APPENDIX II 691 
No. 4. SAFRANIN AND METHYL GREEN 
1. Stain sections with either alcoholic safranin or anilin 
safranin for from 1 to 24 hours or by warming them in the safra- 
nin solution until vapors arise. 
2. Remove excess of stain by moving sections about in a vessel 
of 50 per cent. alcohol until the cellulose walls are pink in color. 
3. Stain with 0.5 to 1 per cent. solution of methyl green for 
from 30 seconds to 5 or 10 minutes. Good results with this stain 
requires experience with the materials to be stained. The 
average hand cut section requires 10 minutes. 
4. Remove excess of stain and dehydrate in 70 per cent. 
alcohol for several seconds. 
5. Dehydrate with 95 per cent. alcohol for 10 seconds. 
6. Further dehydrate by placing material in absolute alcohol 
for one minute. 
7. Clear in cedar oil for one minute. Blot up excess of oil 
from around edge of section. 
8. Mount in Canada balsam. 
9. Label slide. 
If the sections are properly stained, the lignified and suberized 
walls will be stained red and the cellulose walls green. 
METHOD FOR THE PREPARATION OF A GLYCERIN-GELATIN Mount 
1. Stain object with an aqueous solution of eosin. 
2. Transfer to 1 per cent. acetic acid for a minute. 
3. Wash out acid and excess of stain by moving the section 
about in a dish of water. 
4. Transfer object to weak glycerin (glycerin 10 parts, water 
90 parts) for 3 to 5 minutes. 
5. Transfer object to 50 per cent. glycerin for 3 to 5 minutes. 
6. Transfer object to concentrated glycerin for 5 minutes. 
7. Remove excess of glycerin around object and mount in 
glycerin-gelatin. The slide and cover slip should be warmed 
_ before the glycerin-gelatin is dropped over the object and the 
cover slip quickly lowered. The preparation of Glycerin- 
gelatin is as follows: Macerate 14 grams of gelatin in 84 cc. of 
water for 2 hours, add 76 cc. of glycerin and warm; add 2 cc. of 
liquefied phenol, warm and stir for 15 minutes untilclear. Filter 
