[Vol. 6 



20 ANNALS OF THE MISSOURI BOTANICAL GARDEN 



The application of " micro' ' technique to the problem at 

 hand did not seem especially promising, as the nitrogen de- 

 terminations were still accompanied with considerable dif- 

 ficulty, and since the error of the analysis was almost as great, 

 the final value computed to mgs, N per 100 cc. contains an ap- 

 preciably greater error. This point is discussed again be- 

 low. 



Although the data are somewhat erratic, it seems permis- 

 sible to conclude that the addition of colloidal iron to Ashby 's 

 solution produces a beneficial effect and also that calcium 

 carbonate has a beneficial action even though the reaction of 

 the culture medium is carefully adjusted beforehand, and even 

 though it appears to cause a greater flocking out of the col- 

 loidal ferric oxide. 



It seems that we can conclude with reasonable safety from 

 the above admittedly crude results that in a general way the 

 work of Remy and Rosing has been confirmed. The benefi- 

 cial results obtained by the addition of colloidal ferric oxide 

 to culture solutions are much less marked than those of Remy 

 and Rosing, yet there seems to be no reasonable doubt that 

 such action is well worth further study, particularly in the 

 line of the role of the colloidal ferric oxide. Before such a 

 study can be carried on advantageously it is necessary to 

 make a decided improvement in experimental methods. This 

 point will now be considered. 



IMPROVEMENT IN METHODS 



One of the first points to be considered in the improvement 



of experimental methods was that of a suitable method of 

 sugar determination. The exact measurement of the energy 

 consumption in cultures of Azotobacter is worthy of much 

 more study than it has received. Many workers have made 

 computations on the amount of mannite or carbohydrate 

 added to the culture medium, disregarding the residual 

 amount of energy-supplying material. The exact determina- 

 tion of mannite is not feasible, and the determinations that 

 have been made of dextrose have been accomplished with the 



