1921] SPENCER—BRAZIL NUTS 271 
fixative, flooded with water, and allowed to stand over night. 
They were stained with Pianese IIIb, or with jod griin-erythrosin 
in the following manner. They were placed in 95 per cent alcohol 
for five minutes, flooded with jod griin (1 per cent solution in 95 
per cent alcohol) for thirty minutes, washed with 95 per cent 
alcohol, then absolute alcohol, flooded with 1 per cent solution of 
erythrosin in clove oil for forty-five minutes, washed with absolute 
alcohol, cleared with carbol-turpentine clearer, and finally washed 
in xylol and mounted in balsam. This proved to be the most 
satisfactory of any method tried for staining mycelium in the 
tissue. 
PROTEOLYTIC ENZYMES.—The Brazil nut agar serves well to 
show the presence or absence of certain extracellular proteolytic 
enzymes. The proteid precipitates to which the opacity of the 
agar is due are digested by the enzymes, and a transparent halo, 
which enlarges as the thallus or colony enlarges, surrounds the 
growth. All the organisms studied were tested for the presence of 
these enzymes. The enzymes were precipitated from cornmeal 
broth in which an Actinomyces or a Bacillus, the two most active 
enzyme producers, was grown. The broth was poured into Pior- 
kowski culture flasks to a depth of 0.25 inch, about 200 cc. being 
required for each flask, and inoculated. After ten days the culture 
was filtered through paper and enough 95 per cent alcohol added 
to the filtrate to make 80 per cent alcohol. Three days later a 
fluffy white precipitate had collected at the bottom of the precipita- 
tion cones, and the excess alcohol was siphoned off (12). From 
25 to 5occ. of absolute alcohol was added to the precipitate and 
immediately filtered. Before the precipitate had become dry 
it was again washed with 50 cc. of absolute alcohol, and while still 
moist was removed to a desiccator containing sulphuric acid, and 
allowed to remain there for two days. The hard, gray material 
_was then scraped from the paper to be redissolved in sterile water 
when used. 
MorpHOLoGcy oF Brazit NuT.—The kernel of the Brazil nut, 
as it is ordinarily removed on cracking the shell, is covered with 
a thin, dry coat which may be quite loose or may adhere very 
tenaciously. The embryo within, principally radicle, is completely 
