STAINS, ETC. 



1387 



STAINING OF MICROORGANISMS 



tain sections of skin hardened in Miiller's fluid for 5 

 minutes in a mixture of equal parts of anilin-vvater and 

 concentrated aqueous solution of methyl-violet ; wash 

 in water, immerse for a few seconds in Gram's solution, 

 wash again in water, dry with filter-paper, and differ- 

 entiate in a mixture of 1 part of anilin to 2 parts 

 of xylol, and then place in pure xylol This . 

 process demonstrates the ' ' intracellular and inter- 

 cellular fibrils " of epithelia. Mitrophanow s Method. 

 For the study of prickle-cells and intercellular canals. 

 Wash the tail of an axolotl larva in distilled water ; im- 

 merse it for an hour in 0.25 per cent, gold-chlorid so- 

 lution containing one drop of hydrochloric acid to about 

 5 c.c. ; wash, and reduce in a mixture of I part formic 

 acid and 6 parts water. Goblet-cells. Scrape the 

 mucous surface of the stomach of a frog after hardening 

 for 24 hours in dilute alcohol, and press the scrapings 

 between two cover-glasses. Allow the film adhering to 

 each glass to dry, and then stain with the Ehriich-Biondi 

 fluid. Inner Ear. Open the cochlea in Flemming's 

 solution, and fix 4 or 5 hours ; decalcify, if necessary, 

 in I percent, palladium-chlorid solution. Make pr.raffin 

 sections and stain with Renaut's eosin-hematoxyiin, or 

 with safranin. Isolated Mucous and Demilune 

 Cells. Place small fragments of the fresh submaxillary 

 gland of a dog in 5 P er cent, ammonium chromate for 

 4 to 6 days ; then tease a small piece in the same fluid. 

 Each isolated mucous cell has its fibrillar network, 

 a spheric nucleus embedded in protoplasm, and what 

 was the attached end of the cell prolonged into a 

 process. " Mastzellen." Schiefferdecker' s Method. 

 Place a piece of the mesentery of a rat in a solution of 

 gentian-violet in anilin-water for 24 hours ; rinse in 

 water, decolorize in acid alcohol, rinse again in water, 

 counterstain with carmin, and mount in balsam. The 

 nuclei appear red, the granules blue. See Staining of 

 Blood, Ehrlich' s '■'■Mastzellen.' 1 ' 1 Pacinian Corpus- 

 cles. Harden a piece of skin in alcohol or osmic acid. 

 Stain sections in picrocarmin, safranin, or hematoxy- 

 lin, or stain in the mass with borax-carmin. Plasma- 

 cells. 1. Xordmanri 's Method. Stain sections in a 

 solution of vesuvin containing 4 or 5 per cent, of hydro- 

 chloric acid ; after a few minutes' immersion, remove 

 and dehydrate in absolute alcohol. 2. Unna's Method. 

 Add 10 to 15 drops of a solution of methylene-blue I 

 part, caustic potash 0.05 parts, in distilled water loo 

 parts, to a watch -glassful of anilin-water ; stain sections 

 of tissue hardened in alcohol for several hours ; dehy- 

 drate in absolute alcohol, differentiate in cresol, rinse 

 in xylol, and mount in balsam. Red Marrow. I. 

 Expose a cover-glass preparation of red marrow to os- 

 mium vapor for one or two minutes, stain in picrocar- 

 min, and mount in glycerin. 2. Stain a cover-glass pre- 

 paration for 24 hours in the Ehriich-Biondi mixture, and 

 mount in xylol-balsam. Retina. 1. Remove the lens I 

 and the vitreous body, and inject into the cavity of the 

 eye a mixture of equal parts of acetic acid and osmic 

 acid, 2 per cent. ; 3 minutes are required to fix. Wash 

 in alcohol for 15 minutes, and place for 2 hours in 

 Johnson's bichromate and platinic mixture (see Fixing 

 Fluids) ; wash in running water, suspend for 2 days in 

 a large volume of 2.5 per cent, potassium-bichromate 

 solution, and pass through successive alcohols, beginning 

 with 20 per cent, and ending with absolute. Stain in 

 the Ehriich-Biondi mixture, adding to it one-third of 

 20 per cent, solution of nigrosin. The nuclear cells 

 appear pale-brown, the nucleoli a deeper-brown. The 

 Miiller fiber layers, the molecular layers, and the rods 

 are stained a beautiful green. 2. (a) Kill in the dark a 

 frog that has been kept in darkness 36 hours, and 

 harden the eye in alcohol. (b) Kill another frog kept 

 in direct sunlight for a few hours, and harden the retina 



in alcohol. Make sections, and stain with picrocar- 

 min. The pigment-cells covering the rods of the 

 retina in a are retracted, while those in b are pushed 

 out between the segments of the rods. Pin the excised 

 eyeball of a triton (without opening the bulb) to a cork, 

 and expose to osmiiftn vapor for IO minutes. Then di- 

 vide it by an equatorial incision, and place the posterior 

 pole in one-third alcohol for from 6 to IO hours, and 

 then for the same length of time in picrocarmin; 

