2 
AMERICAN JOURNAL OF BOTANY 
[Vol. 8 
appeared, so far as the author is aware, since the above-cited paper. As 
has already been indicated, the results of experiments with pure cultures 
have been pretty generally negative as regards the ability of these forms to 
increase the nitrogen content of the culture. In the light of results pre- 
sented here, some of the previous experimental work will be considered in 
the general discussion to follow. 
Methods 
Seven species of Chlorophyceae were isolated and used in pure culture. 
In making the isolations the plate culture method, as described by Schramm 
(1914 h), was employed. With the exception of one species, Protococcus sp., 
all isolations were made from growths occurring on soil; the material for 
the Protococcus culture was secured from the bark of an elm tree. 
The absolute identity of all the species has not as yet been determined. 
Cultures have been submitted to several authorities on the group, but for 
some of the forms the determinations received have been somewhat at 
variance. For that reason no attempt has been made to apply specific 
names to all the organisms. Moreover, as will be apparent later, the abso- 
lute identity of the forms, though highly desirable, does not become of 
paramount importance because of the very similar way in which all the 
species seem to react. Unless otherwise indicated, therefore, the different 
species will be referred to by number and genus, or by number only, and 
as soon as more satisfactory determinations can be made a list will be pub- 
lished, if possible, in this journal. The forms used in the experiments 
include the following: 
Species number i . Chlorella vulgaris Beyr. There seems to be no 
doubt about the identity of this species. 
Species number 2. Stichococcus sp. 
Species number 3. Protosiphon hotryoides (Kg.) Klebs. 
Species number 5. Chlorella sp. A small form with cup-chaped chro- 
matophore. 
Species number 6. Scenedesmus sp. 
Species number 7. Protococcus sp. 
Species number 11. Chlorella sp. A large form with clathrate chro- 
matophore. 
All these species have been carried along on mineral nutrient agar for 
two or three years; they have been repeatedly transferred to media con- 
taining glucose or sucrose, and have frequently been examined micro- 
scopically. They are known to be free from bacteria and are pure cultures 
in the strict sense. 
Culture Media. — In the experiments Kjeldahl flasks of Pyrex glass and 
of 500 cc. capacity were used as culture flasks, because of the obvious 
advantage of analyzing checks and cultures without transferring the material 
to a digestion flask. Approximately 150 grams of mineral nutrient agar 
were supplied as a medium for each culture. In spite of the difliculties 
