382 BULLETIN OF THE UNITED STATES FISH COMMISSION. 
tions. The difficulty attending the cultivation of the Pleiirococciis-like form was very great, and it 
was not until after a very large number of attempts had been made that vigorous cultures were j 
obtained from which conclusions could be drawn with any degree of certainty. All ordinary solu- 
tions, so commonly used in the cultivation of algee, failed as culture media. In 0.2 to 0.4 per cent . 
Knop’s solution the form simply assumed a yellowish color and passed into a resting condition. 
In Knop’s solution without calcium it remained green for at least six weeks, but showed no indica- | 
tion of reproduction. Organic solutions of various compositions and concentrations proved more 
favorable to its existence, but even in these not more than three generations could usually be I 
obtained in any culture. The only solution tried that seemed really favorable was from a quantity ' 
of decaying Anabcena flos-aquce. The chemical composition of this solution and the degree of 
concentration were not determined, but in it reproduction took place rapidly, and, as far as could 
be determined, normally. This solution proved equally favorable for Ccelastrum. Pure cultures 
were then made of each of the forms and placed in conditions as nearly alike as possible. 
In the course of four weeks an abundance of material of both forms was obtained. These cul- 
tures were then repeated several times, and the results agreed each time. Even externally a differ- 
ence' in the cultures could be detected. The Ccelastrurn form showed as a very thin green covering 
on the bottom of the culture glass, while in the other culture a thick cloudy layer, 8 to 4 mm. deep, 
covered the bottom. A minute examination showed a still more marked difference, and one which 
was identical with the two forms from which the cultures were made. The thick layer on the 
bottom of the Pleurococcus culture was composed of the floating loose compound clusters embedded j 
in jelly exactly as in the plankton, except perhaps that the arrangement of the cells was less regular 
(fig. ix, 2-4). Unless reproduction had just occurretl all the cells were more or less separated 
from one another, sometimes widely so, but all were held in place by the surrounding jelly. In a 
fresh Ccelastrum culture, where there were many hundreds of coenobia, but one instance was 
noticed where any separation of a cell from a coenobium occurred. Each of the other coenobia was 
complete and distinct, without any connection with the other coenobia in the culture (fig. ix, 5). 
In old cultures the disintegration of the coenobia was more frequent, but in no case after disintegra- 
tion had occurred were the individual cells connected by a surrounding gelatinous substance. There 
is, as Senn (1898) has stated, a relatively thin gelatinous envelope to the cells, but after the cells have 
become appreciably separated from one another this envelope no longer connects them. In no case 
did Ccelastrum rnicroporurn form compound clusters. As a result of these experiments, it seems 
evident to the writer that the two forms are distinct species and can not be united. 
Artari (’92), in his description of Pleurococcus regularis, says nothing about the presence of a 
gelatinous envelope, but from his figures it is evidently present, as the cells, though loosely 
arranged, are held in place by some substance not shown. The gelatinous envelope of a cluster, 
when taken from the plankton, as well as of those in the artificial culture, is homogeneous, but 
somewhat denser near the cells. When treated with tannate vesuvin the jelly is colored brown, 
but no prismatic radiations from the cell are shown, as in Staurogenia apiculata Lemm. The 
thickness of the envelope seems to vary somewhat with age, being in young individuals less in 
diameter and denser in consistency than in older compounds (fig. ix, 2, 3). When distilled water 
is added the substance becomes at least partially dissolved. 
The membrane is thin and consists of cellulose, turning blue when iodine and sulphuric acid 
are added. The chloroplast is, as Artari states, a hollow sphere closely lining the membrane, but 
with a circular opening at one side. In each chloroplast is a single pyrenoid. When stained with 
lifematoxylin the single nucleus may be seen. 
The new complexes arise by the division of the cell contents into 2, 4, 8, 16, and possibly 32 
parts (fig. ix, 1). Each of these becomes invested with a cell membrane, and the enveloping 
mother membrane becomes more or less irregularly ruptured and the cell complex is set free. In 
this process of division the first visible step is the division of the pyrenoid, then the division of the 
chloroplast. At just what time the nucleus divides in reference to the division of the other parts 
was not determined. After the chloroplast has undergone one or more divisions the appearance is 
that of a cell with several chloroplasts, each with its own pyrenoid. It is a question whether 
mistakes have not been made at times by investigators in different species of algas in taking these 
portions of the divided chloroplast to be entire chloroplasts and characteristic for the species. The 
mother membrane, after it is cast off, remains for a time embedded in the gelatinous substance 
(fig. ix, 2, 3a), but in the older complexes it is no longer visible (fig. ix, 1,4). 
