STAINS, ETC. 



1405 



ZOOLOGIC METHODS 



potash 0.2 c.c. , distilled water 200 c.c. Kuhne's 

 Carbolic Methylene-blue. Methylene-blue 1.5 gm. , 

 absolute alcohol IO c.c, 5 percent, aqueous solution of 

 carbolic acid 100 c.c. Rub up the dye in the alcohol, 

 and add, slowly, the carbolic acid. Deteriorates with 

 age. Kuhne's Methyl-violet. Methyl-violet 1 gm., 

 distilled water 90 c.c, alcohol 100 c.c. When using 

 add I drop of nitric acid to each 5 c - c - of the stain. 

 Lceffler's Methylene-blue. Thirty c.c. of a concen- 

 trated alcoholic methylene-blue solution are added to 

 IOO c.c of a solution of caustic potash (0.01 : 100). 

 Filter before using. Methyl-violet. I. (Alcoholic.) 

 Methyl-violet 25 gms. , absolute alcohol 100 c.c. 2. 

 (Aqueous.) Methyl- violet I gm. , alcohol 20 c.c, dis- 

 tilled water 80 c.c. Neelsen or Ziehl-Neelsen 

 Carbol-fuchsin. See Fuchsin. Ribbert's Dahlia- 

 violet. See Staining of Microorganisms, Pneumococcus. 

 Rindfleisch's Fuchsin. Distilled water, 90 per cent, 

 alcohol, nitric acid, each 50 c.c. , and enough fuchsin to 

 saturate. Add the fuchsin gradually and stir frequently. 

 Used for staining tubercle-bacilli. Sahli's Methyl- 

 ene-blue. Distilled water 40 c.c, saturated, aque- 

 ous solution of methylene-blue 24 c.c, borax-solu- 

 tion (5 per cent.) 16 c.c. Used when anilin-oil 

 is not available. Trenkmann's Gentian-violet 

 Anilin-water. A drop of a concentrated, alcoholic 

 solution of gentian-violet is let fall into a test-glass 

 and 10 c.c. of water are added. Half of this is then 

 poured away and the glass filled with anilin-water; a 

 solution is thus obtained that remains clear and stains 

 the bacteria deeply, but the ground very slightly. Cover- 

 glasses should remain about x / 2 an hour in the staining 

 fluid. Watson Cheyne's Contrast-stain. Saturated 

 alcoholic solution of methylene-blue 20 c.c, distilled 

 water 100 c.c, formic acid (sp. gr. 1.2) 1 c.c. Used 

 after staining in fuchsin anilin-water. Place sections 

 in the solution for from 1 to 2 hours. Weigert's 

 Differentiating Stain. A. Aqueous solution of gen- 

 tian-violet ; B. acidulated (acetic acid) picro-carmin. 

 Stain sections in A, then in B. The blue of the nuclei 

 will be altered to red and the bacteria will remain 

 blue. Weigert's Gentian-violet. Gentian-violet 2 

 gm. , ammonium hydroxid 0.5 c.c, distilled water 90 

 c.c. , absolute alcohol IO c.c. ; mix and filter. Ziehl's 

 Fuchsin. Filtered, saturated, aqueous solution of 

 carbolic acid 90 parts, saturated alcoholic solution of 

 fuchsin 10 parts. Used for staining the typhoid- 

 bacillus. Ziehl's or Ziehl-Neelsen's Solution. 

 See Staining of Microorganisms, Kiihne ' s Fuchsin. 



ZOOLOGIC METHODS. 

 Vnnelids. The blood-vessels of annelids may be 

 demonstrated by treating them for 2 or 3 hours with 

 aqua regia (2 parts of hydrochloric acid to 4 parts of 

 nitric acid). The animals should be laid open before 

 being put into the mixture. The vessel-walls appear 

 black on a yellow ground. The nerves may be brought 

 to view by staining with methylene-blue (see Staining 

 Reagents). Ehler's fluid (see Fixing Fluids) is 

 recommended for fixing and hardening. The intestine 

 of Lumbricus may be cleansed by putting the animal 

 into a tall glass jar filled with scraps of moistened blot- 

 ting-paper ; they gradually evacuate the earthy contents 

 of the gut and fill it with paper (Kukenthal ). Coffee- 

 grounds may be used instead of paper, and are said to 

 cut better when embedded. The animals may be nar- 

 cotized 1-y exposing them for y 2 an hour to the vapor 

 of chloroform ; if exposed for a longer period they will 

 die in a state of extension. Eyes of Arthropods. 

