448 APPENDIX 



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

 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. 



Nogitchi has used the following modification in demonstrating spirochaetes in 

 brain and cord: Fix ^-inch slabs of tissue in 10% formalin for four or five days. 

 Then place tissues in the following solution: Formalin 10 c.c., pyridin 10 c.c., 

 acetone 25 c.c., absolute alcohol 25 c.c., distilled water 30 c.c. Keep in this 

 solution for five days at room temperature. Then wash in water for one day. 

 Transfer to 95% alcohol for three days and then wash in water for one day. Put 

 tissue in dark bottle in i^% aqueous solution of silver nitrate for five days at room 

 temperature. Wash in distilled water for five to six hours. Transfer to reducing 

 mixture of 95 c.c. of 4% aqueous solution of pyrogallol and 5 c.c. of formalin. Keep 

 in this solution twenty-four hours. Wash in water and put through alcohol and 

 xylol. Imbed in paraffin. 



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 xylol with absolute alcohol, pour on a sufficient number of 

 drops of stain and after one minute 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. 



I now use the panoptic method for staining tissues. In this I stain with Wright's 

 stain as given above but following the washing of the section we treat this with a 

 dilute Giemsa (1-15) for ten to fifteen minutes. Then wash and differentiate in i to 

 1000 acetic acid in water in a small beaker. When the section assumes a pinkish 

 tinge wash in tap water, then in 95% and absolute alcohol and clear in xylol. 

 Then mount in liquid petrolatum, immersion oil or balsam. 



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 solu- 

 tion of paraffin in carbon bisulphide and thence to paraffin. Bisulphide of carbon 



