122 
Harriettc Chick. 
of environment, and it is certain that our knowledge of these 
organisms would be materially advanced if attempts were also 
made to study them when associated with the forms accompanying 
them in nature. Hitherto this has rarely been done, hut the 
following examples may serve to illustrate the point. 
In 1895, Winogradsky isolated from the soil Clostridium pas- 
teurianum, an organism which possessed the specific property of 
fixing free nitrogen from the air. Before the isolation was com¬ 
pleted, a stage was reached when the investigator was workingwith 
a mixture of three microbes only, two bacteria, and the Clostridium 
which proved to be the nitrogen-fixing agent. The culture was 
robust, fixed nitrogen with ease, and continued to do so throughout 
a series of generations. A separation of the mixture was then 
attempted and pure cultures of the two bacteria were easily 
obtained, but neither when together or alone, could either of them 
fix the nitrogen of the air, or develop in a culture fluid deprived of 
combined nitrogen. The Clostridium , on the other hand, was 
isolated with the greatest difficulty; satisfactory pure cultures were 
at last obtained, but only when cultivated under strictly anaerobic 
conditions, and these cultures could only he induced to grow and fix 
free nitrogen if air were excluded and nitrogen were caused to 
bubble continuously through the culture-fluid. This striking 
difference between the external conditions required by Clostridium 
pasteurianum when growing in nature, as constrasted with a pure 
culture, must find explanation in the result of some symbiosis with 
other organisms, and Winogradsky was able, as a consequence of 
work with mixed cultures, to offer the following solution. Clostridium 
pasteurianum, itself a strictly anaerobic organism can flourish and 
do its work under aerobic conditions if associated with an extremely 
aerobic species which absorbs the oxygen of the air with energy, 
thus creating an anaerobic environment for the Clostridium. Such 
associate or associates, moreover, themselves unable to assimilate 
free nitrogen, can in their turn feed upon the nitrogen fixed and 
elaborated by Clostridium pasteurianum. 
The organisms concerned with nitrification in the soil may be 
taken as a second example. These bacteria are of two classes, the 
first attacks the ammonia and converts it to nitrites, while the 
second completes the oxidation to nitrates. Their study in pure 
culture presents great technical difficulties from the surprising fact 
that they are unable on account of the organic matter present, to 
grow upon the culture-media ordinarily employed in bacteriological 
