APPENDIX. 



carmine fluid 'may be employed, the sections, either loose or fixed to 

 the slide, being immersed from 15 to 20 minutes, and then directly 

 transferred to 10 per cent, acidulated 70 per cent, alcohol for about 

 5 minutes, followed by thorough washing in 70 per cent, spirit. 

 Where the tissue is robust, the acid solution for differentiation and 

 fixing may be made with water in place of the alcohol, water being 

 also used for the subsequent washing ; it is an advantage, however, 

 for delicate structures to avoid transfers from alcohol to water, keep- 

 ing as far as possible the sections in alcoholic solutions of about the 

 same strength. 



b. Delafield's Haematoxylin. 



(1) Haematoxylin, crystals 4 gm. 



(2) Alcohol, absolute 25 c.c. 



(3) Ammonia-alum, crystals 52 gm. 



(4) Water 400 c.c. 



(5) Glycerin 100 c.c. 



(6) Methyl-alcohol 100 c.c. 



Dissolve i in 2, and 3 in 4 ; mix, when a slightly-colored fluid is 

 produced ; let stand for 4 days, protected from dust, but with free 

 access to air and light, at the end of which time the fluid has turned 

 to a deep bluish purple. Filter, and add 5 and 6 ; a part of the am- 

 monia-alum falls out in small crystals. After several hours filter 

 again, and then keep in a tightly-stoppered bottle at least four or 

 five weeks before using. 



This tediously-prepared stain possesses the great advantage of 

 penetrating and staining well tissues in bulk, for many purposes 

 being a valuable adjunct to carmine staining. The strong solution 

 above given is diluted with distilled water, and the tissue allowed to 

 remain until of a very dark blue color, when it is placed in distilled 

 water for 24 hours to effect differentiation and remove excess of color ; 

 it is then transferred to 70 per cent, alcohol for subsequent treatment. 

 The action of the stain must be watched, as overstaining may readily 

 occur ; should the coloring be too intense, this may be remedied by 

 soaking in dilute hydrochloric acid, the action of the latter being 

 arrested at the proper time by water, which at the same time restores 

 the tissue to its former blue color, the acid having previously turned 

 it reddish or brown. It is very important to remove every trace 

 of acid, to prevent subsequent fading ; to this end thorough washing 

 after the use of acid is imperative. An avoidance of overstaining in 

 the first place is much more desirable than any subsequent correction. 

 In addition to the purposes of staining in bulk, this haematoxylin 

 fluid works well after fixation in chromic acid or Flemming's solution, 

 yielding excellent preparations of chromatin filaments in such tissues. 



