1914) 
SCHRAMM—PURE CULTURE METHODS IN THE ALGZE 35 
ous exterior characteristic of the cells of both species, 
makes it probable that the pure colonies developed, not from 
mature individuals, but from autocolonies (produced within 
mature cells) which either had just escaped from the mother 
cell or had done so during the vigorous shaking,—in either of 
which cases they are free from adhering bacteria. 
Chlorococcum humicola (Nüg.) Rabenh.—This species was 
isolated in the zoósporie condition. Тһе alga, collected from 
soil, was placed in sterile mineral nutrient solution and after · 
twenty-four hours produced zoóspores in abundance. Platings 
with these yielded numerous pure colonies from which successful 
transfers were made. In this connection it should be men- 
tioned that all zoóspores thus far experimented with—including 
a considerable variety of forms—have been found free from 
bacteria. It is needless to say, therefore, that the presence 
of zoóspores in the life cycle of any alga provides a logical 
point of attack for its isolation in pure culture. While not all 
the attempts to isolate zoósporie forms in pure culture have 
proved successful, it is entirely probable that they will when 
the general technique is more closely adapted to individual 
forms. 
Protosiphon botryoides (Kütz.) Klebs.—The vegetative plant 
of Protosiphon, with its root-like process extending into the 
soil and the large aerial portion, is so persistently covered with 
bacteria that its isolation in pure culture in this condition 
is quite impossible. With slight desiccation, however, large 
numbers of chlamydospores with dry non-gelatinous mem- 
branes appear, which, at least so long as they remain enclosed 
within the mother membrane, are free from bacteria. From 
these, isolations in pure culture can be readily made according 
to the second method suggested for Chlorella—by carefully 
washing an individual plant filled with chlamydospores, liber- 
ating the latter by teasing with needles or by a slight pressure 
of the cover glass, and plating in the usual manner. Another 
method which has yielded pure cultures, but which is not 
to be recommended because it is far less reliable than the one 
just described, is based on the use of the motile gametes. 
When vigorous Protosiphon plants, growing on soil, are covered 
with distilled water, gametes, which congregate in the lighted 
