12 Stopes and Fujii, The nutritive relations of the surrounding tissues etc. 
Even in the fresh nnstained condition most of the detailed structure 
of these “protein vacuoles” and the grains in the cytoplasm are to 
be clearly seen, particularly with the oil immersion, and they show 
a nnmber of granules in eacli which react towards reagents as 
protein suhstance; thus we see that they are by no means the result 
of fixing as was suggested by Blackman 1 ). Digestion experiments 
however, which yielded such satisfactory results with Macrozamia etc., 
were technically very difficult with Pinus, for on using sections of 
fresh material the vacuoles ran together so that the cytoplasm 
becarne uniformly granulär even when left in pure water only for 
a short time. With prolonged treatment with the warm digestive 
fluid it was soon impossible to distinguish the granules of the 
protein vacuoles owing to the indistinct homogeneous appearance 
of the whole mass of cytoplasm, so that we were unable to determine 
the effects of digestion on the protein granules themselves. With 
fixed material on the other hand the granules appeared to have 
been rendered more resistent, and the chief granules in a protein 
vacuole were not digested even after a long time in the fluid. 
With acetic methyl green however, we found that the nuclei of the 
endosperm, jacket cells, and egg cell stained quite strouglv, but 
that the protein granules in the egg cytoplasm and in the “protein 
vacuoles’’ were hardly stained, if at all. This shows that these protein 
granules differ from nuclei and have no nuclein as their constituent 
part. 2 ) 
In some cases, and this appeared to be quite erratic, there 
were grains present in the protein vacuoles which were exceptionally 
refractive and which proved on staining to be starch grains (see 
fig. 10). Thus in the “protein vacuoles“ we get not only protein 
suhstance, but also carbohydrate in the form of starch grains. For 
the present we propose the term “nutritive vacuoles” to replace 
“protein vacuoles”, but we will have more to say later on this 
subject. It is interesting to note that in Hofmeister’s 3 ) original 
paper he figures the „Keimbläschen“ so accurately as to appear 
as if he had observed not only the protein bodies but also the 
starch grains without recognising them. The starch grains we have 
observed in these vacuoles are always small, sometimes there being 
only one or two, sometimes several together. Though they are not 
by any means always present we have not been able to observe as 
yet any regulär periodicity in their appearance corresponding to that 
in the earlier stages described. 
In that stage of the ovule in which the contents of the 
pollentube are about to be discharged into the egg cell we found 
the following conditions. The nucellus tip was quite crammed with 
*) Blackman, Y. H., „Cytol. feat. of fertil. in Pinus sylvestris (Pliil. 
Trans. Roy. Soc. Lond. Ser. B. 1898. see p. 417.) 
2 ) Zacharias, E., „Ueb. Rachweis u. Vorkommen v. Nuclein“. (Bericht, 
d. D. Bot. Ges. Bd. XVI. p. 194—197.) 
3 ) Hofmeister, W., „Vergleich. Unters, d. Keimung etc. höherer Krypto¬ 
gamen ... u. d. Samenbildung d. Coniferenk Leipzig 1851. cf. plate XXIX. 
fig. 1, 3 and 4. 
