CHAPTER III 



ON TECHNIQUE 



In the work here reported, two things were desired: To obtain 

 as accurate as possible a measurement of the rate of growth of the 

 cultures studied, and also to obtain, as far as possible clear micro- 

 scopic preparations of the cells, from which reasonably accurate 

 measurements of their size and form could be made, since the pur- 

 pose of the work has been to study morphologic variations of bac- 

 teria quantitatively, and to correlate these variations as far as 

 possible with the rate of growth. For various reasons it was neces- 

 sary to use solid media for the growth of the organisms. First, 

 because it is difficult to obtain clear microscopic preparations from 

 liquid media, because of the presence of peptone or other material 

 which obscures the picture; and second, because it is necessary to 

 obtain considerable numbers of cells for the purpose of microscopic 

 observation during the early hours of growth when they are relatively 

 few in number in the medium. If liquid media were used, it would 

 be necessary to centrifuge, wash, and recentrifuge, in order to clear 

 the organisms of the accompanying debris of the medium, and to 

 sufficiently concentrate them. In addition to being time consuming, 

 it was thought likely that this process would be apt to lead to some 

 artificial variations in the morphology of the cells. 



Broth cultures were, however, used for certain studies of B. 

 megatherium described in Chapter IV (Cultures II-VI). Since this 

 organism grows very slowly in liquid media because it grows only 

 at the bottom and therefore lacks oxygen, some method had to 

 be used for providing aeration. In Cultures II and III this was 

 accomplished by inoculating 1,500 c.c. quantities of broth, and 

 then distributing this in 60 c.c. amounts into Kolle flasks, so that 

 the broth was disposed in a thin layer with a large surface exposed 

 to the air. A flask was removed for sampling after each hour, assum- 

 ing that the conditions of growth were identical in all of the flasks. 

 In Cultures IV, V, and VI, 1,500 c.c. flasks of broth were also inocu- 



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