[VOL.6 



28 ANNALS OP THE MISSOURI BOTANICAL GARDEN 



would reveal irregularities in the method of inoculation bet- 

 ter than would a longer period. The results reported as mgs. 

 N per culture are shown in table vi. 



Method A, in which the suspension was incubated 24 hours 

 on a shaking machine, gave slightly higher results, yet growth 

 was certain and reasonably uniform in those inoculated ac- 

 cording to method B. It is rather difficult to decide between 

 these two methods on the basis of the above experiment. In 

 subsequent work method A was favored, but if for any reason 

 it was undesirable to delay the experiment 24 hours method B 

 was used. The significant thing is that growth took place in 

 every flask seeded. The difficulty encountered in the ' ' orienta- 

 tion experiments" was probably due to the fact that the sus- 

 pensions were too dilute. No more difficulty was experienced 

 in obtaining growth from the heavy suspensions used. How- 

 ever, some rather surprising failures of growths on plates 

 from dilutions from the suspensions were observed. The point 

 seemed to be that in working with Azotobacter heavier sus- 

 pensions must be used than are needed with most other 

 bacteria. This is possibly to be explained by their heavy 

 slime production. 



FINAL EXPERIMENTS 



The few experiments which we were able to carry out after 

 revising our methods were designed to see how far the facts 

 would fit the hypothesis suggested above ; that is, to what ex- 

 tent certain variations with regard to the presence of a second 

 phase in the nutrient solution would affect the growth and de- 

 velopment of the microorganism under study. Experi- 

 ments were conducted on the following points: (1) removal 

 of the solid phase ; (2) restoring of the solid phase ; (3) homog- 

 enous nutrient solutions; and (4) the action of protective 

 colloids designed to partially or wholly prevent the flocking 

 out of the phosphate precipitate. These experiments will 

 now be considered in the order named. 



REMOVAL OF SOLID PHASE 



For the study of this point Ashby tap water dextrose 1 me- 



1 Whenever dextrose was used the culture medium was sterilized by the inter- 

 mittent method. 



