370 
Spratt. — Some Observations on the 
produced such as Klebs and others have used in cultivating various Algae. 
Here, however, exceedingly slight growth of the Anabaena was obtained, 
although bacterial colonies appeared. Blotting-paper soaked in dilute sap 
from the tubercles, and kept moist in a Petri dish, was found to provide 
a suitable medium for the artificial culture of the Alga, small green patches of 
Anabaena appearing after a time in various parts of the blotting-paper. 
They appeared to lodge themselves in the pores of the substratum and 
there grow and divide. In the case of all the above media, several positions 
both with regard to light and temperature were tried, and the most favour- 
able for the development of the organism appeared to be one illuminated 
by diffuse light, with a temperature of about 35 0 C. 
In view of the fact that during some period of its life-history this 
Anabaena must continue its existence in the soil around the Cycas roots in 
order that it may infect the newly formed tubercles, a kind of artificial soil, 
placed in a Petri dish and kept damp, was inoculated with some of the 
Alga. Here, however, arose a new difficulty, namely, the manipulation of 
such a medium for the examination of the growth therein. Further, as 
will be shown later, it is exceedingly improbable that the organism exists 
in the soil in its easily recognizable vegetative condition. 
The various stages in the life cycle were repeatedly obtained directly 
from the nodules, kept for varying periods, as described above. In order to 
determine the connexion between the stages thus observed, a number of 
hanging-drop preparations were employed. 
When studying the action of various stains and reagents on the 
organism, it was found most successful and convenient to use albuminized 
slides, and then allow the drop containing the Alga to dry on the slide 
slowly at the atmospheric temperature. With this method very little con- 
traction of cells or tlieir contents took place, and when dry, the organisms 
were adhering to the slide, so that any reagent could be readily applied for 
any length of time. When it was desired to use a fixative, a small quantity 
of 2 % osmic acid was added to the drop containing the Alga. Flemming’s 
fluid and Bouin’s fixative have also been used with considerable success. 
The various stains and chemicals used, together with their reactions, will be 
described later, as they emphasize particular portions of the life cycle. 
Vegetative Structure. 
The species of Anabaena found in the Cycas tubercle (PI. XXXI I, Fig. 1) 
is a typical member of that group, each filament having around it a sheath 
of highly transparent mucilage. The trichomes are more or less straight 
or circinnate, and heterocysts are very abundant. The vegetative cells are 
small spherical cells, slightly flattened at each end. Their contents are blue- 
green in colour, usually very granular, but they may assume a homogeneous 
appearance. 
