GLYCOGEN IN THE NERVOUS SYSTEM 461 
gram). The tissue is fixed in this twelve to twenty-four hours and then 
transferred to 67 per cent alcohol the same time; and then for a day or 
more in 82 per cent alcohol before the final imbedding. Plenty of 
fixer and alcohol should be used. 
Other fixers have been recommended for glycogen, and many dif- 
ferent ones preserve a part of the glycogen, but as the purpose of any 
investigation on glycogen is to find all the elements in which it is 
present either in large or in small amounts, a fixer should be used 
which experience has shown to be the most precise and certain, and that 
fixer is alcohol. 
For large embryos, small animals, limbs, etc. containing bone it 
was found entirely practicable to decalcify the bone without in any 
way disturbing the glycogen. The embryo, animal or part is fixed 
with alcohol as usual for glycogen, then it is placed in the nitric acid 
decalcifier composed of 67 per cent alcohol to which has been added 3 
per cent nitric acid. After the decalcification is complete, the embryo 
remains a day cr twe in 67 per cent alcohol, changed two or three times. 
It is then transferred to 82 per cent alcohol for a day or more before 
dehydrating and imbedding. 
' Imbedding and sectioning. Either the collodion or the paraffin 
method can be used. The paraffin method or the combined collodion 
and paraffin method’has proved most satisfactory in my work. Scme 
of the sections should be moderately thick, 10 to 15 u. Sections less 
than 5 uv are not serviceable for glycogen investigations. 
Staining glycogen. The only fully reliable and satisfactory stain 
for glycogen is iodin. As some glycogen is very soluble, a glycogen 
stain containing alcohol was found most generally useful (95 per cent 
alcohol, 150 ece.; water, 150 cc.; iedin crystals, 1.5 grams or 15 ce. of a 
10 per cent alcoholic solution of iodin; iodid of potassium, 3 grams; 
sodium chlorid, 1.5 grams). For the aqueous stain, water is used 
instead of the alcohol mixture. 
For staining, spread the paraffin sections with the iodin stain in- 
stead of water. The glycogen in the sections will stain a mahogany 
red and the stain will remain in the spread sections for years (10 to 
15). If care is taken not to melt the paraffin when spreading the sec- 
tions, they can be restained at any time by immersing the slide in the 
stain or placing some of the stain on the sections. 
Permanent preparations. The permanence of the iodin stain in the 
spread paraffin sections gave the clue. For low powers the sections in 
paraffin show very well without further preparation, but for high 
powers the crystals of paraffin interfere. Various paraffin media 
were tried, and finally ordinary yellow vaseline was settled upon 
as best. For mounting, the sections are restained by immersing the 
slide in the iodin stain for two to three minutes or longer; they are 
then dried half an hour or more in the air or in a drying oven, then 
immersed in xylene to dissolve off the paraffin. Some melted yellow 
vaseline is then put on the sections and a cover-glass added exactly 