 Hickson's Method. Remove the posterior wall of 

 the head of a fly, and expose the rest to osmium- 

 vapor for 20 minutes ; wash in 60 per cent. 



alcohol, harden in absolute alcohol, and make 

 sections. Fix sections to the slide with Mayer's albu- 

 min (see Fixatives), remove the paraffin with turpen- 

 tine, wash with absolute alcohol, and decolorize by 

 inverting the slide over a capsule containing 90 per 

 cent, alcohol to which a few drops of nitric acid have 

 been added ; wash with pure alcohol. Parker's 

 Method. For the eye of Homarus. Make paraffin 

 sections and fix them to the slide with Schallibaums 

 collodion, treat with alcohol, then with water, and then 

 for % a minute with a o. 1 per cent. caustic-pota»h 

 solution ; wash thoroughly, and stain for 3 hours in 

 Weigert's hematoxylin (see Staining Reagents) at a 

 temperature of 50 C. ; wash, dehydrate, and mount in 

 balsam. Eyes of Gastropods. Carriere's Method. 

 Excise the eye and a part of the tentacle ; expose them 

 to osmium vapor for a few minutes, and prepare sections 

 in the usual way. Fix the sections to the slide with 

 Schallibaum's collodion (see Fixatives), decolorize 

 with very dilute Javelle water, stain with picro-carmin, 

 and mount in dammar. Gelatin Casts. A method for 

 making anatomic models, etc., from a combination of 

 gelatin 2 oz., glycerin 4 oz., the best Irish glue 4 oz. , 

 and boiled linseed-oil *£ oz. Soften the glue in 60 per 

 cent, alcohol, melt it, stir in the glycerin and oil, and 

 add a few drops of carbolic acid. This mixture has 

 the merit of resisting ordinary temperature-changes, 

 and models made of it are not softened by heat or 

 rendered brittle by cold. In making a cast, melt the 

 mixture and pour it into the mold, which should be 

 previously warmed. If it is desired to color the ca»ts, 

 dry or tube colors may be used, and should be well 

 mixed with the glycerin before being added to the glue 

 (J. W. Scollick). Infusoria. Asa mechanical means 

 of slowing the movements of minute organisms, 

 Eismond directs that a drop of a thick, aqueous solu- 

 tion of cherry-tree gum be added to the water con- 

 taining them, and an intra vitam stain may be 

 obtained by adding methylene-blue to the gum-solution. 

 Jensen prepares a solution of 3 gm. of gelatin in 100 

 c.c of water, which is a jelly at the ordinary tempera- 

 ture ; for use it is warmed, and a drop of it is mixed in 

 a watch-glass with a drop of water containing the 

 organisms. This method inhibits movement while 

 preserving life, and is recommended for vivisection. 

 Living Infusoria may be stained in very dilute solutions 

 of cyanin, methylene-blue, and other anilins, in the 

 medium that constitutes their natural habitat. They 

 may also be examined in a colored medium which does 

 not stain them, but which simply serves as a dark back- 

 ground. For this purpose a solution of anilin black is 

 recommended ; the organisms will live in this for several 

 weeks. Cattaneo fixes the organisms for a few minutes 

 in a 0.33 per cent, aqueous solution of palladium chlorid, 

 or with the double chlorid of gold and cadmium, which 

 demonstrates the nuclei better than the palladium. 

 Brass treats protozoa that are opaque through accumula- 

 tion of nutritive material for a few minutes with 

 Kleinenberg's fluid and then with boiling water ; from 

 this they are brought into water containing a little 

 ammonia. To stain, neutralize the ammonia by adding 

 acetic acid, and bring into borax -carmin ; wash, and 

 mount in dilute glycerin. The method of Certes is as 

 follows : Expose the organisms to osmium-vapor for 

 from 10 to 30 minutes, cover, and remove the excess 

 of liquid with bibulous paper. Prepare a solution of 

 picrocarmin, 1 part, in I part each of water and 

 glycerin, and place a little of this stain at the edge of 

 the cover-glass; place the slide in a moist chamber, 

 and when the water has evaporated and the glycerin 

 taken its place, strong glycerin is added and gradually 

 substituted for the dilute. The organisms thus prepared 



