No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 
into the nucleus. And that in the prophase the single nucle¬ 
olus of the nucleus derives the material necessary for its 
further growth from the cell substance, seems highly probable 
when we recall the fact that at the time of its most rapid 
growth it is usually apposed to the nuclear membrane, which 
would denote that it is taking up a substance which penetrates 
that membrane from the side of the cell body. 
We have alluded to certain chemical changes which occur in 
the nucleolar substance when discharged from the nucleus 
during the metaphase of the latter. These staining differentia¬ 
tions and the coloration of the cytoplasm as observed on five 
different preparations are as follows (the first preparation was 
fixed with Flemming’s fluid, the others with corrosive subli¬ 
mate). 
First preparation (Ehrlich’s haematoxylin, two hours ; eosin, 
ten minutes): cytoplasm pale lilac; nucleoli in the nucleus, and 
when first discharged from it, reddish or rusty brown; nucleo¬ 
lar substance at the end of the metaphase lighter in color. 
Second preparation (gentian violet in aqueous solution, twenty- 
five minutes ; eosin, four and one-half minutes) : cytoplasm 
very faintly stained ; nucleoli in the nucleus deep violet, those 
in the cytoplasm yellowish red. 
Third preparation (Ehrlich’s haematoxylin, one hour ; eosin, 
five minutes): cytoplasm pale pink ; nucleoli in the nucleus, 
and when first discharged from it, purple ; nucleolar substance 
in the cytoplasm at the end of the metaphase pure blue. 
Fourth preparation (Ehrlich’s haematoxylin, forty minutes ; 
eosin, five minutes) : nucleolar substance within and without 
the nucleus yellowish red ; cytoplasm of a paler red. 
Fifth preparation (Mayer’s acid carmine, twenty minutes ; 
Lyons blue, five minutes): cytoplasm unstained ; nucleoli in the 
nucleus, and, when first discharged, bluish green; nucleolar 
substance at the end of the metaphase reddish purple in the 
cytoplasm. These methods of double staining show that the 
nucleolar substance, when discharged from the nucleus, under¬ 
goes some chemical change in the cytoplasm ; and they 
serve to distinguish, further, this substance from the true 
cytoplasm. 
