STAINS 



505 



STAINS 



Rabl's Mixture for fixing embryos : One volume 

 each of saturated solutions of sublimate and picric acid 

 and 2 volumes of distilled water. After 12 hours' fix- 

 ing wash in water and transfer to dilute alcohol. 

 Ranvier's Method for the study of the clasmatocytes 

 of amphibia and mammals: Stretch the mesentery. 

 fix with osmic acid, stain with violet 5 B, and mount 

 in glycerol. Result : nuclei, blue ; cytoplasm, violet. 

 Ravenel's Medium for bacterial cultures: 1. (a) 

 Add 10 gm. of dried peptone, 5 g™- each of salt and 

 Liebig's extract, to 500 c.c. of water; boil for 3 min- 

 utes and neutralize. (b) Add 12 gm. of chopped 

 agar-agar to 530 c.c. of water and place in the auto- 

 clave. Run autoclave up to two atmospheres of 

 pressure, giving 121. 4 C. of heat. As soon as this 

 pressure is reached, turn out the flame and allow the 

 autoclave to cool until below 100 C. before opening. 

 Mix a and b, cool to 6o° C, add the white of 2 eggs 

 beaten in 5 c - c - of water, boil, and filter through 

 paper. Blood-serum may be added instead of the egg 

 albumin. 2. (<z) To make a clear and permanent 

 agar medium chop 500 gm. of fresh meat, add 500 c.c. 

 of water, stand in a cool place overnight, and strain 

 through a towel. (b) The same as b in No. I. 

 When cooled to 7 s C. mix a and b, add 10 gm. of 

 dried peptone, 5 g m - °f s 3 '*, boil for 3 minutes, neu- 

 tralize, and filter. Rawitz's Aqueous Carmin : 

 Dissolve 2 gm. carminic acid and 20 gm. ammonia 

 alum in 150 cc. each of water and glycerol. Raw- 

 itz's Artificial Alizarin, a process by means of which 

 a double stain is obtained, staining cytoplasm and 

 chromatin different colors. Prepare a 2. 5 % suspen- 

 sion of alizarin RX. in distilled water and add a few 

 drops of I ^ calcium acetate. Stain for 24 hours at a 

 temperature of 40 C. The sections, which should 

 be of material fixed in chromic acid or in Hemming" s 

 mixture, must be treated with cbrombeize G A I before 

 they are put into the stain. Rawitz's Fluid : I. 

 Four parts of I % chromic acid and one part of picro- 

 nitric acid. 2. One part of 2% osmic acid and 6 

 parts of picronitric acid. Wash in 70',- alcohol. 

 Rawitz's Inversion Stain : Put sections fixed in 

 Flemming's or in Hermann's fluid for 24 hours into 

 20% aqueous solution of tannin (prepared cold); 

 wash and put them for 2 or 3 hours into a I or 2 ' r 

 solution of tartar emetic, at a temperature of 37 C, 

 or for 24 hours at room-temperature ; wash and stain 

 for 24 hours with safranin, fuchsin, methyl -violet, 

 gentian-violet, or emerald green ; differentiate with 

 alcohol I or with 2. 5 ^ solution of tannin) ; clear and 

 mount in the usual way. Successful preparations 

 show nuclei colorless, cytoplasm and intercellular sub- 

 stance stained. In sections of testicle, centrosome and 

 astrosphere are intensely stained. By this method an 

 inversion of nuclear stains is obtained and they behave 

 as plasmatic stains. Rawitz's Mucicarminic Acid : 

 Dissolve I gm. of carminic acid and 2 gm. of alumi- 

 num chlorid in ioo c.c. of 50^ alcohol; evaporate to 

 dryness on a sand-bath and dissolve the residue in loo 

 c.c. of 50 <£ alcohol. For application and technic see 

 mucicarmin. Red from Methylene-blue. Accord- 

 ing to Nochte, a red pigment can be isolated by chlo- 

 roform from old alcoholic solution of methylene-blue. 

 This pigment makes a red-violet solution in water and 

 is not identical with methylene-red or methylene- 

 violet. Nochte names it "Rothaus Mefhylenblau." 