 harden in osmic acid, embed and cut in soft paraffin. 

 Salivary Glands. 1 . Heidenhaiii's Method. Harden 

 small pieces for I hour in 75 per cent, alcohol, 5 hours 

 in absolute alcohol, 24 hours in a fresh supply of abso- 

 lute alcohol. Stain 6 to 8 hours in 10 c.c. of a 1 per 

 cent, aqueous solution of hematoxylin, and differentiate 

 6 to 8 hours in 1 per cent, solution of potassium bichro- 

 mate. Embed in paraffin. The nuclei appear bluish- 

 black, the cell-substance steel-gray, and the demilunes 

 very distinct. 2. Schiefferdecker s Method. Stain sec- 

 tions, hardened as described, for half an hour in a watch- 

 glassful of alcohol, to which a few drops of a 5 per cent, 

 alkaline alcoholic solution of eosin have been added ; 

 then place them for a few minutes in a I per cent, aque- 

 ous solution of anilin-green ; dehydrate and mount. 

 Tactile Corpuscles. Impregnate pieces of skin with 

 gold chlorid, according to Lowit's method (see Stain- 

 ing Feagents, Metallic Stains), harden in alcohol, sec- 

 tion, and stain in picrocarmin, hematoxylin, or purpu- 

 rin. Tendon. 1. Take the tendon of the anterior 

 and superior insertion of the gemini muscles of a rabbit, 

 remove as far as possible the adherent muscle-fibers, 

 treat according to Ranvier's formic -acid-gold method 

 (see Staining Reagents, Metallic Stains) ; after reduc- 

 tion of the metal scrape with a fine scalpel, to remove 

 the muscle -tissue that masks the corpuscles of Golgi, 

 which this method is intended to demonstrate. 2. 

 Harden a rat's tail, denuded of integument, for 3 hours 

 in 5 per cent, corrosive-sublimate solution, and wash 

 well in alcohol. Stain in bulk in borax-carmin, de- 

 calcify in dilute hydrochloric acid, embed in paraf- 

 fin, and make transverse sections. Terminal Discs 

 in Tongue of Frog. Curarize or etherize the frog, 

 and inject through the abdominal vein a solution of 

 methylene-blue I part in 800 parts of 0.6 percent, salt- 

 solution, and secure access of air to the mouth. Good 

 results are also obtained by simply pouring the stain into 

 the. mouth. Test for Non-striped Muscle. Fix 

 the tissue in a mixture of 10 volumes of 90 per cent, 

 alcohol and I volume of formic acid ; wash, and stain 

 for 24 hours in alum-carmin. The connective-tissue 

 cells are swollen and unstained. The cytoplasm of the 

 muscle-cells appears red. 



STAINING OF MICROORGANISMS. 

 General Methods. Ahrens' Method for Bacteria 

 in Milk or Fatty Substances. Dilute the milk 

 with an equal quantity of water or, in case of denser 

 substances, with a larger volume. Spread on a cover- 

 glass, and fix by heating after it has become dry. Stain 

 for 5 minutes in 12 or 15 drops of methyl-blue to which 

 3 or 4 drops of chloroform have been added. Then re- 

 move, and allow the chloroform to evaporate ; wash in 

 water; mount. Bizzozero's Method, for microorgan- 

 isms in the vermiform appendix. Stain the preparation 

 in a gentian-violet solution, wash in absolute alcohol for 

 half a minute, transfer to Gram's solution for two min- 

 utes ; then wash alternately in I per cent, chromic 

 acid and absolute alcohol, allowing the preparation to 

 remain half an hour or more in each fluid ; repeat the 

 chromic acid and alcohol, clear, and mount. The surplus 

 stain must be well removed by the alcohol. Ehrlich- 

 Weigert Method. Float the cover-glass (film-surface 



