APPENDIX. 



vation of the tissue and precedes the embedding and sectioning, this 

 arrangement being based on the supposition that the tissue is to be 

 stained in bulk and cut in paraffin : with this sequence in view, the 

 specimen is removed from the 80 per cent, spirit and placed directly 

 in the staining solution, which, for all the ordinary purposes for which 

 carmine is employed, is best made up as : 



a. Borax-Carmine (Grenacher). 



Carmine, best 2.5 gm. 



Borax 4.0 gm. 



Water 100 c.c. 



Alcohol (70 per cent.) 100 c.c. 



The carmine and borax are thoroughly rubbed up in a mortar and 

 dissolved as far as possible in the previously-heated water, the alco- 

 hol being subsequently added. The fluid may then be filtered, but 

 it is preferable not to do so ; the solution is set aside for at least two 

 weeks, and then carefully decanted. 



The exact length of time required to stain sufficiently a block of 

 tissue throughout evidently depends upon the size and density of 

 the specimen ; it is, however, seldom safe to trust to an immersion 

 of less than 24 hours' duration, and if the object be of large size and 

 compact texture, say a piece of kidney 2 cm. in thickness, it should 

 be allowed to remain in an ample quantity of the stain for at least 

 three days. The vessel containing the fluid and tissue must be well 

 stoppered, a wide-mouthed bottle or tightly-covered capsule being 

 the most suitable receptacle. 



From the carmine the tissue is directly transferred, without the 

 slightest washing in water, into a large quantity of acid alcohol, 

 made by adding strong hydrochloric acid to 70 per cent, alcohol in 

 the proportion of 5 drops of acid to every 100 c.c. of spirit. The 

 object of the acid solution is to effect differentiation and fixation of 

 the color ; for this purpose the tissue should remain at least 24. hours, 

 and, if of the size and character above supposed, twice as long 

 until the frequently-changed acid alcohol no longer becomes tinged. 

 If the staining has been successful, the block of tissue now appears 

 of a brilliant deep uniform red ; if inspection shows inequality of 

 tint or insufficient color in the central parts of the mass, the staining 

 will not be satisfactory and should be repeated. Failure in bulk 

 staining is due to an unfavorable condition of the tissue or to an 

 improperly-prepared staining fluid, and not to the method, which 

 extended experience shows is always capable of yielding the most 

 gratifying results, whose brilliancy and differentiation compare favor- 

 ably with those of any carmine staining of individual sections. 



Where it is preferable to stain the sections after cutting, the same 