 It is said to be a specific stain for the young forms of 

 the malarial parasite. It can also be isolated from so- 

 lutions of borax-methylene-blue that have been kept 

 for several days at 50 to 6o° C. Rees' Method for 

 the preservation of mosquitos : Narcotize or kill the 

 insect and place it ventral side up on a slide ; cover it 



with a large drop of thick xylol -balsam, arrange the 

 legs and wings, and pour on some thin balsam, which 

 will straighten the proboscis and antennae. When the 

 balsam is hard, cut off the excess, make a cell with a 

 glass ring, so fill with balsam that the surface is con- 

 vex, and apply a cover-glass. Regaud's Method 

 for the study of the cells of Sertoli : Fix the testicle 

 in the liquid of Tellyesniczky. Stain the sections 

 deeply in alum hematoxylin, decolor in an aqueous 

 solution of formic acid I : 100, wash in water and stain 

 in safranin ; treat with very dilute acid alcohol, then 

 with neutral 90^- alcohol, absolute alcohol and xylol, 

 and mount in balsam. Result : cytoplasm, pale rose- 

 violet; chromatin, purple- violet to red-purple. Re- 

 gressive Staining of Heidenhain : A method of 

 overstaining followed by partial decolorization. It is 

 the same as the indirect method of Flemming. Cf. 

 Progressive Staining. Rehm-Nissl Method for the 

 connective-tissue elements of the central nervous sys- 

 tem : Fix in absolute alcohol and stain the celloidin 

 sections for one minute in hot aqueous solution of 

 methylene-blue; wash in 95% alcohol and stain for 

 from 15 to 30 minutes in O.I ^ solution of magenta in 

 95 fy alcohol ; wash in alcohol and clear in clove oil. 

 Nerve-cells reddish blue with colorless nuclei and blue 

 nucleoli ; nuclei of connective-tissue elements red. 

 Reid's Method for mounting mosquitos : Paralyze in 

 a drop of glycerin and then arrange with dissecting 

 needles. Reinbach-Ehrlich Stain: Mix 120, 80, 

 and 100 volumes respectively of saturated aqueous so- 

 lutions of orange G, acid fuchsin, and methyl green 

 and add 300 volumes of distilled water, 180 volumes 

 of absolute alcohol, and 50 volumes of glycerol. Do 

 not stir, a-, t-, and ,3- granules stain in mixture. 

 Reinke-Flemming Method for kinetic nuclei : 

 Treat sections of tissue fixed in Hermann's mixture 

 for 24 hours with saturated solution of potassium sul- 

 fite ; wash ; stain for I to 2 hours in saturated alco- 

 holic solution of safranin diluted with anilin-water ; 

 wash; stain for 24 hours in Reinke's gentian-orange. 

 Reinke's Gentian-orange : Add a few drops of a 

 saturated aqueous solution of orange G to a saturated 

 solution of gentian-violet. A drop on blotting-paper 

 should make a violet or brown spot with a narrow 

 orange border. For the application of this mixture- 

 see Rein ke- Flemming Method. Reinke's Method 

 for dissociating the cortical cells of hairs, the epithelial 

 cells of salamandra, and the spermatozoa of the rat : 

 Treat the object with a. \of f solution of lysol in dis- 

 tilled water, to which alcohol and glycerol may be 

 added. Its action is said to be instantaneous and to 

 be destructive to chromatin. Renaut's Method for 

 nerve-fibers : To 4 c.c. of a mixture of equal parts of 

 I fy osmic acid and saturated solution of picric acid add 

 I c.c. of \ r r silver nitrate and inject with a gold or 

 platinum needle into the still warm tissue. Harden 

 in alcohol and stain. Retterer's Method for the 

 study of developing cartilage in reticular connective 

 tissue : Fix the object in aqueous solution of platinum 

 chlorid I : iooo and without decalcifying embed in 

 paraffin and stain in safranin in anilin-water; wash in 

 water and stain in alum hematoxylin ; wash in alco- 

 hol containing a very little picric acid. Reuter's 

 Stain for blood : Preparation of the solution of poly- 

 chrome methylene-blue: To 100 c.c. of 1% aqueous 

 solution of methylene-blue — blue med. puriss. 

 (Hochst) add 0.5 gm. NajCO,. Keep this solution, 

 for 2 or 3 days at 40 to 6o° C. Filter. Preparation 

 of the neutral stain : Without previously neutralizing 

 the polychrome methylene-blue, add to it a saturated 

 aqueous solution of eosin (Hochst). Filter off the 

 precipitate formed, and wash it with distilled water. 



