APPENDIX. 401 



obtained with "water soluble" eosin (not "alcohol soluble"), of which 

 a .5 per cent, solution in 70 alcohol is preferable, the sections remaining 

 until decidedly rosy, usually a matter of 1-2 minutes. They are then 

 transferred to 70 alcohol, in which they remain so long as clouds of eosin 

 are discharged; after one or two changes of 70 alcohol and no further 

 color is given off, the sections are passed as rapidly as thorough dehy- 

 dration will permit through 95 alcohol, then cleared in carbol-xylol, and 

 mounted in balsam, as presently described. 



Grenadier's Borax-Carmine. 



Carmine (No. 40) ... 3 gm. 



Borax 4 gm. 



Distilled water 100 cc. 



70 alcohol 100 cc. 



The carmine and borax are rubbed together in a mortar in the warmed 

 distilled water until dissolved. After cooling, the alcohol is added and the 

 unfiltered solution placed in a stoppered bottle. The stain is not ready for 

 use until it has stood for several weeks. The quantity required for staining, 

 which may be repeatedly employed, should be decanted and filtered before 

 using. Although sections may be stained with .the solution, its chief value 

 is for mass-staining, since its powers of penetration and uniform coloration 

 are excellent. Assuming that the solution is to be put to this use, the 

 object is transferred from 70 alcohol to the undiluted stain, in which it 

 remains 24-48 hours and is then directly placed, that is without washing, 

 into acid alcohol (8 drops of hydrochloric acid to 100 cc. of 70 alcohol) 

 for 8-24 hours, or longer, depending upon the size and density of the 

 object. The purpose of the acid bath is to differentiate the nuclei and fix 

 the stain. Deep staining and thorough differentiation produce the most 

 satisfactory preparations. After two changes of 70 alcohol, the object 

 remaining from 2-3 hours in each, it is dehydrated in 95 and absolute 

 alcohol preparatory to embedding. 



Contrast staining after carmine may be carried out with .5-1 per 

 cent, alcoholic solutions of such aniline dyes as methylene blue, methyl 

 violet, or methyl green. The labor and time involved in staining serial sec- 

 tions on the slide, as of course must be done, as well as the limited endurance 

 of the aniline tint, ordinarily deter from double staining. Although the 

 second dye yields pleasing preparations, unless there is some special reason 

 to warrant the additional labor, the contrast color may be omitted with serial 

 sections of tissues stained in bulk. 



Staining sections on the slide is necessary when it is desirable to 

 tinge uncolored paraffin sections, or to add a contrast tint to those stained 

 en masse before cutting. Since the paraffin must be removed from the sec- 

 tion before the stain can act, it is evident that the support afforded by the 

 embedding mass must be replaced by that of the slide before the paraffin may 

 be removed with safety. The sections must, therefore, be fixed to the slide, 

 so that subsequent manipulations will not disturb even the most delicate and 

 isolated parts of the sectioned object. 



All slides and cover-glasses used for mounting preparations must be 

 thoroughly cleaned. This is readily accomplished by soaking the slides and 

 cover-glasses in strong sulphuric acid for 5-10 minutes, care being taken to 

 immerse the pieces separately and not to allow them to adhere. They are 

 transferred, piece by piece, to a large vessel containing water and washed 

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