For vertebrates : 

 Ammonium molybdate I gm., distilled water 10 c.c, 

 hydrochloric acid I drop, hydrogen dioxid I c.c. (0) 

 For invertebrates: Ammonium molybdate 1 gm., dis- 

 tilled water 10 c.c, hydrogen dioxid 0.5 c.c. The 

 tissue should remain in the ice-cold fluid from 2 to 4 

 hours; wash for 2 hours in cold water ; harden for 1 5 

 minutes in each of the ascending series of alcohol up 

 to absolute — all ice-cold ; transfer for 2 hours to fresh 

 absolute alcohol at freezing temperature. Stain in any 

 alcoholic solution, at room -temperature, dehydrate, 

 clear for from 12 to 24 hours in xylol several times re- 

 newed, and embedded in paraffin. Bethe's Method. 

 I. For tissue stained in methylene-blue : Treat very 

 small pieces for from 10 to 15 minutes with concen- 

 trated aqueous solution of ammonium picrate and 

 then place them for one hour in Bethe's Fluid, No. I 

 or 2, — for 5 hours if the solution contains osmic acid. 

 Wash in water and dehydrate in alcohol — cooled to 

 1 5° C. if the solution containing the sodium salt was 

 used. 2. For demonstrating the primitive fibrils of 

 nerves : Fix in osmic acid for 24 hours, wash 6 horns, 

 harden in 90^ alcohol 10 hours; then treat with 

 water 4 hours and transfer into a mixture of hydro- 

 chloric acid and 2^ sodium sulfate solution (5:2); 

 after from 6 to 12 hours, wash, dehydrate, clear, and 

 embed in paraffin. Cut very thin sections, attach them 

 to the slide with albumin fixative, stain for 10 minutes 

 in 0.1$ solution of ammonium molybdate, and mount 

 in balsam. Betz's Method for hardening the brain 

 and spinal cord : (a) Divide the cerebrum along the 

 median line and place it in iodin alcohol; altera few 

 hours remove the pia from the callosum and the syl- 

 vian fissure, also remove the choroid plexus, and return 

 to the iodin-alcohol ; after 24 or 48 hours remove the 

 pia from the fissures and gyri and place in fresh iodin- 

 alcohol ; renew the liquid again in 2 or 3 days and 

 after IO or 24 days transfer the cerebrum into 4', 

 potassium bichromate. (0) Remove the membranes 

 and vessels from the cerebellum and place it in the 

 iodin-alcohol ; on a support of cotton wool ; frequently 

 renew the solution and after about 14 days transfer into 

 5% potassium bichromate. (<■) Remove the dura from 

 the cord and suspend it in a cylinder containing iodin- 

 alcohol ; after 2 or 3 days remove the pia and return 

 into the alcohol ; when the alcohol no longer fades (after 

 about 6 days) the preliminary hardening is completed ; 

 then place in 3% potassium bichromate. The color 

 of the iodin-alcohol must be restored as often as it 

 fades by the addition of fresh tincture of iodin and the 



