1888.] MICROSCOPICAL JOURNAL. 231 



Notes on Histological Technique. 



By GEORGE C. FREEBORN, M. D., 



INSTRUCTOK IN NORMAL HISTOLOGY IN THE COLLEGE OF PHYSICIANS AND SURGEONS, N. Y. 



A Selective Stain for Connective Tissue. — The dye employed is one 

 of the aniline dyes, known in commerce as nigrosine, induline, or aniline blue- 

 black. It comes in two forms, one soluble in water, the other in alcohol. 

 The first variety is the one used. The quality of this dye varies greatly ; with 

 some samples I was unable to obtain any constant results, but with the nigro- 

 sine obtained from Dr. Griibler, in Leipsic, m}^ results have been uniform. 



The staining fluid is prepared as follows :^To 45 c.c. of a saturated aqueous 

 solution of picric acid 5 c.c. of a i per cent, aqueous solution of nigrosine 

 ai'e added. This makes a dark olive-green fluid. 



Sections of tissues, hardened in any of the usual media, are placed in the 

 staining fluid from water, and allowed to remain in it for from three to five 

 minutes, the exact time depending upon the thickness and density of the sec- 

 tions. They are then removed and washed in water vmtil their color changes 

 from a yellowish-green to a deep blue. 



The sections are now dehydrated, cleared in oil of cloves, and mounted in 

 Canada balsam. The oil of cloves is to be preferred for clearing as it dissolves 

 out the celloidin, which stains deeply with the dye, and, if allowed to remain 

 in the sections, detracts from the sharpness of the picture. Or, after dehy- 

 drating, the sections are stained for from five to six minutes in a mixture of 

 I c.c. of a saturated alcoholic solution of eosin and 49 c.c. of 97 per cent, alco- 

 hol, then cleared and mounted in balsam. A too prolonged action of the 

 eosin-alcohol will remove the primary stain from the finer fibres of connec- 

 tive tissue. 



Sections stained by the first method show all connective tissue fibres stained 

 bright blue, nuclei blackish, all other elements greenish-yellow. In the second 

 method the yellow color is replaced by red. 



Carminic Acid. — This dye was first recommended by Dimmock* as a 

 staining agent for histological work in place of carmine, the varying quality of 

 the latter rendering it very unsatisfactory. Dimmock used it in a f percent, 

 solution in 8=^ per cent, alcohol ; stained sections in it for two to five minutes, 

 and, after clearing, mounted in balsam. If a pvu^e nuclei stain was wanted 

 he washed the sections in a i per cent, aqueous solution of hydric chloride. 



I have found this dye when used in the following manner an excellent stain 

 for ganglionic cells : — Sections of the central nervous system are overstained 

 in Dimmock's solution and then washed in a 10 per cent, aqueous or alco- 

 holic solution of the officinal solution of the chloride of iron. In this flviid 

 the color of the section changes from a deep red to black, clouds of color 

 being given off. As soon as the section begins to show a yellowish color it 

 is transferred to a large dish of water, washed well, dehydrated, cleared in 

 oil of origanum, and mounted in balsam. 



vSections treated by this method show the nerve cells and their processes 

 stained black, the intercellular substance yellowish. 



Macerating Fluid for Nerve Cells. — Thin slices of the spinal cord, 

 cerebral cortex, or cerebellum, not over one-sixteenth of an inch thick, are 

 placed in fifty times their volume of a 5 per cent, aqueous solution of potas- 

 sium chromate for twenty-four hours. At the end of this time the gray mat- 

 ter will be found to have become jelly-like in consistency and quite transpar- 

 ent. It is to be cut away from the white matter and the bits placed in a long, 

 narrow tube — aMohr's burette with the lower end closed with a cork answers 

 the purpose perfectly. The burette is then filled up to within one inch of the 



* Proceed. Soc. Amer. Natrl. of the Eastern U. S., Amer. Nat., xviii, p. 324. 



