APPENDIX 375 



Mix equal parts of number one and number two. The mixture only keeps about 

 three days. The HC1 prevents overstaining. 



This stain followed by Van Giesen's stain gives more perfect results than any 

 common method of staining. The iron haematoxylin intensifies the sharpness of 

 the Van Geisen differentiation. 



Van Giesen's Stain. Take of i% aqueous solution acid fuchsin from 5 to 15 

 c.c. Saturated aqueous solution picric acid 100 c.c. The method of using is to first 

 stain with haematoxylin in the usual way. Then pour on the picric-acid fuchsin 

 solution and allow to stain for one to five minutes. Wash, pass through alcohols 

 and xylol and mount in balsam. 



Connective-tissue fibers, axis cylinders, and ganglion cells are stained a bright 

 garnet red. Myelin, muscle fibers, and cells generally are stained yellow. Nuclear 

 staining is that of hasmatoxylin. The stronger stain is used for nerve tissue; the 

 weaker, for demonstrating connective tissue in tumors. 



Levaditi's Method. Take small pieces of tissue, about 2 mm. in thickness, and 

 harden in 10% formalin for twenty-four hours and then in alcohol for the same period; 

 then wash in water for a short period. They are stained in a freshly made solution 

 of silver nitrate 1.5% for three successive days, changing the solution each day, 

 maintaining the blood temperature, and excluding light. The tissue is then placed 

 in a 2% solution of pyrogallic acid, with the addition of 5% formalin. After remain- 

 ing in this for twenty-four hours, light being excluded, they are passed through 

 85%, 95%, and absolute alcohol, respectively; embedded in paraffin; and cut in 

 about 5 micron sections. Equally good results may be obtained by allowing the 

 silver nitrate to act at room temperature and embedding in celloidin. 



Romanowsky. Staining sections with Romanowsky stains is not very satis- 

 factory. The differential staining seems to fade out in passing through the alcohols. 

 This may be avoided by blotting the section after staining and differentiation and 

 then applying the xylol to the blotted section. After staining with Giemsa's stain 

 for ten to fifteen minutes, differentiate with i to 500 acetic acid. When the section 

 has a pinkish tinge, wash in water, dry, clear in xylol, and mount. 



Good tissue staining may be gotten with Wright's stain. After removing the 

 paraffin with xylol and the rylol with absolute alcohol, pour on a sufficient number of 

 drops of stain and immediately dilute with an equal number of drops of water. 

 Allow the diluted stain to remain for three to five minutes. Next wash in water, 

 differentiate, until the tissue has a pinkish tinge, in i to 500 acetic acid. This 

 differentiation is best done in a tumbler of the dilute acetic acid. 



After washing in water, quickly pass through 95% and absolute alcohol, clear in 

 xylol, and mount. 



Skin Sectioning. Of all tissues that of skin offers the greatest difficulty in pre- 

 paring sections. The best results can probably be obtained by fixation in picro- 

 sublimate (saturated aqueous solution picric acid i part; saturated aqueous solu- 

 tion bichloride of mercury one part); to this stock mixture add 5% glacial acetic 

 acid just before using. Fix small pieces of skin six to eighteen hours. Transfer 

 direct to 70% alcohol in which the tissue may be kept indefinitely. 



For sectioning run through alcohols to absolute and then to a mixture of absolute 

 alcohol and carbon bisulphide (equal parts). Leave until tissue sinks, then transfer 

 to pure carbon bisulphide until tissue sinks. Then transfer to a saturated 



