44 Wager.— The Nucleolus and Nuclear Division 
colouration. Thus in gentian violet and safranin the nucleolus stains red, 
the chromosomes deep red, whilst the cytoplasm and nuclear network are 
stained violet. In safranin and picro-nigrosin, well washed out, the larger 
part of the nucleolus stains deep red, but a thin peripheral layer stains blue, 
sometimes giving to the nucleolus the appearance of being surrounded 
by a membrane. The chromosomes stain light red or reddish-blue, the 
spindle deep blue, and the cytoplasm and nuclear network blue. 
In gentian violet the chromosomes are usually more deeply stained 
than the nucleoli of the resting nucleus ; but this appears to be due partly 
to the fact that the stain is more easily washed out of the nucleoli. 
The chromatin-like substance thus appears to occur mainly in the 
outer more deeply stained portion of the nucleolus. This is further sub- 
stantiated by the following observations. 
In sections cut by hand from fresh root-apices and stained in dilute 
acetic acid solution of methyl green the protoplasm and nucleus both stain 
bluish-green, but the nucleolus is the most deeply stained, especially in 
the actively growing cells, and the outer layers of the vacuolate nucleoli 
are the most deeply stained. If fresh sections are placed in pepsin solu- 
tion for from three to six hours, at a temperature of 36° C., a contraction 
and distinct diminution of the cell contents are shown. The nucleolus 
becomes much smaller and presents a bright glistening appearance. On 
staining in the methyl green solution, the protoplasmic remnant stains 
green, while the nucleolar remnant stains bright green, and in some cases 
a few granules, probably chromatin-granules in the nuclear network, take a 
similar bright green colour. The nuclear network stains like the cytoplasm. 
Again, if sections, either fresh or in spirit, be treated according to the 
method of Macallum, we get a strong reaction for phosphorus in the 
nucleoli and chromosomes, but very little in the nuclear reticulum. In 
vacuolar nucleoli this reaction is confined mainly to the outer layers ; we 
get very little in the vacuolar portion. And further, we get an intense 
reaction for phosphorus in the nucleolar remnant which is left after treat- 
ment with digestive fluid. 
In gentian violet-stained specimens the nucleolar remnant left in 
contact with the chromosomes at the time of the formation of the nuclear 
plate (Fig. 21) is often much less deeply stained than the chromosomes, and 
in some cases probably represents that inner portion of the resting nucleolus 
which does not become stained deeply in nuclear stains. It is impossible 
to definitely decide this point, however, by microscopic examination. 
The observations just described all point to the conclusion that there 
are at least two different substances in the nucleolus, one of which at any 
rate possesses the reaction of chromatin. It seems to me probable that, as 
Cavara suggests, the explanation of the different accounts which have been 
1 Loc. cit., 1898. 
